Sample records for lrrk2 kinase inhibitors
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Achieving neuroprotection with LRRK2 kinase inhibitors in Parkinson disease.
West, Andrew B
2017-12-01
In the translation of discoveries from the laboratory to the clinic, the track record in developing disease-modifying therapies in neurodegenerative disease is poor. A carefully designed development pipeline built from discoveries in both pre-clinical models and patient populations is necessary to optimize the chances for success. Genetic variation in the leucine-rich repeat kinase two gene (LRRK2) is linked to Parkinson disease (PD) susceptibility. Pathogenic mutations, particularly those in the LRRK2 GTPase (Roc) and COR domains, increase LRRK2 kinase activities in cells and tissues. In some PD models, small molecule LRRK2 kinase inhibitors that block these activities also provide neuroprotection. Herein, the genetic and biochemical evidence that supports the involvement of LRRK2 kinase activity in PD susceptibility is reviewed. Issues related to the definition of a therapeutic window for LRRK2 inhibition and the safety of chronic dosing are discussed. Finally, recommendations are given for a biomarker-guided initial entry of LRRK2 kinase inhibitors in PD patients. Four key areas must be considered for achieving neuroprotection with LRRK2 kinase inhibitors in PD: 1) identification of patient populations most likely to benefit from LRRK2 kinase inhibitors, 2) prioritization of superior LRRK2 small molecule inhibitors based on open disclosures of drug performance, 3) incorporation of biomarkers and empirical measures of LRRK2 kinase inhibition in clinical trials, and 4) utilization of appropriate efficacy measures guided in part by rigorous pre-clinical modeling. Meticulous and rational development decisions can potentially prevent incredibly costly errors and provide the best chances for LRRK2 inhibitors to slow the progression of PD. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.
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Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond.
Göring, Stefan; Taymans, Jean-Marc; Baekelandt, Veerle; Schmidt, Boris
2014-10-01
The most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation G2019S is associated with Parkinson's disease (PD). It enhances kinase activity and has been identified in both familial and sporadic cases. Kinase activity was reported to be required for LRRK2 mutants to exert their toxic effects. Hence LRRK2 kinase inhibition may be a promising therapeutic target for PD. Here we report on the discovery and characterization of indolinone based LRRK2 inhibitors. Indolinone 15b, the most potent and selective inhibitor of the present series, is characterized by an IC50 of 15nM against wild-type LRRK2 and 10nM against the LRRK2 G2019S mutant, respectively. Compound 15b was further evaluated in a kinase panel including 46 human protein kinases and in a zebrafish embryo phenotype assay, which enabled toxicity determination in whole organisms. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Leucine-rich repeat kinase 2 inhibitors: a review of recent patents (2011 - 2013).
Kethiri, Raghava R; Bakthavatchalam, Rajagopal
2014-07-01
Leucine-rich repeat kinase 2 (LRRK2) is a large (2527 residues) complex multi-domain protein that has GTPase and kinase domains. Autosomal dominant missense mutations in LRRK2 have been found in individuals with Parkinson's disease (PD) and are considered responsible for 1% of all cases of PD. Among the mutations confirmed to contribute to PD pathogenicity, G2019S is the most common cause of PD and it increases the kinase activity of LRRK2 by around threefold. LRRK2 has received considerable attention as a therapeutic target for PD, and LRRK2 inhibitors may help prevent and/or treat the disease. LRRK2 inhibitors are being investigated by various industrial and academic institutions. The present review covers patents literature on small-molecule LRRK2 inhibitors patented between 2011 and 2013. Currently, wild-type and mutant LRRK2 are being examined as therapeutic targets for PD. In testimony to the significance of these novel targets, over 20 patent applications related to LRRK2 have been filed in the last 3 years. Several distinct chemotypes have been reported to be LRRK2 inhibitors with very good potency. These compounds are being used to elucidate the physiological and pathophysiological functions of LRRK2, and some may even emerge as therapeutics for PD.
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Cirnaru, Maria D.; Marte, Antonella; Belluzzi, Elisa; Russo, Isabella; Gabrielli, Martina; Longo, Francesco; Arcuri, Ludovico; Murru, Luca; Bubacco, Luigi; Matteoli, Michela; Fedele, Ernesto; Sala, Carlo; Passafaro, Maria; Morari, Michele; Greggio, Elisa; Onofri, Franco; Piccoli, Giovanni
2014-01-01
Mutations in Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains executing several functions, including GTP hydrolysis, kinase activity, and protein binding. Robust evidence suggests that LRRK2 acts at the synaptic site as a molecular hub connecting synaptic vesicles to cytoskeletal elements via a complex panel of protein-protein interactions. Here we investigated the impact of pharmacological inhibition of LRRK2 kinase activity on synaptic function. Acute treatment with LRRK2 inhibitors reduced the frequency of spontaneous currents, the rate of synaptic vesicle trafficking and the release of neurotransmitter from isolated synaptosomes. The investigation of complementary models lacking LRRK2 expression allowed us to exclude potential off-side effects of kinase inhibitors on synaptic functions. Next we studied whether kinase inhibition affects LRRK2 heterologous interactions. We found that the binding among LRRK2, presynaptic proteins and synaptic vesicles is affected by kinase inhibition. Our results suggest that LRRK2 kinase activity influences synaptic vesicle release via modulation of LRRK2 macro-molecular complex. PMID:24904275
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Small molecule kinase inhibitors for LRRK2 and their application to Parkinson's disease models.
Kramer, Thomas; Lo Monte, Fabio; Göring, Stefan; Okala Amombo, Ghislaine Marlyse; Schmidt, Boris
2012-03-21
Parkinson's disease (PD) is the second most common neurodegenerative disorder. Several single gene mutations have been linked to this disease. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) indicate LRRK2 as promising therapeutic target for the treatment of PD. LRRK2 mutations were observed in sporadic as well as familial PD patients and have been investigated intensively. LRRK2 is a large and complex protein, with multiple enzymatic and protein-interaction domains, each of which is effected by mutations. The most common mutation in PD patients is G2019S. Several LRRK2 inhibitors have been reported already, although the crystal structure of LRRK2 has not yet been determined. This review provides a summary of known LRRK2 inhibitors and will discuss recent in vitro and in vivo results of these inhibitors.
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Chen, Jinhua; Chen, Ying; Pu, Jiali
2018-04-27
Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons in the midbrain. The pathogenesis of PD is not fully understood but is likely caused by a combination of genetic and environmental factors. Several genes are associated with the onset and progression of familial PD. There is increasing evidence that leucine-rich repeat kinase 2 (LRRK2) plays a significant role in PD pathophysiology. Many studies have been conducted to elucidate the functions of LRRK2 and identify effective LRRK2 inhibitors for PD treatment. In this review, we discuss the role of LRRK2 in PD and recent progress in the use of LRRK2 inhibitors as therapeutic agents. Key Messages: LRRK2 plays a significant role in the pathophysiology of PD, and pharmacological inhibition of LRRK2 has become one of the most promising potential therapies for PD. Further research is warranted to determine the functions of LRRK2 and expand the applications of LRRK2 inhibitors in PD treatment. © 2018 S. Karger AG, Basel.
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Liu, Min; Bender, Samantha A.; Cuny, Gregory D; Sherman, Woody; Glicksman, Marcie; Ray, Soumya S.
2014-01-01
A number of well-known type II inhibitors (ATP non-competitive) that bind kinases in their DFG-out conformation were tested against wild-type LRRK2 and the most common Parkinson’s disease-linked mutation G2019S. We found that traditional type II inhibitors exhibit surprising variability in their inhibition mechanism between wild type (WT) and the G2019S mutant of LRRK2. The type II kinase inhibitors were found to work by an ATP-competitive fashion against the G2019S mutant, whereas they appear to follow the expected non-competitive mechanism against WT. Since the G2019S mutation lies in the DXG-motif (DYG in LRRK2 but DFG in most other kinases) of the activation loop, we explored the structural consequence of the mutation on loop dynamics using an enhanced sampling method called metadynamics. The simulations suggest that the G2019S mutation stabilizes the DYG-in state of LRRK2 through a series of hydrogen bonds, leading to an increase in the conformational barrier between the active and inactive forms of the enzyme and a relative stabilization of the active form. The conformational bias toward the active form of LRRK2 mutants has two primary consequences: 1) the mutant enzyme becomes hyperactive, a known contributor to the Parkinsonian phenotype, as a consequence of being “locked” into the activated state and 2) the mutation creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. Our results suggest that developing type II inhibitors, which are generally considered superior to type I inhibitors due to desirable selectivity profiles, might be especially challenging for the G2019S LRRK2 mutant. PMID:23379419
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Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D
2015-11-01
Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Schapansky, Jason; Khasnavis, Saurabh; DeAndrade, Mark P; Nardozzi, Jonathan D; Falkson, Samuel R; Boyd, Justin D; Sanderson, John B; Bartels, Tim; Melrose, Heather L; LaVoie, Matthew J
2018-03-01
Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD. Copyright © 2017 Elsevier Inc. All rights reserved.
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Leucine-rich repeat kinase 2 inhibitors: a patent review (2014-2016).
Galatsis, Paul
2017-06-01
Leucine-rich repeat kinase 2 (LRRK2) is a member of the Tyrosine Kinase-Like (TKL) branch of the kinome tree and is a multi-domain protein that includes GTPase and kinase activity. While genome-wide association studies (GWAS) has linked LRRK2 with Crohn's disease and leprosy, it has received the greatest attention due to it being implicated as one of the genetic loci associated with autosomal dominant inheritance in Parkinson's disease (PD). Areas covered: In this review, the small molecule patent literature from 2014-2016 with a focus on composition of matter and use patents was surveyed. Scifinder was primarily searched using 'LRRK2' as the query to identify all relevant literature and then triaged for small molecule patents. Expert opinion: The patent landscape around LRRK2 continues to develop. The early patents covered using existing kinase inhibitors for use against LRRK2. This evolved to compounds specifically designed for selectivity against LRRK2, but key exemplified compounds lacked sufficient brain exposure to affect sufficient efficacy. More recent compounds have addressed this deficiency and show greater potential for treating PD. While potency will be necessary to generate medicines with low human daily doses, brain penetration and safety will be the key differentiators for ultimately determining the most effective LRRK2 disease-modifying treatment for PD.
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Longo, Francesco; Russo, Isabella; Shimshek, Derya R.; Greggio, Elisa; Morari, Michele
2014-01-01
The leucine-rich repeat kinase 2 mutation G2019S in the kinase-domain is the most common genetic cause of Parkinson's disease. To investigate the impact of the G2019S mutation on motor activity in vivo, a longitudinal phenotyping approach was developed in knock-in (KI) mice bearing this kinase-enhancing mutation. Two cohorts of G2019S KI mice and wild-type littermates (WT) were subjected to behavioral tests, specific for akinesia, bradykinesia and overall gait ability, at different ages (3, 6, 10, 15 and 19 months). The motor performance of G2019S KI mice remained stable up to the age of 19 months and did not show the typical age-related decline in immobility time and stepping activity of WT. Several lines of evidence suggest that enhanced LRRK2 kinase activity is the main contributor to the observed hyperkinetic phenotype of G2019S KI mice: i) KI mice carrying a LRRK2 kinase-dead mutation (D1994S KD) showed a similar progressive motor decline as WT; ii) two LRRK2 kinase inhibitors, H-1152 and Nov-LRRK2-11, acutely reversed the hyperkinetic phenotype of G2019S KI mice, while being ineffective in WT or D1994S KD animals. LRRK2 target engagement in vivo was further substantiated by reduction of LRRK2 phosphorylation at Ser935 in the striatum and cortex at efficacious doses of Nov-LRRK2-11, and in the striatum at efficacious doses of H-1152. In summary, expression of the G2019S mutation in the mouse LRRK2 gene confers a hyperkinetic phenotype that is resistant to age-related motor decline, likely via enhancement of LRRK2 kinase activity. This study provides an in vivo model to investigate the effects of LRRK2 inhibitors on motor function. PMID:25107341
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Delbroek, Lore; Van Kolen, Kristof; Steegmans, Liesbeth; da Cunha, Raquel; Mandemakers, Wim; Daneels, Guy; De Bock, Pieter-Jan; Zhang, Jinwei; Gevaert, Kris; De Strooper, Bart; Alessi, Dario R.; Verstreken, Patrik; Moechars, Diederik W.
2014-01-01
After the discovery of kinase activating mutations in leucine-rich repeat kinase 2 (LRRK2) as associated with autosomal dominant forms of Parkinson’s disease, inhibition of the kinase is being extensively explored as a disease modifying strategy. As signaling properties and substrate(s) of LRRK2 are poorly documented, autophosphorylation has been an important readout for the enzyme’s activity. Western blotting using anti-phospho-S910 or S935 LRRK2 antibodies showed effectiveness in demonstrating inhibitory effects of compounds. In this communication we describe two types of enzyme-linked immunosorbent assays (ELISA) to determine LRRK2 protein levels and kinase activity. Both assays take advantage of the sensitivity of the earlier described total and pS935 antibodies for detection (Nichols et al., Biochem. J. 2010) [10]. The first assay is based on anti-GFP-based capturing of overexpressed LRRK2 and is highly suitable to show cellular effects of kinase inhibitors in a 96-well format. In the other platform anti-LRRK2-based capturing allows detection of endogenously expressed LRRK2 in rat tissue with no significant signal in tissue from LRRK2 knockout rats. Furthermore, both assays showed a significant reduction in pS935 levels on cellular and transgenic R1441C/G LRRK2. With the anti-LRRK2 ELISA we were able to detect LRRK2 phosphorylation in human peripheral blood mononuclear cells (PBMC). To conclude, we report two sensitive assays to monitor LRRK2 expression and kinase activity in samples coming from cellular and in vivo experimental settings. Both can show their value in drug screening and biomarker development but will also be useful in the elucidation of LRRK2-mediated signaling pathways. PMID:23313773
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2014-01-01
Background Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. Results Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. Conclusions Given DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs. PMID:24885928
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G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization
Papkovskaia, Tatiana D.; Chau, Kai-Yin; Inesta-Vaquera, Francisco; Papkovsky, Dmitri B.; Healy, Daniel G.; Nishio, Koji; Staddon, James; Duchen, Michael R.; Hardy, John; Schapira, Anthony H.V.; Cooper, J. Mark
2012-01-01
The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD. PMID:22736029
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Mir, Rafeeq; Tonelli, Francesca; Lis, Pawel; Macartney, Thomas; Polinski, Nicole K.; Martinez, Terina N.; Chou, Meng-Yun; Howden, Andrew J.M.; König, Theresa; Hotzy, Christoph; Milenkovic, Ivan; Brücke, Thomas; Zimprich, Alexander; Sammler, Esther; Alessi, Dario R.
2018-01-01
Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans. PMID:29743203
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Ray, Soumya; Bender, Samantha; Kang, Stephanie; Lin, Regina; Glicksman, Marcie A.; Liu, Min
2014-01-01
The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtideS. Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtideS. As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtideS phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3–7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance to ATP competitive inhibitors. Two well known DFG-out or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYG motif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. PMID:24695735
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Tozzi, Alessandro; Tantucci, Michela; Marchi, Saverio; Mazzocchetti, Petra; Morari, Michele; Pinton, Paolo; Mancini, Andrea; Calabresi, Paolo
2018-02-12
Parkinson's disease (PD) is a neurodegenerative disorder in which genetic and environmental factors synergistically lead to loss of midbrain dopamine (DA) neurons. Mutation of leucine-rich repeated kinase2 (Lrrk2) genes is responsible for the majority of inherited familial cases of PD and can also be found in sporadic cases. The pathophysiological role of this kinase has to be fully understood yet. Hyperactivation of Lrrk2 kinase domain might represent a predisposing factor for both enhanced striatal glutamatergic release and mitochondrial vulnerability to environmental factors that are observed in PD. To investigate possible alterations of striatal susceptibility to mitochondrial dysfunction, we performed electrophysiological recordings from the nucleus striatum of a G2019S Lrrk2 mouse model of PD, as well as molecular and morphological analyses of G2019S Lrrk2-expressing SH-SY5Y neuroblastoma cells. In G2019S mice, we found reduced striatal DA levels, according to the hypothesis of alteration of dopaminergic transmission, and increased loss of field potential induced by the mitochondrial complex I inhibitor rotenone. This detrimental effect is reversed by the D2 DA receptor agonist quinpirole via the inhibition of the cAMP/PKA intracellular pathway. Analysis of mitochondrial functions in G2019S Lrrk2-expressing SH-SY5Y cells revealed strong rotenone-induced oxidative stress characterized by reduced Ca 2+ buffering capability and ATP synthesis, production of reactive oxygen species, and increased mitochondrial fragmentation. Importantly, quinpirole was able to prevent all these changes. We suggest that the G2019S-Lrrk2 mutation is a predisposing factor for enhanced striatal susceptibility to mitochondrial dysfunction induced by exposure to mitochondrial environmental toxins and that the D2 receptor stimulation is neuroprotective on mitochondrial function, via the inhibition of cAMP/PKA intracellular pathway. We suggest new possible neuroprotective strategies for patients carrying this genetic alteration based on drugs specifically targeting Lrrk2 kinase domain and mitochondrial functionality.
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Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R.
2014-01-01
By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration. PMID:24403142
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Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa
2016-01-13
Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.
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Li, Xianting; Wang, Qing Jun; Pan, Nina; Lee, Sangkyu; Zhao, Yingming; Chait, Brian T.; Yue, Zhenyu
2011-01-01
Background Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown. Methodology/Principal Findings Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2. Conclusions/Significance LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD. PMID:21390248
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Mamais, Adamantios; Chia, Ruth; Beilina, Alexandra; Hauser, David N.; Hall, Christine; Lewis, Patrick A.; Cookson, Mark R.; Bandopadhyay, Rina
2014-01-01
Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are a common genetic cause of Parkinson disease, but the mechanisms whereby LRRK2 is regulated are unknown. Phosphorylation of LRRK2 at Ser910/Ser935 mediates interaction with 14-3-3. Pharmacological inhibition of its kinase activity abolishes Ser910/Ser935 phosphorylation and 14-3-3 binding, and this effect is also mimicked by pathogenic mutations. However, physiological situations where dephosphorylation occurs have not been defined. Here, we show that arsenite or H2O2-induced stresses promote loss of Ser910/Ser935 phosphorylation, which is reversed by phosphatase inhibition. Arsenite-induced dephosphorylation is accompanied by loss of 14-3-3 binding and is observed in wild type, G2019S, and kinase-dead D2017A LRRK2. Arsenite stress stimulates LRRK2 self-association and association with protein phosphatase 1α, decreases kinase activity and GTP binding in vitro, and induces translocation of LRRK2 to centrosomes. Our data indicate that signaling events induced by arsenite and oxidative stress may regulate LRRK2 function. PMID:24942733
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Phosphorylation of p53 by LRRK2 induces microglial tumor necrosis factor α-mediated neurotoxicity
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ho, Dong Hwan, E-mail: ethan2887@gmail.com; Seol, Wongi; Eun, Jin Hwan
Leucine-rich repeat kinase (LRRK2), a major causal gene of Parkinson's disease (PD), functions as a kinase. The most prevalent mutation of LRRK2 is G2019S. It exhibits increased kinase activity compared to the wildtype LRRK2. Previous studies have shown that LRRK2 can phosphorylate p53 at T304 and T377 of threonine-X-arginine (TXR) motif in neurons. Reduction of LRRK2 expression or inhibition of LRRK2 kinase activity has been shown to be able to alleviate LPS-induced neuroinflammation in microglia cells. In this study, we found that LRRK2 could also phosphorylate p53 in microglia model BV2 cells. Transfection of BV2 with phosphomimetic p53 T304/377D significantlymore » increased the secretion of pro-inflammatory cytokine TNFα compared to BV2 transfected with p53 wild type after LPS treatment. In addition, conditioned media from these transfected cells increased the death of dopaminergic neuronal SN4741 cells. Moreover, such neurotoxic effect was rescued by co-treatment with the conditioned media and etanercept, a TNFα blocking antibody. Furthermore, TNFα secretion was significantly increased in primary microglia derived from G2019S transgenic mice treated with LPS compared to that in cells derived from their littermates. These results suggest that LRRK2 kinase activity in microglia can contribute to neuroinflammation in PD via phosphorylating p53 at T304 and T377 site. - Highlights: • LPS stimulates LRRK2-mediated p53 phosphorylation and its nuclear localization. • Phosphorylation of p53 by LRRK2 in microglia enhances TNFα expression. • Microglial TNFα via LRRK2-induced p53 phosphorylation decreases neuronal survival.« less
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Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.
2016-01-01
Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524
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Interplay between Leucine-Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy.
Park, Sangwook; Han, Seulki; Choi, Insup; Kim, Beomsue; Park, Seung Pyo; Joe, Eun-Hye; Suh, Young Ho
2016-01-01
The deposit of polyubiquitinated aggregates has been implicated in the pathophysiology of Parkinson's disease (PD), and growing evidence indicates that selective autophagy plays a critical role in the clearance of ubiquitin-positive protein aggregates by autophagosomes. The selective autophagic receptor p62/SQSTM-1, which associates directly with both ubiquitin and LC3, transports ubiquitin conjugates to autophagosomes for degradation. Leucine-rich repeat kinase 2 (LRRK2), a PD-associated protein kinase, is tightly controlled by autophagy-lysosome degradation as well as by the ubiquitin-proteasome pathway. However, little is known about the degradation of ubiquitinated LRRK2 via selective autophagy. In the present study, we found that p62/SQSTM-1 physically interacts with LRRK2 as a selective autophagic receptor. The overexpression of p62 leads to the robust degradation of LRRK2 through the autophagy-lysosome pathway. In addition, LRRK2 indirectly regulates Ser351 and Ser403 phosphorylation of p62. Of particular interest, the interaction between phosphorylated p62 and Keap1 is reduced by LRRK2 overexpression. Therefore, we propose that the interplay between LRRK2 and p62 may contribute to the pathophysiological function and homeostasis of LRRK2 protein.
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Kritzinger, Astrid; Ferger, Boris; Gillardon, Frank; Stierstorfer, Birgit; Birk, Gerald; Kochanek, Stefan; Ciossek, Thomas
2018-06-01
Mutations in leucine-rich repeat kinase 2 (LRRK2) age-dependently cause Parkinson's disease and are associated with several inflammatory diseases. So far, the potential role of LRRK2 expression in glial cells as mediators of neuroinflammation and the influence of aging have not been investigated in viral vector-based LRRK2 animal models. In this study, we compared the effect of striatal injection of high-capacity adenoviral vectors expressing either a kinase-overactive LRRK2 with the familial G2019S mutation or a kinase-inactive LRRK2 variant in young and old C57BL/6J mice. The intrinsic adenovirus tropism guided preferentially glial transduction, and the vector design led to stable expression for at least 6 months. In histopathological analysis, young mice expressing either LRRK2 variant presented with transient vacuolization of striatal white fiber tracts accompanied by accumulation of microglial cells and astrogliosis, but inflammation resolved without permanent damage. Old mice had a stronger and prolonged inflammatory reaction and experienced permanent damage in form of partial neuron loss after 3 months exclusively in case of LRRK2_G2019S expression. The autophagic receptor p62 accumulated in cells with high levels of either LRRK2 variant, even more so in old mice. We conclude that the aging mouse brain is more susceptible to LRRK2-associated pathology, and in this model, glial LRRK2 expression significantly contributed to neuroinflammation, ultimately causing neurodegeneration. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
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Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.
Madero-Pérez, Jesús; Fdez, Elena; Fernández, Belén; Lara Ordóñez, Antonio J; Blanca Ramírez, Marian; Gómez-Suaga, Patricia; Waschbüsch, Dieter; Lobbestael, Evy; Baekelandt, Veerle; Nairn, Angus C; Ruiz-Martínez, Javier; Aiastui, Ana; López de Munain, Adolfo; Lis, Pawel; Comptdaer, Thomas; Taymans, Jean-Marc; Chartier-Harlin, Marie-Christine; Beilina, Alexandria; Gonnelli, Adriano; Cookson, Mark R; Greggio, Elisa; Hilfiker, Sabine
2018-01-23
Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
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Guaitoli, Giambattista; Raimondi, Francesco; Gilsbach, Bernd K.; Gómez-Llorente, Yacob; Deyaert, Egon; Renzi, Fabiana; Li, Xianting; Schaffner, Adam; Jagtap, Pravin Kumar Ankush; Boldt, Karsten; von Zweydorf, Felix; Gotthardt, Katja; Lorimer, Donald D.; Yue, Zhenyu; Burgin, Alex; Janjic, Nebojsa; Sattler, Michael; Versées, Wim; Ueffing, Marius; Ubarretxena-Belandia, Iban; Kortholt, Arjan; Gloeckner, Christian Johannes
2016-01-01
Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson’s disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity—together with the LRR domain—to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. PMID:27357661
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[Advance of the study on LRRK2 gene in Parkinson's disease].
Zhang, Yu; Chen, Shengdi
2008-12-01
The leucine-rich repeat kinase2 (LRRK2) has been identified to be the gene causing autosomal dominant inherited Parkinson's disease(PD)8. The clinical features of this type of PD are similar to those of idiopathic PD, but the pathological changes are diverse. The mutation types and frequencies of the LRRK2 distribute unevenly in different populations. LRRK2 is a large complex protein with multiple functions and expresses widely in human body. Sequence alignment shows that LRRK2 might be a multiple function kinase for substrate phosphorylation and might also act as a scaffolding protein. Further study on the physiological function and pathogenic mechanism of LRRK2 will help to find out the possible pathogenesis and new treatment for PD.
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LRRK2 modulates vulnerability to mitochondrial dysfunction in C. elegans
Saha, Shamol; Guillily, Maria; Ferree, Andrew; Lanceta, Joel; Chan, Diane; Ghosh, Joy; Hsu, Cindy H.; Segal, Lilach; Raghavan, Kesav; Matsumoto, Kunihiro; Hisamoto, Naoki; Kuwahara, Tomoki; Iwatsubo, Takeshi; Moore, Landon; Goldstein, Lee; Cookson, Mark; Wolozin, Benjamin
2009-01-01
Summary Mutations in leucine rich repeat kinase 2 (LRRK2) cause autosomal dominant familial Parkinson’s disease. We generated lines of C. elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 expression increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C or kinase dead LRRK2 was less than protection by wild type LRRK2. Knockdown of lrk-1, the endogenous orthologue of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT∷GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the WT line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing WT LRRK2; however loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different than that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition, and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson’s disease. PMID:19625511
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Meixner, Andrea; Boldt, Karsten; Van Troys, Marleen; Askenazi, Manor; Gloeckner, Christian J.; Bauer, Matthias; Marto, Jarrod A.; Ampe, Christophe; Kinkl, Norbert; Ueffing, Marius
2011-01-01
Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. PMID:20876399
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Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke
2015-01-01
Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680
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Rivero-Ríos, Pilar; Gómez-Suaga, Patricia; Fernández, Belén; Madero-Pérez, Jesús; Schwab, Andrew J; Ebert, Allison D; Hilfiker, Sabine
2015-06-01
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene comprise the most common cause of familial Parkinson's disease (PD), and variants increase the risk for sporadic PD. LRRK2 displays kinase and GTPase activity, and altered catalytic activity correlates with neurotoxicity, making LRRK2 a promising therapeutic target. Despite the importance of LRRK2 for disease pathogenesis, its normal cellular function, and the mechanism(s) by which pathogenic mutations cause neurodegeneration remain unclear. LRRK2 seems to regulate a variety of intracellular vesicular trafficking events to and from the late endosome in a manner dependent on various Rab proteins. At least some of those events are further regulated by LRRK2 in a manner dependent on two-pore channels (TPCs). TPCs are ionic channels localized to distinct endosomal structures and can cause localized calcium release from those acidic stores, with downstream effects on vesicular trafficking. Here, we review current knowledge about the link between LRRK2, TPC- and Rab-mediated vesicular trafficking to and from the late endosome, highlighting a possible cross-talk between endolysosomal calcium stores and Rab proteins underlying pathomechanism(s) in LRRK2-related PD.
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Vancraenenbroeck, Renée; Lobbestael, Evy; Weeks, Stephen D; Strelkov, Sergei V; Baekelandt, Veerle; Taymans, Jean-Marc; De Maeyer, Marc
2012-03-01
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions. Copyright © 2011 Elsevier B.V. All rights reserved.
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Li, Xianting; Patel, Jyoti C; Wang, Jing; Avshalumov, Marat V; Nicholson, Charles; Buxbaum, Joseph D; Elder, Gregory A; Rice, Margaret E; Yue, Zhenyu
2010-02-03
PARK8/LRRK2 (leucine-rich repeat kinase 2) was recently identified as a causative gene for autosomal dominant Parkinson's disease (PD), with LRRK2 mutation G2019S linked to the most frequent familial form of PD. Emerging in vitro evidence indicates that aberrant enzymatic activity of LRRK2 protein carrying this mutation can cause neurotoxicity. However, the physiological and pathophysiological functions of LRRK2 in vivo remain elusive. Here we characterize two bacterial artificial chromosome (BAC) transgenic mouse strains overexpressing LRRK2 wild-type (Wt) or mutant G2019S. Transgenic LRRK2-Wt mice had elevated striatal dopamine (DA) release with unaltered DA uptake or tissue content. Consistent with this result, LRRK2-Wt mice were hyperactive and showed enhanced performance in motor function tests. These results suggest a role for LRRK2 in striatal DA transmission and the consequent motor function. In contrast, LRRK2-G2019S mice showed an age-dependent decrease in striatal DA content, as well as decreased striatal DA release and uptake. Despite increased brain kinase activity, LRRK2-G2019S overexpression was not associated with loss of DAergic neurons in substantia nigra or degeneration of nigrostriatal terminals at 12 months. Our results thus reveal a pivotal role for LRRK2 in regulating striatal DA transmission and consequent control of motor function. The PD-associated mutation G2019S may exert pathogenic effects by impairing these functions of LRRK2. Our LRRK2 BAC transgenic mice, therefore, could provide a useful model for understanding early PD pathological events.
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Short- and long-term effects of LRRK2 on axon and dendrite growth.
Sepulveda, Bryan; Mesias, Roxana; Li, Xianting; Yue, Zhenyu; Benson, Deanna L
2013-01-01
Mutations in leucine-rich repeat kinase 2 (LRRK2) underlie an autosomal-dominant form of Parkinson's disease (PD) that is clinically indistinguishable from idiopathic PD. The function of LRRK2 is not well understood, but it has become widely accepted that LRRK2 levels or its kinase activity, which is increased by the most commonly observed mutation (G2019S), regulate neurite growth. However, growth has not been measured; it is not known whether mean differences in length correspond to altered rates of growth or retraction, whether axons or dendrites are impacted differentially or whether effects observed are transient or sustained. To address these questions, we compared several developmental milestones in neurons cultured from mice expressing bacterial artificial chromosome transgenes encoding mouse wildtype-LRRK2 or mutant LRRK2-G2019S, Lrrk2 knockout mice and non-transgenic mice. Over the course of three weeks of development on laminin, the data show a sustained, negative effect of LRRK2-G2019S on dendritic growth and arborization, but counter to expectation, dendrites from Lrrk2 knockout mice do not elaborate more rapidly. In contrast, young neurons cultured on a slower growth substrate, poly-L-lysine, show significantly reduced axonal and dendritic motility in Lrrk2 transgenic neurons and significantly increased motility in Lrrk2 knockout neurons with no significant changes in length. Our findings support that LRRK2 can regulate patterns of axonal and dendritic growth, but they also show that effects vary depending on growth substrate and stage of development. Such predictable changes in motility can be exploited in LRRK2 bioassays and guide exploration of LRRK2 function in vivo.
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Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.
Sancho, Rosa M; Law, Bernard M H; Harvey, Kirsten
2009-10-15
Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.
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Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways
Sancho, Rosa M.; Law, Bernard M.H.; Harvey, Kirsten
2009-01-01
Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2–DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2–DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2–DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2–DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease. PMID:19625296
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Piccoli, Giovanni; Onofri, Franco; Cirnaru, Maria Daniela; Kaiser, Christoph J. O.; Jagtap, Pravinkumar; Kastenmüller, Andreas; Pischedda, Francesca; Marte, Antonella; von Zweydorf, Felix; Vogt, Andreas; Giesert, Florian; Pan, Lifeng; Antonucci, Flavia; Kiel, Christina; Zhang, Mingjie; Weinkauf, Sevil; Sattler, Michael; Sala, Carlo; Matteoli, Michela; Ueffing, Marius
2014-01-01
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function. PMID:24687852
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Gómez-Suaga, Patricia; Rivero-Ríos, Pilar; Fdez, Elena; Blanca Ramírez, Marian; Ferrer, Isidro; Aiastui, Ana; López De Munain, Adolfo; Hilfiker, Sabine
2014-12-20
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Venderova, Katerina; Kabbach, Ghassan; Abdel-Messih, Elizabeth; Zhang, Yi; Parks, Robin J; Imai, Yuzuru; Gehrke, Stephan; Ngsee, Johnny; Lavoie, Matthew J; Slack, Ruth S; Rao, Yong; Zhang, Zhuohua; Lu, Bingwei; Haque, M Emdadul; Park, David S
2009-11-15
Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.
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Wong, Alicia Yoke Wei; Oikonomou, Vasilis; Paolicelli, Giuseppe; De Luca, Antonella; Pariano, Marilena; Fric, Jan; Tay, Hock Soon; Ricciardi-Castagnoli, Paola; Zelante, Teresa
2018-01-01
The Parkinson's disease-associated protein, Leucine-rich repeat kinase 2 (LRRK2), a known negative regulator of nuclear factor of activated T cells (NFAT), is expressed in myeloid cells such as macrophages and dendritic cells (DCs) and is involved in the host immune response against pathogens. Since, the Ca 2+ /NFAT/IL-2 axis has been previously found to regulate DC response to the fungus Aspergillus , we have investigated the role played by the kinase LRRK2 during fungal infection. Mechanistically, we found that in the early stages of the non-canonical autophagic response of DCs to the germinated spores of Aspergillus , LRRK2 undergoes progressive degradation and regulates NFAT translocation from the cytoplasm to the nucleus. Our results shed new light on the complexity of the Ca 2+ /NFAT/IL-2 pathway, where LRRK2 plays a role in controlling the immune response of DCs to Aspergillus .
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Law, Bernard M H; Spain, Victoria A; Leinster, Veronica H L; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J; Taymans, Jean-Marc; Urban, Mary K; Sancho, Rosa M; Blanca Ramírez, Marian; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R; Berwick, Daniel C; Harvey, Kirsten
2014-01-10
Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.
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Verma, Manish; Callio, Jason; Otero, P Anthony; Sekler, Israel; Wills, Zachary P; Chu, Charleen T
2017-11-15
Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis. SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD. Copyright © 2017 the authors 0270-6474/17/3711152-15$15.00/0.
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Kim, Kwang Soo; Marcogliese, Paul C; Yang, Jungwoo; Callaghan, Steve M; Resende, Virginia; Abdel-Messih, Elizabeth; Marras, Connie; Visanji, Naomi P; Huang, Jana; Schlossmacher, Michael G; Trinkle-Mulcahy, Laura; Slack, Ruth S; Lang, Anthony E; Park, David S
2018-05-14
Leucine-rich repeat kinase 2 ( LRRK2 ) has been implicated in both familial and sporadic Parkinson's disease (PD), yet its pathogenic role remains unclear. A previous screen in Drosophila identified Scar/WAVE (Wiskott-Aldrich syndrome protein-family verproline) proteins as potential genetic interactors of LRRK2 Here, we provide evidence that LRRK2 modulates the phagocytic response of myeloid cells via specific modulation of the actin-cytoskeletal regulator, WAVE2. We demonstrate that macrophages and microglia from LRRK2-G2019S PD patients and mice display a WAVE2-mediated increase in phagocytic response, respectively. Lrrk2 loss results in the opposite effect. LRRK2 binds and phosphorylates Wave2 at Thr470, stabilizing and preventing its proteasomal degradation. Finally, we show that Wave2 also mediates Lrrk2 - G2019S-induced dopaminergic neuronal death in both macrophage-midbrain cocultures and in vivo. Taken together, a LRRK2-WAVE2 pathway, which modulates the phagocytic response in mice and human leukocytes, may define an important role for altered immune function in PD.
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Hui, Ken Y; Fernandez-Hernandez, Heriberto; Hu, Jianzhong; Schaffner, Adam; Pankratz, Nathan; Hsu, Nai-Yun; Chuang, Ling-Shiang; Carmi, Shai; Villaverde, Nicole; Li, Xianting; Rivas, Manual; Levine, Adam P; Bao, Xiuliang; Labrias, Philippe R; Haritunians, Talin; Ruane, Darren; Gettler, Kyle; Chen, Ernie; Li, Dalin; Schiff, Elena R; Pontikos, Nikolas; Barzilai, Nir; Brant, Steven R; Bressman, Susan; Cheifetz, Adam S; Clark, Lorraine N; Daly, Mark J; Desnick, Robert J; Duerr, Richard H; Katz, Seymour; Lencz, Todd; Myers, Richard H; Ostrer, Harry; Ozelius, Laurie; Payami, Haydeh; Peter, Yakov; Rioux, John D; Segal, Anthony W; Scott, William K; Silverberg, Mark S; Vance, Jeffery M; Ubarretxena-Belandia, Iban; Foroud, Tatiana; Atzmon, Gil; Pe'er, Itsik; Ioannou, Yiannis; McGovern, Dermot P B; Yue, Zhenyu; Schadt, Eric E; Cho, Judy H; Peter, Inga
2018-01-10
Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10 -10 ) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10 -8 ). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
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Kinase inhibitors for CNS diseases: an analysis of the recent patent literature.
Amigoni, Federica; Legnaghi, Elena; Pevarello, Paolo
2012-05-01
Protein kinases (PKs), as members of an important target class in current pharmaceutical research, have been mostly exploited so far in therapeutic areas such as oncology and inflammation. However, basic research on some PKs as key components of molecular mechanisms underlying neurodegeneration and neuroprotection may translate into new medicines for CNS diseases in the next few years. This review is an account of recent patents dealing with kinase inhibitors primarily designed for CNS indications. CNS-directed patents on kinase modulators published after 2008 were surveyed using SciFinder(®) and public patent search engines. Some PK targets, such as GSK-3β, CDK5, ROCK and p38α MAPK, continue to attract interest even though a clinical proof-of-concept is yet to be attained in a CNS setting. Less established PKs such as LRRK2, MLK, PAK and DAPK-1 hold promise as valuable targets of the future.
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Beilina, Alexandria; Rudenko, Iakov N.; Kaganovich, Alice; Civiero, Laura; Chau, Hien; Kalia, Suneil K.; Kalia, Lorraine V.; Lobbestael, Evy; Chia, Ruth; Ndukwe, Kelechi; Ding, Jinhui; Nalls, Mike A.; Olszewski, Maciej; Hauser, David N.; Kumaran, Ravindran; Lozano, Andres M.; Baekelandt, Veerle; Greene, Lois E.; Taymans, Jean-Marc; Greggio, Elisa; Cookson, Mark R.; Nalls, Mike A.; Plagnol, Vincent; Martinez, Maria; Hernandez, Dena G; Sharma, Manu; Sheerin, Una-Marie; Saad, Mohamad; Simón-Sánchez, Javier; Schulte, Claudia; Lesage, Suzanne; Sveinbjörnsdóttir, Sigurlaug; Arepalli, Sampath; Barker, Roger; Ben-Shlomo, Yoav; Berendse, Henk W; Berg, Daniela; Bhatia, Kailash; de Bie, Rob M A; Biffi, Alessandro; Bloem, Bas; Bochdanovits, Zoltan; Bonin, Michael; Bras, Jose M; Brockmann, Kathrin; Brooks, Janet; Burn, David J; Charlesworth, Gavin; Chen, Honglei; Chong, Sean; Clarke, Carl E; Cookson, Mark R; Cooper, J Mark; Corvol, Jean Christophe; Counsell, Carl; Damier, Philippe; Dartigues, Jean-François; Deloukas, Panos; Deuschl, Günther; Dexter, David T; van Dijk, Karin D; Dillman, Allissa; Durif, Frank; Dürr, Alexandra; Edkins, Sarah; Evans, Jonathan R; Foltynie, Thomas; Gao, Jianjun; Gardner, Michelle; Gibbs, J Raphael; Goate, Alison; Gray, Emma; Guerreiro, Rita; Gústafsson, Ómar; Harris, Clare; van Hilten, Jacobus J; Hofman, Albert; Hollenbeck, Albert; Holton, Janice; Hu, Michele; Huang, Xuemei; Huber, Heiko; Hudson, Gavin; Hunt, Sarah E; Huttenlocher, Johanna; Illig, Thomas; München, Helmholtz Zentrum; Jónsson, Pálmi V; Lambert, Jean-Charles; Langford, Cordelia; Lees, Andrew; Lichtner, Peter; München, Helmholtz Zentrum; Limousin, Patricia; Lopez, Grisel; Lorenz, Delia; McNeill, Alisdair; Moorby, Catriona; Moore, Matthew; Morris, Huw R; Morrison, Karen E; Mudanohwo, Ese; O’Sullivan, Sean S; Pearson, Justin; Perlmutter, Joel S; Pétursson, Hjörvar; Pollak, Pierre; Post, Bart; Potter, Simon; Ravina, Bernard; Revesz, Tamas; Riess, Olaf; Rivadeneira, Fernando; Rizzu, Patrizia; Ryten, Mina; Sawcer, Stephen; Schapira, Anthony; Scheffer, Hans; Shaw, Karen; Shoulson, Ira; Sidransky, Ellen; Smith, Colin; Spencer, Chris C A; Stefánsson, Hreinn; Steinberg, Stacy; Stockton, Joanna D; Strange, Amy; Talbot, Kevin; Tanner, Carlie M; Tashakkori-Ghanbaria, Avazeh; Tison, François; Trabzuni, Daniah; Traynor, Bryan J; Uitterlinden, André G; Velseboer, Daan; Vidailhet, Marie; Walker, Robert; van de Warrenburg, Bart; Wickremaratchi, Mirdhu; Williams, Nigel; Williams-Gray, Caroline H; Winder-Rhodes, Sophie; Stefánsson, Kári; Hardy, John; Heutink, Peter; Brice, Alexis; Gasser, Thomas; Singleton, Andrew B; Wood, Nicholas W; Chinnery, Patrick F; Arepalli, Sampath; Cookson, Mark R; Dillman, Allissa; Ferrucci, Luigi; Gibbs, J Raphael; Hernandez, Dena G; Johnson, Robert; Longo, Dan L; Majounie, Elisa; Nalls, Michael A; O’Brien, Richard; Singleton, Andrew B; Traynor, Bryan J; Troncoso, Juan; van der Brug, Marcel; Zielke, H Ronald; Zonderman, Alan B
2014-01-01
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. PMID:24510904
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Chen, Zhongcan; Cao, Zhen; Zhang, Wei; Gu, Minxia; Zhou, Zhi Dong; Li, Baojie; Li, Jing; Tan, Eng King; Zeng, Li
2017-11-15
Pathogenic leucine-rich repeat kinase 2 (LRRK2) mutations are recognized as the most common cause of familial Parkinson's disease in certain populations. Recently, LRRK2 mutations were shown to be associated with a higher risk of hormone-related cancers. However, how LRRK2 itself contributes to cancer risk remains unknown. DNA damage causes cancer, and DNA damage responses are among the most important pathways in cancer biology. To understand the role of LRRK2 in DNA damage response pathway, we induced DNA damage by applying genotoxic stress to the cells with Adriamycin. We found that DNA damage enhances LRRK2 phosphorylation at Serine 910, Serine 935 and Serine 1292. We further showed that LRRK2 phosphorylation is abolished in the absence of ATM, suggesting that LRRK2 phosphorylation requires ATM. It should also be noted that LRRK2 interacts with ATM. In contrast, overexpression or knockdown of LRRK2 does not affect ATM phosphorylation, indicating that LRRK2 is the downstream target of ATM in response to DNA damage. Moreover, we demonstrated that LRRK2 increases the expression of p53 and p21 by increasing the Mdm2 phosphorylation in response to DNA damage. Loss-of-function in LRRK2 has the opposite effect to that of LRRK2. In addition, FACS analysis revealed that LRRK2 enhances cell cycle progression into S phase in response to DNA damage, a finding that was confirmed by 5-bromo-2'-deoxyuridine immunostaining. Taken together, our findings demonstrate that LRRK2 plays an important role in the ATM-Mdm2-p53 pathway that regulates cell proliferation in response to DNA damage. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Mestre, Tiago A; Pont-Sunyer, Claustre; Kausar, Farah; Visanji, Naomi P; Ghate, Taneera; Connolly, Barbara S; Gasca-Salas, Carmen; Kern, Drew S; Jain, Jennifer; Slow, Elizabeth J; Faust-Socher, Achinoam; Kasten, Meike; Wadia, Pettarusp M; Zadikoff, Cindy; Kumar, Prakash; de Bie, Ronald M; Thomsen, Teri; Lang, Anthony E; Schüle, Birgitt; Klein, Christine; Tolosa, Eduardo; Marras, Connie
2018-04-17
The objective of this study was to determine phenotypic features that differentiate nonparkinsonian first-degree relatives of PD leucine-rich repeat kinase 2 (LRRK2) G2019S multiplex families, regardless of carrier status, from healthy controls because nonparkinsonian individuals in multiplex families seem to share a propensity to present neurological features. We included nonparkinsonian first-degree relatives of LRRK2 G2019S familial PD cases and unrelated healthy controls participating in established multiplex family LRRK2 cohorts. Study participants underwent neurologic assessment including cognitive screening, olfaction testing, and questionnaires for daytime sleepiness, depression, and anxiety. We used a multiple logistic regression model with backward variable selection, validated with bootstrap resampling, to establish the best combination of motor and nonmotor features that differentiates nonparkinsonian first-degree relatives of LRRK2 G2019S familial PD cases from unrelated healthy controls. We included 142 nonparkinsonian family members and 172 unrelated healthy controls. The combination of past or current symptoms of anxiety (adjusted odds ratio, 4.16; 95% confidence interval, 2.01-8.63), less daytime sleepiness (adjusted odds ratio [1 unit], 0.90; 95% confidence interval, 0.83-0.97], and worse motor UPDRS score (adjusted odds ratio [1 unit], 1.4; 95% confidence interval, 1.20-1.67) distinguished nonparkinsonian family members, regardless of LRRK2 G2019S mutation status, from unrelated healthy controls. The model accuracy was good (area under the curve = 79.3%). A set of motor and nonmotor features distinguishes first-degree relatives of LRRK2 G2019S probands, regardless of mutation status, from unrelated healthy controls. Environmental or non-LRRK2 genetic factors in LRRK2-associated PD may influence penetrance of the LRRK2 G2019S mutation. The relationship of these features to actual PD risk requires longitudinal observation of LRRK2 familial PD cohorts. © 2018 International Parkinson and Movement Disorder Society. © 2018 International Parkinson and Movement Disorder Society.
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Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest
2017-07-11
Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson's disease.
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2012-01-01
Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713
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Yuan, Yiyuan; Cao, Pengxiu; Smith, Mark A.; Kramp, Kristopher; Huang, Ying; Hisamoto, Naoki; Matsumoto, Kunihiro; Hatzoglou, Maria; Jin, Hui; Feng, Zhaoyang
2011-01-01
Mutation of leucine-rich repeat kinase 2 (LRRK2) is the leading genetic cause of Parkinson's Disease (PD), manifested as age-dependent dopaminergic neurodegeneration, but the underlying molecular mechanisms remain unclear. Multiple roles of LRRK2 may contribute to dopaminergic neurodegeneration. Endoplasmic reticulum (ER) stress has also been linked to PD pathogenesis, but its interactive mechanism with PD genetic factors is largely unknown. Here, we used C. elegans, human neuroblastoma cells and murine cortical neurons to determine the role of LRRK2 in maintaining dopaminergic neuron viability. We found that LRRK2 acts to protect neuroblastoma cells and C. elegans dopaminergic neurons from the toxicity of 6-hydroxydopamine and/or human α-synuclein, possibly through the p38 pathway, by supporting upregulation of GRP78, a key cell survival molecule during ER stress. A pathogenic LRRK2 mutant (G2019S), however, caused chronic p38 activation that led to death of murine neurons and age-related dopaminergic-specific neurodegeneration in nematodes. These observations establish a critical functional link between LRRK2 and ER stress. PMID:21857923
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CSF lamp2 concentrations are decreased in female Parkinson's disease patients with LRRK2 mutations.
Klaver, Andrea C; Coffey, Mary P; Aasly, Jan O; Loeffler, David A
2018-03-15
Lysosome-associated membrane glycoprotein 2 (lamp2) plays critical roles in chaperone-mediated autophagy (CMA) and macroautophagy. Its isoform lamp2a is decreased in Parkinson's disease (PD) substantia nigra. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most known common cause of late-onset PD; although LRRK2 is thought to regulate macroautophagy, the influence of LRRK2 mutations on lamp2 concentrations in the CNS is unknown. To examine this issue we compared lamp2 levels in cerebrospinal fluid (CSF) between sporadic PD (sPD) patients (n = 31), LRRK2 PD patients (n = 20), and healthy control subjects with or without LRRK2 mutations (LRRK2 CTL = 30, CTL = 27). We also examined lamp2's correlations with age, oxidative stress, PD progression, and PD duration. Median lamp2 concentrations (pg/mL) were LRRK2 PD = 127, sPD = 333, CTL = 436, and LRRK2 CTL = 412. Log-transformed lamp2 concentrations, adjusting for gender effects (and excluding male LRRK2 PD patients because of low number), were lower in female LRRK2 PD patients than in LRRK2 CTL (p = 0.002) and CTL (p = 0.005) subjects (p = 0.06 for lamp2 comparison between female LRRK2 PD patients and sPD patients). Lamp2 did not appear to be associated with age, PD progression, or PD duration; however, three of four Spearman rho values for correlations between lamp2 and oxidative stress markers in PD subjects were ≥0.30. These findings suggest that CSF lamp2 concentrations may be decreased in female LRRK2 PD patients compared to healthy individuals with or without LRRK2 mutations. Copyright © 2018 Elsevier B.V. All rights reserved.
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Park, Jeongho; Lee, Jang-Won; Cooper, Scott C; Broxmeyer, Hal E; Cannon, Jason R; Kim, Chang H
2017-10-01
Parkinson's disease (PD) is a neurodegenerative disease, whereas Crohn's disease is an inflammatory bowel disease. Interestingly, polymorphisms in the LRRK2 gene have been identified as risk factors for both diseases. LRRK2 G2019S is the most prevalent mutation found in PD. To gain insights into the role of the LRRK2 G2019S gene on the development and activation of the immune system in the brain-gut axis, we investigated the effect of LRRK2 G2019S on bone marrow myeloid progenitors and myeloid cell function in the periphery. We used bacterial artificial chromosome transgenic rats harboring the human LRRK2 G2019S gene. LRRK2 G2019S transgene decreased the numbers of monocytic and granulocytic progenitors in the bone marrow. However, the numbers of peripheral, immature myeloid cells with suppressive activity were increased in the gut and blood circulation of LRRK2 G2019S compared with control rats in various acute and chronic inflammatory responses. In inflammatory conditions, Th17 cell activity was suppressed, but tissue-associated phylum Bacteroidetes was abnormally increased in the intestine of LRRK2 G2019S rats. The abnormally expanded myeloid cells because of the LRRK2 G2019S gene were highly suppressive on Th17 cell differentiation. Moreover, we found that inhibition of LRRK2 kinase affects myeloid progenitors and myeloid cell differentiation. Taken together, the results indicate that abnormal LRRK2 activity can alter bone marrow myelopoiesis, peripheral myeloid cell differentiation, and intestinal immune homeostasis. These findings may have ramifications in immune and inflammatory responses in patients with LRRK2 abnormalities. © Society for Leukocyte Biology.
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Analysis of Lrrk2 R1628P as a risk factor for Parkinson's disease.
Ross, Owen A; Wu, Yih-Ru; Lee, Mei-Ching; Funayama, Manabu; Chen, Meng-Ling; Soto, Alexandra I; Mata, Ignacio F; Lee-Chen, Guey-Jen; Chen, Chiung Mei; Tang, Michelle; Zhao, Yi; Hattori, Nobutaka; Farrer, Matthew J; Tan, Eng-King; Wu, Ruey-Meei
2008-07-01
Common genetic variants that increase the risk for Parkinson's disease may differentiate patient subgroups and influence future individualized therapeutic strategies. Herein we show evidence for leucine-rich repeat kinase 2 (LRRK2) c.4883G>C (R1628P) as a risk factor in ethnic Chinese populations. A study of 1,986 individuals from 3 independent centers in Taiwan and Singapore demonstrates that Lrrk2 R1628P increases risk for Parkinson's disease (odds ratio, 1.84; 95% confidence interval, 1.20-2.83; p = 0.006). Haplotype analysis suggests an ancestral founder for carriers approximately 2,500 years ago. These findings support the importance of LRRK2 variants in sporadic Parkinson's disease. Ann Neurol 2008.
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Functional properties of LRRK2 mutations in Taiwanese Parkinson disease.
Chang, Kuo-Hsuan; Chen, Chiung-Mei; Lin, Chih-Hsin; Chang, Wen-Teng; Jiang, Pei-Ru; Hsiao, Ya-Chin; Wu, Yih-Ru; Lee-Chen, Guey-Jen
2017-03-01
Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7. Copyright © 2016. Published by Elsevier B.V.
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Loeffler, David A; Klaver, Andrea C; Coffey, Mary P; Aasly, Jan O; LeWitt, Peter A
2017-01-01
Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are the most frequent cause of inherited Parkinson's disease (PD). The most common PD-associated LRRK2 mutation, G2019S, induces increased production of reactive oxygen species in vitro . We therefore hypothesized that individuals with PD-associated LRRK2 mutations might have increased concentrations of oxidative stress markers and/or decreased total antioxidant capacity (TAC) in their cerebrospinal fluid (CSF). We measured two oxidative stress markers, namely 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), and TAC in CSF from LRRK2 mutation-bearing PD patients ( LRRK2 PD = 19), sporadic PD patients (sPD = 31), and healthy control subjects with or without these mutations ( LRRK2 CTL = 30, CTL = 27). 8-OHdG and 8-ISO levels were increased in LRRK2 CTL subjects, while TAC was similar between groups. 8-ISO was negatively correlated, and TAC was positively correlated, with Montreal Cognitive Assessment scores in LRRK2 PD, LRRK2 CTL, and CTL subjects. Correlations in both groups of PD patients between the two oxidative stress markers and Unified Parkinson Disease Rating Scale Total scores were weak, while TAC was negatively correlated with these scores. These findings suggest that oxidative stress may be increased in the CNS in healthy individuals with PD-associated LRRK2 mutations. Further, TAC may decrease in the CNS with the progression of PD, and when cognitive impairment is present regardless of the presence or absence of PD.
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Loeffler, David A.; Klaver, Andrea C.; Coffey, Mary P.; Aasly, Jan O.; LeWitt, Peter A.
2017-01-01
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most frequent cause of inherited Parkinson’s disease (PD). The most common PD-associated LRRK2 mutation, G2019S, induces increased production of reactive oxygen species in vitro. We therefore hypothesized that individuals with PD-associated LRRK2 mutations might have increased concentrations of oxidative stress markers and/or decreased total antioxidant capacity (TAC) in their cerebrospinal fluid (CSF). We measured two oxidative stress markers, namely 8-hydroxy-2′-deoxyguanosine (8-OHdG) and 8-isoprostane (8-ISO), and TAC in CSF from LRRK2 mutation-bearing PD patients (LRRK2 PD = 19), sporadic PD patients (sPD = 31), and healthy control subjects with or without these mutations (LRRK2 CTL = 30, CTL = 27). 8-OHdG and 8-ISO levels were increased in LRRK2 CTL subjects, while TAC was similar between groups. 8-ISO was negatively correlated, and TAC was positively correlated, with Montreal Cognitive Assessment scores in LRRK2 PD, LRRK2 CTL, and CTL subjects. Correlations in both groups of PD patients between the two oxidative stress markers and Unified Parkinson Disease Rating Scale Total scores were weak, while TAC was negatively correlated with these scores. These findings suggest that oxidative stress may be increased in the CNS in healthy individuals with PD-associated LRRK2 mutations. Further, TAC may decrease in the CNS with the progression of PD, and when cognitive impairment is present regardless of the presence or absence of PD. PMID:28420983
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Giesert, F; Glasl, L; Zimprich, A; Ernst, L; Piccoli, G; Stautner, C; Zerle, J; Hölter, S M; Vogt Weisenhorn, D M; Wurst, W
2017-09-01
The aim of the present study was to further explore the in vivo function of the Leucine-rich repeat kinase 2 (LRRK2)-gene, which is mutated in certain familial forms of Parkinson's disease (PD). We generated a mouse model harboring the disease-associated point mutation R1441C in the GTPase domain of the endogenous murine LRRK2 gene (LRRK2 R1441C line) and performed a comprehensive analysis of these animals throughout lifespan in comparison with an existing knockdown line of LRRK2 (LRRK2 knockdown line). Animals of both lines do not exhibit severe motor dysfunction or pathological signs of neurodegeneration neither at young nor old age. However, at old age the homozygous LRRK2 R1441C animals exhibit clear phenotypes related to the prodromal phase of PD such as impairments in fine motor tasks, gait, and olfaction. These phenotypes are only marginally observable in the LRRK2 knockdown animals, possibly due to activation of compensatory mechanisms as suggested by in vitro studies of synaptic transmission. Thus, at the organismal level the LRRK2 R1441C mutation does not emerge as a loss of function of the protein, but induces mutation specific deficits. Furthermore, judged by the phenotypes presented, the LRRK2-R1441C knock-in line is a valid preclinical model for the prodromal phase of PD. Copyright © 2017. Published by Elsevier Inc.
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Bravo-San Pedro, José M.; Gómez-Sánchez, Rubén; Niso-Santano, Mireia; Pizarro-Estrella, Elisa; Aiastui-Pujana, Ana; Gorostidi, Ana; Climent, Vicente; López de Maturana, Rakel; Sanchez-Pernaute, Rosario; López de Munain, Adolfo; Fuentes, José M.; González-Polo, Rosa A.
2012-01-01
The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway. PMID:22914360
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Clinical Correlations With Lewy Body Pathology in LRRK2-Related Parkinson Disease
Kalia, Lorraine V.; Lang, Anthony E.; Hazrati, Lili-Naz; Fujioka, Shinsuke; Wszolek, Zbigniew K.; Dickson, Dennis W.; Ross, Owen A.; Van Deerlin, Vivianna M.; Trojanowski, John Q.; Hurtig, Howard I.; Alcalay, Roy N.; Marder, Karen S.; Clark, Lorraine N.; Gaig, Carles; Tolosa, Eduardo; Ruiz-Martínez, Javier; Marti-Masso, Jose F.; Ferrer, Isidre; de Munain, Adolfo López; Goldman, Samuel M.; Schüle, Birgitt; Langston, J. William; Aasly, Jan O.; Giordana, Maria T.; Bonifati, Vincenzo; Puschmann, Andreas; Canesi, Margherita; Pezzoli, Gianni; De Paula, Andre Maues; Hasegawa, Kazuko; Duyckaerts, Charles; Brice, Alexis; Stoessl, A. Jon; Marras, Connie
2015-01-01
IMPORTANCE Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of genetic Parkinson disease (PD) known to date. The clinical features of manifesting LRRK2 mutation carriers are generally indistinguishable from those of patients with sporadic PD. However, some PD cases associated with LRRK2 mutations lack Lewy bodies (LBs), a neuropathological hallmark of PD. We investigated whether the presence or absence of LBs correlates with different clinical features in LRRK2-related PD. OBSERVATIONS We describe genetic, clinical, and neuropathological findings of 37 cases of LRRK2-related PD including 33 published and 4 unpublished cases through October 2013. Among the different mutations, the LRRK2 p.G2019S mutation was most frequently associated with LB pathology. Nonmotor features of cognitive impairment/dementia, anxiety, and orthostatic hypotension were correlated with the presence of LBs. In contrast, a primarily motor phenotype was associated with a lack of LBs. CONCLUSIONS AND RELEVANCE To our knowledge, this is the first report of clinicopathological correlations in a series of LRRK2-related PD cases. Findings from this selected group of patients with PD demonstrated that parkinsonian motor features can occur in the absence of LBs. However, LB pathology in LRRK2-related PD may be a marker for a broader parkinsonian symptom complex including cognitive impairment. PMID:25401511
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CSF Nrf2 and HSPA8 in Parkinson's disease patients with and without LRRK2 gene mutations.
Loeffler, David A; Smith, Lynnae M; Coffey, Mary P; Aasly, Jan O; LeWitt, Peter A
2016-03-01
Leucine-rich repeat kinase 2 (LRRK2) gene mutations are the most common genetic cause of Parkinson's disease (PD). CSF specimens from LRRK2 + PD patients and healthy LRRK2 mutation carriers are, therefore, useful for biomarker studies. This study examined the hypothesis that differences are present between subjects with sporadic PD (sPD), PD carriers of LRRK2 mutations (LRRK2 + PD), healthy control subjects lacking LRRK2 mutations (CTL), and LRRK2 mutation-carrying healthy controls (LRRK2 + CTL) for CSF concentrations of six potential PD biomarkers. Two of these proteins, nuclear factor (erythroid-derived 2)-like 2 ("Nrf2") and heat shock 70 kDa protein 8 ("HSPA8"), were detected in preliminary ELISAs, then measured in a larger cohort (60 sPD, 10 LRRK2 + PD, 23 CTL, 31 LRRK2 + CTL). No statistically significant differences were found between the groups (Nrf2 p = 0.13, HSPA8 p = 0.21). Nrf2 concentrations in LRRK2 + PD subjects were strongly positively associated with Unified Parkinson's Disease Rating Scale (UPDRS) total and motor scores [Spearman rho = 0.77 (p = 0.012) and 0.83 (p = 0.005)] and negatively associated with Montreal Cognitive Assessment (MoCA) scores (rho = -0.57; p = 0.11). Partial correlation coefficient calculations indicated that disease duration contributed to the associations of Nrf2 levels with UPDRS scores and with MoCA scores in this group. While CSF Nrf2 and HSPA8 do not appear to offer diagnostic biomarkers for PD, the associations between Nrf2 levels and UPDRS scores in LRRK2 + PD patients merit further investigation.
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Beccano-Kelly, Dayne A; Kuhlmann, Naila; Tatarnikov, Igor; Volta, Mattia; Munsie, Lise N; Chou, Patrick; Cao, Li-Ping; Han, Heather; Tapia, Lucia; Farrer, Matthew J; Milnerwood, Austen J
2014-01-01
Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.
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Rubio, Justin P.; Topp, Simon; Warren, Liling; St Jean, Pamela L.; Wegmann, Daniel; Kessner, Darren; Novembre, John; Shen, Judong; Fraser, Dana; Aponte, Jennifer; Nangle, Keith; Cardon, Lon R.; Ehm, Margaret G.; Chissoe, Stephanie L.; Whittaker, John C.; Nelson, Matthew R.; Mooser, Vincent E.
2012-01-01
Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype-phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single nucleotide variants (SNVs), 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC-COR-kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Amongst Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. PMID:22415848
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Rubio, Justin P; Topp, Simon; Warren, Liling; St Jean, Pamela L; Wegmann, Daniel; Kessner, Darren; Novembre, John; Shen, Judong; Fraser, Dana; Aponte, Jennifer; Nangle, Keith; Cardon, Lon R; Ehm, Margaret G; Chissoe, Stephanie L; Whittaker, John C; Nelson, Matthew R; Mooser, Vincent E
2012-07-01
Genetic variation in LRRK2 predisposes to Parkinson disease (PD), which underpins its development as a therapeutic target. Here, we aimed to identify novel genotype-phenotype associations that might support developing LRRK2 therapies for other conditions. We sequenced the 51 exons of LRRK2 in cases comprising 12 common diseases (n = 9,582), and in 4,420 population controls. We identified 739 single-nucleotide variants, 62% of which were observed in only one person, including 316 novel exonic variants. We found evidence of purifying selection for the LRRK2 gene and a trend suggesting that this is more pronounced in the central (ROC-COR-kinase) core protein domains of LRRK2 than the flanking domains. Population genetic analyses revealed that LRRK2 is not especially polymorphic or differentiated in comparison to 201 other drug target genes. Among Europeans, we identified 17 carriers (0.13%) of pathogenic LRRK2 mutations that were not significantly enriched within any disease or in those reporting a family history of PD. Analysis of pathogenic mutations within Europe reveals that the p.Arg1628Pro (c4883G>C) mutation arose independently in Europe and Asia. Taken together, these findings demonstrate how targeted deep sequencing can help to reveal fundamental characteristics of clinically important loci. © 2012 Wiley Periodicals, Inc.
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Peng, Fang; Sun, Yi-Min; Chen, Chen; Luo, Su-Shan; Li, Da-Ke; Wang, Yi-Xuan; Yang, Ke; Liu, Feng-Tao; Zuo, Chuan-Tao; Ding, Zheng-Tong; An, Yu; Wu, Jian-Jun; Wang, Jian
2017-02-15
Leucine-rich repeat kinase 2 gene (LRRK2) was recognized associated with both familial and sporadic Parkinson Disease (PD). Seven missense mutations (G2019S, R1441C, R1441G, R1441H, Y1699C, I2020T, N1437H) of it have been confirmed disease- causing. They were common among Caucasian PD patients, but rarely reported in Asian, especially in Chinese Han population. We aimed to identify the frequencies of these seven mutations of LRRK2 in Chinese early-onset PD (EOPD) patients and analyze the phenotypes. One hundred and thirty seven EOPD patients were enrolled for genetic testing. The seven disease-causing mutations of LRRK2 were carried out by target sequencing using Illumina HiSeq 2000 Sequencer. The identified variants were further confirmed by Sanger sequence. The clinical materials were investigated retrospectively. Only one patient (0.73%) was found carrying pathogenetic LRRK2 mutation of R1441C. The age at onset of the female patient was 44. She manifested typical motor symptoms of PD and responded well to levodopa therapy. Longitudinal evaluation showed progression of motor symptoms and depression but no cognitive impairment. The dopamine transporter (DAT) imaging via [11C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) and Positron emission computed tomography (PET) revealed typical dopamine transporter uptake reduction. The LRRK2 R1441C mutation was found in a Chinese EOPD patient for the first time. The manifestations of LRRK2-R1441C carriers were indistinguishable from sporadic PD patients. Copyright © 2016 Elsevier B.V. All rights reserved.
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Manzoni, Claudia; Mamais, Adamantios; Dihanich, Sybille; McGoldrick, Phillip; Devine, Michael J; Zerle, Julia; Kara, Eleanna; Taanman, Jan-Willem; Healy, Daniel G; Marti-Masso, Jose-Felix; Schapira, Anthony H; Plun-Favreau, Helene; Tooze, Sharon; Hardy, John; Bandopadhyay, Rina; Lewis, Patrick A
2013-11-29
LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
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Dodson, Mark W; Leung, Lok K; Lone, Mohiddin; Lizzio, Michael A; Guo, Ming
2014-12-01
Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling. © 2014. Published by The Company of Biologists Ltd.
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Carrion, Maria Dolores Perez; Marsicano, Silvia; Daniele, Federica; Marte, Antonella; Pischedda, Francesca; Di Cairano, Eliana; Piovesana, Ester; von Zweydorf, Felix; Kremmer, Elisabeth; Gloeckner, Christian Johannes; Onofri, Franco; Perego, Carla; Piccoli, Giovanni
2017-07-14
Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.
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Heckman, Michael G.; Elbaz, Alexis; Soto-Ortolaza, Alexandra I.; Serie, Daniel J.; Aasly, Jan O.; Annesi, Grazia; Auburger, Georg; Bacon, Justin A.; Boczarska-Jedynak, Magdalena; Bozi, Maria; Brighina, Laura; Chartier-Harlin, Marie-Christine; Dardiotis, Efthimios; Destée, Alain; Ferrarese, Carlo; Ferraris, Alessandro; Fiske, Brian; Gispert, Suzana; Hadjigeorgiou, Georgios M.; Hattori, Nobutaka; Ioannidis, John P. A.; Jasinska-Myga, Barbara; Jeon, Beom S.; Kim, Yun Joong; Klein, Christine; Kruger, Rejko; Kyratzi, Elli; Lin, Chin-Hsien; Lohmann, Katja; Loriot, Marie-Anne; Lynch, Timothy; Mellick, George D.; Mutez, Eugénie; Opala, Grzegorz; Park, Sung Sup; Petrucci, Simona; Quattrone, Aldo; Sharma, Manu; Silburn, Peter A.; Sohn, Young Ho; Stefanis, Leonidas; Tadic, Vera; Tomiyama, Hiroyuki; Uitti, Ryan J.; Valente, Enza Maria; Vassilatis, Demetrios K.; Vilariño-Güell, Carles; White, Linda R.; Wirdefeldt, Karin; Wszolek, Zbigniew K.; Wu, Ruey-Meei; Xiromerisiou, Georgia; Maraganore, Demetrius M.; Farrer, Matthew J.; Ross, Owen A.
2013-01-01
The best validated susceptibility variants for Parkinson’s disease (PD) are located in the alpha-synuclein (SNCA) and microtubule-associated protein tau (MAPT) genes. Recently, a protective p.N551K-R1398H-K1423K haplotype in the leucine-rich repeat kinase 2 (LRRK2) gene was identified, with p.R1398H appearing to be the most likely functional variant. To date, the consistency of the protective effect of LRRK2 p.R1398H across MAPT and SNCA variant genotypes has not been assessed. To address this, we examined four SNCA variants (rs181489, rs356219, rs11931074, rs2583988), the MAPT H1-haplotype defining variant rs1052553, and LRRK2 p.R1398H (rs7133914) in Caucasian (N=10,322) and Asian (N=2,289) series. There was no evidence of an interaction of LRRK2 p.R1398H with MAPT or SNCA variants (all P≥0.10); the protective effect of p.R1398H was observed at similar magnitude across MAPT and SNCA genotypes, and the risk effects of MAPT and SNCA variants were observed consistently for LRRK2 p.R1398H genotypes. Our results indicate that the association of LRRK2 p.R1398H with PD is independent of SNCA and MAPT variants, and vice versa, in Caucasian and Asian populations. PMID:23962496
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Healy, Daniel G; Falchi, Mario; O'Sullivan, Sean S; Bonifati, Vincenzo; Durr, Alexandra; Bressman, Susan; Brice, Alexis; Aasly, Jan; Zabetian, Cyrus P; Goldwurm, Stefano; Ferreira, Joaquim J; Tolosa, Eduardo; Kay, Denise M; Klein, Christine; Williams, David R; Marras, Connie; Lang, Anthony E; Wszolek, Zbigniew K; Berciano, Jose; Schapira, Anthony HV; Lynch, Timothy; Bhatia, Kailash P; Gasser, Thomas; Lees, Andrew J; Wood, Nicholas W
2008-01-01
Summary Background Mutations in LRRK2, the gene that encodes leucine-rich repeat kinase 2, are a cause of Parkinson's disease (PD). The International LRRK2 Consortium was established to answer three key clinical questions: can LRRK2-associated PD be distinguished from idiopathic PD; which mutations in LRRK2 are pathogenic; and what is the age-specific cumulative risk of PD for individuals who inherit or are at risk of inheriting a deleterious mutation in LRRK2? Methods Researchers from 21 centres across the world collaborated on this study. The frequency of the common LRRK2 Gly2019Ser mutation was estimated on the basis of data from 24 populations worldwide, and the penetrance of the mutation was defined in 1045 people with mutations in LRRK2 from 133 families. The LRRK2 phenotype was defined on the basis of 59 motor and non-motor symptoms in 356 patients with LRRK2-associated PD and compared with the symptoms of 543 patients with pathologically proven idiopathic PD. Findings Six mutations met the consortium's criteria for being proven pathogenic. The frequency of the common LRRK2 Gly2019Ser mutation was 1% of patients with sporadic PD and 4% of patients with hereditary PD; the frequency was highest in the middle east and higher in southern Europe than in northern Europe. The risk of PD for a person who inherits the LRRK2 Gly2019Ser mutation was 28% at age 59 years, 51% at 69 years, and 74% at 79 years. The motor symptoms (eg, disease severity, rate of progression, occurrence of falls, and dyskinesia) and non-motor symptoms (eg, cognition and olfaction) of LRRK2-associated PD were more benign than those of idiopathic PD. Interpretation Mutations in LRRK2 are a clinically relevant cause of PD that merit testing in patients with hereditary PD and in subgroups of patients with PD. However, this knowledge should be applied with caution in the diagnosis and counselling of patients. Funding UK Medical Research Council; UK Parkinson's Disease Society; UK Brain Research Trust; Internationaal Parkinson Fonds; Volkswagen Foundation; National Institutes of Health: National Institute of Neurological Disorders and Stroke and National Institute of Aging; Udall Parkinson's Disease Centre of Excellence; Pacific Alzheimer Research Foundation Centre; Italian Telethon Foundation; Fondazione Grigioni per il Morbo di Parkinson; Michael J Fox Foundation for Parkinson's Research; Safra Global Genetics Consortium; US Department of Veterans Affairs; French Agence Nationale de la Recherche. PMID:18539534
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Catelani, Tiziano; Setzu, Maria Dolores; Solla, Paolo; Marrosu, Francesco; Sanna, Enrico; Kasture, Sanjay; Acquas, Elio
2016-01-01
The common fruit fly Drosophila melanogaster (Dm) is a simple animal species that contributed significantly to the development of neurobiology whose leucine-rich repeat kinase 2 mutants (LRRK2) loss-of-function in the WD40 domain represent a very interesting tool to look into physiopathology of Parkinson’s disease (PD). Accordingly, LRRK2 Dm have also the potential to contribute to reveal innovative therapeutic approaches to its treatment. Withania somnifera Dunal, a plant that grows spontaneously also in Mediterranean regions, is known in folk medicine for its anti-inflammatory and protective properties against neurodegeneration. The aim of this study was to evaluate the neuroprotective effects of its standardized root methanolic extract (Wse) on the LRRK2 loss-of-function Dm model of PD. To this end mutant and wild type (WT) flies were administered Wse, through diet, at different concentrations as larvae and adults (L+/A+) or as adults (L-/A+) only. LRRK2 mutants have a significantly reduced lifespan and compromised motor function and mitochondrial morphology compared to WT flies 1% Wse-enriched diet, administered to Dm LRRK2 as L-/A+and improved a) locomotor activity b) muscle electrophysiological response to stimuli and also c) protected against mitochondria degeneration. In contrast, the administration of Wse to Dm LRRK2 as L+/A+, no matter at which concentration, worsened lifespan and determined the appearance of increased endosomal activity in the thoracic ganglia. These results, while confirming that the LRRK2 loss-of-function in the WD40 domain represents a valid model of PD, reveal that under appropriate concentrations Wse can be usefully employed to counteract some deficits associated with the disease. However, a careful assessment of the risks, likely related to the impaired endosomal activity, is required. PMID:26727265
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Revisiting the Roco G-protein cycle.
Terheyden, Susanne; Ho, Franz Y; Gilsbach, Bernd K; Wittinghofer, Alfred; Kortholt, Arjan
2015-01-01
Mutations in leucine-rich-repeat kinase 2 (LRRK2) are the most frequent cause of late-onset Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins which share a conserved Ras-like G-domain (Roc) and a C-terminal of Roc (COR) domain tandem. The nucleotide state of small G-proteins is strictly controlled by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because of contradictory structural and biochemical data, the regulatory mechanism of the LRRK2 Roc G-domain and the RocCOR tandem is still under debate. In the present study, we solved the first nucleotide-bound Roc structure and used LRRK2 and bacterial Roco proteins to characterize the RocCOR function in more detail. Nucleotide binding induces a drastic structural change in the Roc/COR domain interface, a region strongly implicated in patients with an LRRK2 mutation. Our data confirm previous assumptions that the C-terminal subdomain of COR functions as a dimerization device. We show that the dimer formation is independent of nucleotide. The affinity for GDP/GTP is in the micromolar range, the result of which is high dissociation rates in the s-1 range. Thus Roco proteins are unlikely to need GEFs to achieve activation. Monomeric LRRK2 and Roco G-domains have a similar low GTPase activity to small G-proteins. We show that GTPase activity in bacterial Roco is stimulated by the nucleotide-dependent dimerization of the G-domain within the complex. We thus propose that the Roco proteins do not require GAPs to stimulate GTP hydrolysis but stimulate each other by one monomer completing the catalytic machinery of the other.
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Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet
2016-04-01
Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p<0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p<0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD.
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Sánchez-Danés, Adriana; Richaud-Patin, Yvonne; Carballo-Carbajal, Iria; Jiménez-Delgado, Senda; Caig, Carles; Mora, Sergio; Di Guglielmo, Claudia; Ezquerra, Mario; Patel, Bindiben; Giralt, Albert; Canals, Josep M; Memo, Maurizio; Alberch, Jordi; López-Barneo, José; Vila, Miquel; Cuervo, Ana Maria; Tolosa, Eduard; Consiglio, Antonella; Raya, Angel
2012-01-01
Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type. PMID:22407749
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Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo; Fuentes, José M; González-Polo, Rosa A
2014-10-03
Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.
Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming
2012-03-15
LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.
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Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning
Dodson, Mark W.; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming
2012-01-01
LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2G2019S allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD. PMID:22171073
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Liu, Min; Kang, Stephanie; Ray, Soumya; Jackson, Justin; Zaitsev, Alexandra D; Gerber, Scott A; Cuny, Gregory D; Glicksman, Marcie A
2011-11-01
Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.
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Inflexible ethanol intake: A putative link with the Lrrk2 pathway.
da Silva E Silva, Daniel Almeida; Frozino Ribeiro, Andrea; Damasceno, Samara; Rocha, Cristiane S; Berenguer de Matos, Alexandre H; Boerngen-Lacerda, Roseli; Correia, Diego; Brunialti Godard, Ana Lúcia
2016-10-15
Alcoholism is a complex multifactorial disorder with a strong genetic influence. Although several studies have shown the impact of high ethanol intake on the striatal gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with the two major concerns in addiction: the excessive drug consumption and relapsing. In this study, we used a chronic three-bottle free-choice murine model to address striatal transcript regulation among animals with different ethanol intakes and preferences: Light Drinkers (preference for water throughout the experiment), Heavy Drinkers (preference for ethanol with a non-compulsive intake) and Inflexible Drinkers (preference for ethanol and simultaneous loss of control over the drug intake). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the striatum, a brain region critical for the development of alcohol addiction. We found that the transcripts of the Lrrk2 gene, which encodes a multifunctional protein with kinase and GTPase activities, is upregulated only in Inflexible Drinkers suggesting, for the first time, that the Lrrk2 pathway plays a major role in the compulsive ethanol intake behaviour of addicted subjects. Copyright © 2016 Elsevier B.V. All rights reserved.
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Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J
2017-12-01
The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.
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Genetic analysis of LRRK2 functional domains in Brazilian patients with Parkinson's disease.
Abdalla-Carvalho, C B; Santos-Rebouças, C B; Guimarães, B C; Campos, M; Pereira, J S; de Rosso, A L Zuma; Nicaretta, D H; Marinho e Silva, M; dos Santos, Mendonça J; Pimentel, M M G
2010-12-01
Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been associated with Parkinson's disease (PD), and the majority of the pathogenic variants are located in the ROC and MAPKKK domains. Exons 29-31 and 38-44 (ROC and MAPKKK domains) were sequenced in 204 patients with PD, mostly Brazilian. We identified four polymorphisms, a novel silent variant p.R1398R and four substitutions: p.T1410M, p.G2019S, p.Y2189C and the novel variant p.C2139S. The most prevalent mutation was the p.G2019S (2.4%). We consider that the p.T1410M and the p.Y2189C variants are probably polymorphisms and that the p.C2139S mutation is potentially pathogenic. © 2010 The Author(s). European Journal of Neurology © 2010 EFNS.
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Role of LRRK2 and SNCA in autosomal dominant Parkinson's disease in Turkey.
Kessler, Christoph; Atasu, Burcu; Hanagasi, Hasmet; Simón-Sánchez, Javier; Hauser, Ann-Kathrin; Pak, Meltem; Bilgic, Basar; Erginel-Unaltuna, Nihan; Gurvit, Hakan; Gasser, Thomas; Lohmann, Ebba
2018-03-01
Mutations in the LRRK2 and alpha-synuclein (SNCA) genes are well-established causes of autosomal dominant Parkinson's disease (PD). However, their frequency differs widely between ethnic groups. Only three studies have screened all coding regions of LRRK2 and SNCA in European samples so far. In Turkey, the role of LRRK2 in Parkinson's disease has been studied fragmentarily, and the incidence of SNCA copy number variations is unknown. The purpose of this study is to determine the frequency of LRRK2 and SNCA mutations in autosomal dominant PD in Turkey. We performed Sanger sequencing of all coding LRRK2 and SNCA exons in a sample of 91 patients with Parkinsonism. Copy number variations in SNCA, PRKN, PINK1, DJ1 and ATP13A2 were assessed using the MLPA method. All patients had a positive family history compatible with autosomal dominant inheritance. Known mutations in LRRK2 and SNCA were found in 3.3% of cases: one patient harbored the LRRK2 G2019S mutation, and two patients carried a SNCA gene duplication. Furthermore, we found a heterozygous deletion of PRKN exon 2 in one patient, and four rare coding variants of unknown significance (LRRK2: A211V, R1067Q, T2494I; SNCA: T72T). Genetic testing in one affected family identified the LRRK2 R1067Q variant as a possibly pathogenic substitution. Point mutations in LRRK2 and SNCA are a rare cause of autosomal dominant PD in Turkey. However, copy number variations should be considered. The unclassified variants, especially LRRK2 R1067Q, demand further investigation. Copyright © 2017. Published by Elsevier Ltd.
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Sleep Disorders in Parkinsonian and Nonparkinsonian LRRK2 Mutation Carriers
Pont-Sunyer, Claustre; Iranzo, Alex; Gaig, Carles; Fernández-Arcos, Ana; Vilas, Dolores; Valldeoriola, Francesc; Compta, Yaroslau; Fernández-Santiago, Ruben; Fernández, Manel; Bayés, Angels; Calopa, Matilde; Casquero, Pilar; de Fàbregues, Oriol; Jaumà, Serge; Puente, Victor; Salamero, Manel; José Martí, Maria; Santamaría, Joan; Tolosa, Eduard
2015-01-01
Objective In idiopathic Parkinson disease (IPD) sleep disorders are common and may antedate the onset of parkinsonism. Based on the clinical similarities between IPD and Parkinson disease associated with LRRK2 gene mutations (LRRK2-PD), we aimed to characterize sleep in parkinsonian and nonmanifesting LRRK2 mutation carriers (NMC). Methods A comprehensive interview conducted by sleep specialists, validated sleep scales and questionnaires, and video-polysomnography followed by multiple sleep latency test (MSLT) assessed sleep in 18 LRRK2-PD (17 carrying G2019S and one R1441G mutations), 17 NMC (11 G2019S, three R1441G, three R1441C), 14 non-manifesting non-carriers (NMNC) and 19 unrelated IPD. Results Sleep complaints were frequent in LRRK2-PD patients; 78% reported poor sleep quality, 33% sleep onset insomnia, 56% sleep fragmentation and 39% early awakening. Sleep onset insomnia correlated with depressive symptoms and poor sleep quality. In LRRK2-PD, excessive daytime sleepiness (EDS) was a complaint in 33% patients and short sleep latencies on the MSLT, which are indicative of objective EDS, were found in 71%. Sleep attacks occurred in three LRRK2-PD patients and a narcoleptic phenotype was not observed. REM sleep behavior disorder (RBD) was diagnosed in three LRRK2-PD. EDS and RBD were always reported to start after the onset of parkinsonism in LRRK2-PD. In NMC, EDS was rarely reported and RBD was absent. When compared to IPD, sleep onset insomnia was more significantly frequent, EDS was similar, and RBD was less significantly frequent and less severe in LRRK2-PD. In NMC, RBD was not detected and sleep complaints were much less frequent than in LRRK2-PD. No differences were observed in sleep between NMC and NMNC. Conclusions Sleep complaints are frequent in LRRK2-PDand show a pattern that when compared to IPD is characterized by more frequent sleep onset insomnia, similar EDS and less prominent RBD. Unlike in IPD, RBD and EDS seem to be not markers of the prodromal stage of LRRK2-PD. PMID:26177462
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Role of LRRK2 in the regulation of dopamine receptor trafficking
Sanna, Simona; Taymans, Jean Marc; Morari, Michele; Brugnoli, Alberto; Frassineti, Martina; Masala, Alessandra; Esposito, Sonia; Galioto, Manuela; Valle, Cristiana; Carri, Maria Teresa; Biosa, Alice; Greggio, Elisa; Crosio, Claudia
2017-01-01
Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson’s disease (PD). Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1) and D2 (DRD2) trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD. PMID:28582422
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Gatto, Emilia Mabel; Parisi, Virginia; Converso, Daniela Paola; Poderoso, Juan José; Carreras, María Cecilia; Martí-Massó, José Felix; Paisán-Ruiz, Coro
2013-03-14
To determine clinical characteristics and frequency of leucine-rich repeat kinase 2 gene (LRRK2) mutations in a cohort of patients with Parkinson's disease (PD) from Argentina. Variation in the LRRK2 gene represents the most common genetic determinant of PD, only few data are available from Latin-America. Informed consent was obtained and all studies were approved by the Institutional Review Boards. Fifty five consecutive PD patients were recruited. A structured interview and neurological examination were used to collect demographic and clinical information. Blood samples were obtained and DNA extracted from patient venous blood. All LRRK2 exons from 25 exon to 51 exon were screened in all patients. Clinical and molecular data of 55 patients with PD were analyzed. Mean age was 68.8±10.6 years. Jewish and Basque ancestries were found positive in 9 and 7 patients, respectively; family history of PD was identified in 16 patients. The G2019S mutation was present in 3 Ashkenazi Jewish subjects (5.45%); all of them reported family history of PD in first-degree relatives. Although Argentina possesses one of the most important Basque communities outside Spain, non R1414G mutation was identified in this cohort. Eleven single polymorphisms (SNP) were identified in this cohort. The mean age at onset was higher in G2019S mutation carriers than non-carriers (66.67 vs 58.78 years). Asymmetrical tremor as initial symptom and non-motor symptoms occurred at similar frequencies in both groups. The G2019S mutation carriers showed a non significant increase in dyskinesias, and 2/3 developed Dopamine Dysregulation Syndrome and visual hallucinations. Systemic disorder identified in G2019S mutation carriers included: celiac disease, hypothyroidism, Hashimoto's Thyroiditis and arterial hypertension. The prevalence of LRRK2 G2019S mutation in this Argentinean cohort was similar to other international series, with a higher prevalence in Ashkenazi Jewish. The phenotype was indistinguishable from patients with idiopathic PD. Interestingly, we identified immune mediated disorders in two PD patients carrying the G2019S mutation. Within this context, recent studies have identified full-length LRRK2 as a relatively common constituent of many cell types in the immune system including human peripheral blood mononuclear cells. Nevertheless, a casual association could not be excluded and the analysis of more extensive series is required. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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Lrrk2 and alpha-synuclein are co-regulated in rodent striatum.
Westerlund, Marie; Ran, Caroline; Borgkvist, Anders; Sterky, Fredrik H; Lindqvist, Eva; Lundströmer, Karin; Pernold, Karin; Brené, Stefan; Kallunki, Pekka; Fisone, Gilberto; Olson, Lars; Galter, Dagmar
2008-12-01
LRRK2, alpha-synuclein, UCH-L1 and DJ-1 are implicated in the etiology of Parkinson's disease. We show for the first time that increase in striatal alpha-synuclein levels induce increased Lrrk2 mRNA levels while Dj-1 and Uch-L1 are unchanged. We also demonstrate that a mouse strain lacking the dopamine signaling molecule DARPP-32 has significantly reduced levels of both Lrrk2 and alpha-synuclein, while mice carrying a disabling mutation of the DARPP-32 phosphorylation site T34A or lack alpha-synuclein do not show any changes. To test if striatal dopamine depletion influences Lrrk2 or alpha-synuclein expression, we used the neurotoxin 6-hydroxydopamine in rats and MitoPark mice in which there is progressive degeneration of dopamine neurons. Because striatal Lrrk2 and alpha-synuclein levels were not changed by dopamine depletion, we conclude that Lrrk2 and alpha-synuclein mRNA levels are possibly co-regulated, but they are not influenced by striatal dopamine levels.
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Anfossi, Maria; Colao, Rosanna; Gallo, Maura; Bernardi, Livia; Conidi, M Elena; Frangipane, Francesca; Vasso, Franca; Puccio, Gianfranco; Clodomiro, Alessandra; Mirabelli, Maria; Curcio, Sabrina A M; Torchia, Giusi; Smirne, Nicoletta; Di Lorenzo, Raffaele; Maletta, Raffaele; Bruni, Amalia C
2014-01-01
LRRK2 mutations are common in familial and sporadic Parkinson's disease (PD) cases. We present a screening of the most frequently mutated exons of LRRK2 in Calabrian population. Eighty-eight PD patients diagnosed according to standard criteria, underwent screening for LRRK2 mutations in exons 19, 21, 24, 25, 27, 29, 31, 32, 33, 35, 38, 40, 41, and 48. Eight LRRK2 variations were identified in nine patients affected by PD, including three novel missense variations (p.Phe1227Leu, p.Gly1520Ala, p.Ile2020Ser) and five previously identified mutations (p.Ala1151Thr, IVS31+3A>G, p.Arg1514Gln, p.Gly2019Ser, p.Thr2356Ile). LRRK2 frequency mutations were approximately 10.2% in all PD patients, 12% in familial, 8% in sporadic cases. The p.Gly2019Ser mutation was found in 2.3% of the total cohort and in 3.2% of sporadic cases. The clinical features of LRRK2-associated with PD in our patients were similar to those of idiopathic PD although most LRRK2 mutated patients presented with bradykinesia instead of tremor; 33.3% developed dementia. We identified three novel LRRK2 mutations and reported a higher frequency in Calabria compared to previously reported data possibly due to the relative genetic isolation of the Calabrian population. These findings contribute to the understanding of the role of LRKK2 variations in PD and provide additional genetic insight into this disease.
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123I-MIBG cardiac uptake and smell identification in parkinsonian patients with LRRK2 mutations.
Valldeoriola, Francesc; Gaig, Carles; Muxí, Africa; Navales, Ignacio; Paredes, Pilar; Lomeña, Francisco; De la Cerda, Andres; Buongiorno, Mariateresa; Ezquerra, Mario; Santacruz, Pilar; Martí, Maria Jose; Tolosa, Eduardo
2011-06-01
Reduced uptake of (123)I- metaiodobenzylguanidine (MIBG) on cardiac gammagraphy and impaired odor identification are markers of neurodegenerative diseases with Lewy bodies (LB) as a pathological hallmark, such as idiopathic Parkinson's disease (IPD). LRRK2 patients present with a clinical syndrome indistinguishable from IPD, but LB have not been found in some cases. Patients with such mutations could behave differently than patients with IPD with respect to MIBG cardiac uptake and olfaction. We studied 14 LRRK2 patients, 14 IPD patients matched by age, gender, disease duration and severity, and 13 age and gender matched control subjects. Olfaction was analyzed through the University of Pennsylvania Smell Identification Test (UPSIT). MIBG cardiac uptake was evaluated through the H/M ratio. The late H/M was 1.44 ± 0.31 for LRRK2 patients, 1.19 ± 0.15 for PD patients, and 1.67 ± 0.16 for control subjects. LRRK2 patients presented lower but not statistically significant MIBG cardiac uptake than controls (p = 0.08) and significant higher uptake than PD patients (p = 0.04). UPSIT mean scores were 21.5 ± 7.3 for LRRK2 patients, 18.7 ± 6.2 for IPD patients and 29.7 ± 5.7 for control subjects. UPSIT score was lower in both LRRK2 and PD than in controls. In LRRK2 patients a positive correlation was found between myocardial MIBG uptake and UPSIT scores, (R = 0.801, p < 0.001). In LRRK2 patients, MIBG cardiac uptake was less impaired than in PD; a positive correlation between MIBG cardiac uptake and UPSIT scores was observed. As MIBG cardiac reduced uptake and impaired odor identification are markers of LB pathology, this findings may represent neuropathological heterogeneity among LRRK2 patients.
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Liu, Shu-Ying; Wile, Daryl J; Fu, Jessie Fanglu; Valerio, Jason; Shahinfard, Elham; McCormick, Siobhan; Mabrouk, Rostom; Vafai, Nasim; McKenzie, Jess; Neilson, Nicole; Perez-Soriano, Alexandra; Arena, Julieta E; Cherkasova, Mariya; Chan, Piu; Zhang, Jing; Zabetian, Cyrus P; Aasly, Jan O; Wszolek, Zbigniew K; McKeown, Martin J; Adam, Michael J; Ruth, Thomas J; Schulzer, Michael; Sossi, Vesna; Stoessl, A Jon
2018-04-01
Markers of neuroinflammation are increased in some patients with LRRK2 Parkinson's disease compared with individuals with idiopathic Parkinson's disease, suggesting possible differences in disease pathogenesis. Previous PET studies have suggested amplified dopamine turnover and preserved serotonergic innervation in LRRK2 mutation carriers. We postulated that patients with LRRK2 mutations might show abnormalities of central cholinergic activity, even before the diagnosis of Parkinson's disease. Between June, 2009, and December, 2015, we recruited participants from four movement disorder clinics in Canada, Norway, and the USA. Patients with Parkinson's disease were diagnosed by movement disorder neurologists on the basis of the UK Parkinson's Disease Society Brain Bank criteria. LRRK2 carrier status was confirmed by bidirectional Sanger sequencing. We used the PET tracer N- 11 C-methyl-piperidin-4-yl propionate to scan for acetylcholinesterase activity. The primary outcome measure was rate of acetylcholinesterase hydrolysis, calculated using the striatal input method. We compared acetylcholinesterase hydrolysis rates between groups using ANCOVA, with adjustment for age based on the results of linear regression analysis. We recruited 14 patients with LRRK2 Parkinson's disease, 16 LRRK2 mutation carriers without Parkinson's disease, eight patients with idiopathic Parkinson's disease, and 11 healthy controls. We noted significant between-group differences in rates of acetylcholinesterase hydrolysis in cortical regions (average cortex p=0·009, default mode network-related regions p=0·006, limbic network-related regions p=0·020) and the thalamus (p=0·008). LRRK2 mutation carriers without Parkinson's disease had increased acetylcholinesterase hydrolysis rates compared with healthy controls in the cortex (average cortex, p=0·046). Patients with LRRK2 Parkinson's disease had significantly higher acetylcholinesterase activity in some cortical regions (average cortex p=0·043, default mode network-related regions p=0·021) and the thalamus (thalamus p=0·004) compared with individuals with idiopathic disease. Acetylcholinesterase hydrolysis rates in healthy controls were correlated inversely with age. LRRK2 mutations are associated with significantly increased cholinergic activity in the brain in mutation carriers without Parkinson's disease compared with healthy controls and in LRRK2 mutation carriers with Parkinson's disease compared with individuals with idiopathic disease. Changes in cholinergic activity might represent early and sustained attempts to compensate for LRRK2-related dysfunction, or alteration of acetylcholinesterase in non-neuronal cells. Michael J Fox Foundation, National Institutes of Health, and Pacific Alzheimer Research Foundation. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Wile, Daryl J; Agarwal, Pankaj A; Schulzer, Michael; Mak, Edwin; Dinelle, Katherine; Shahinfard, Elham; Vafai, Nasim; Hasegawa, Kazuko; Zhang, Jing; McKenzie, Jessamyn; Neilson, Nicole; Strongosky, Audrey; Uitti, Ryan J; Guttman, Mark; Zabetian, Cyrus P; Ding, Yu-Shin; Adam, Mike; Aasly, Jan; Wszolek, Zbigniew K; Farrer, Matthew; Sossi, Vesna; Stoessl, A Jon
2017-05-01
People with Parkinson's disease can show premotor neurochemical changes in the dopaminergic and non-dopaminergic systems. Using PET, we assessed whether dopaminergic and serotonin transporter changes are similar in LRRK2 mutation carriers with Parkinson's disease and individuals with sporadic Parkinson's disease, and whether LRRK2 mutation carriers without motor symptoms show PET changes. We did two cross-sectional PET studies at the Pacific Parkinson's Research Centre in Vancouver, BC, Canada. We included LRRK2 mutation carriers with or without manifest Parkinson's disease, people with sporadic Parkinson's disease, and age-matched healthy controls, all aged 18 years or older. People with Parkinson's disease were diagnosed by a neurologist with movement disorder training, in accordance with the UK Parkinson's Disease Society Brain Bank criteria. LRRK2 carrier status was confirmed by bidirectional Sanger sequencing. In the first study, LRRK2 mutation carriers with or without manifest Parkinson's disease who were referred for investigation between July, 1999, and January, 2012, were scanned with PET tracers for the membrane dopamine transporter, and dopamine synthesis and storage ( 18 F-6-fluoro-L-dopa; 18 F-FDOPA). We compared findings with those in people with sporadic Parkinson's disease and age-matched healthy controls. In the second study, distinct groups of LRRK2 mutation carriers, individuals with sporadic Parkinson's disease, and age-matched healthy controls seen from November, 2012, to May, 2016, were studied with tracers for the serotonin transporter and vesicular monoamine transporter 2 (VMAT2). Striatal dopamine transporter binding, VMAT2 binding, 18 F-FDOPA uptake, and serotonin transporter binding in multiple brain regions were compared by ANCOVA, adjusted for age. Between January, 1997, and January, 2012, we obtained data for our first study from 40 LRRK2 mutation carriers, 63 individuals with sporadic Parkinson's disease, and 35 healthy controls. We identified significant group differences in striatal dopamine transporter binding (all age ranges in caudate and putamen, p<0·0001) and 18 F-FDOPA uptake (in caudate: age ≤50 years, p=0·0002; all other age ranges, p<0·0001; in putamen: all age ranges, p<0·0001). LRRK2 mutation carriers with manifest Parkinson's disease (n=15) had reduced striatal dopamine transporter binding and 18 F-FDOPA uptake, comparable with amounts seen in individuals with sporadic Parkinson's disease of similar duration. LRRK2 mutation carriers without manifest Parkinson's disease (n=25) had greater 18 F-FDOPA uptake and dopamine transporter binding than did individuals with sporadic Parkinson's disease, with 18 F-FDOPA uptake comparable with controls and dopamine transporter binding lower than in controls. Between November, 2012, and May, 2016, we obtained data for our second study from 16 LRRK2 mutation carriers, 13 individuals with sporadic Parkinson's disease, and nine healthy controls. Nine LRRK2 mutation carriers without manifest Parkinson's disease had significantly elevated serotonin transporter binding in the hypothalamus (compared with controls, individuals with LRRK2 Parkinson's disease, and people with sporadic Parkinson's disease, p<0·0001), striatum (compared with people with sporadic Parkinson's disease, p=0·02), and brainstem (compared with LRRK2 mutation carriers with manifest Parkinson's disease, p=0·01), after adjustment for age. Serotonin transporter binding in the cortex did not differ significantly between groups after age adjustment. Striatal VMAT2 binding was reduced in all individuals with manifest Parkinson's disease and reduced asymmetrically in one LRRK2 mutation carrier without manifest disease. Dopaminergic and serotonergic changes progress in a similar fashion in LRRK2 mutation carriers with manifest Parkinson's disease and individuals with sporadic Parkinson's disease, but LRRK2 mutation carriers without manifest Parkinson's disease show increased serotonin transporter binding in the striatum, brainstem, and hypothalamus, possibly reflecting compensatory changes in serotonergic innervation preceding the motor onset of Parkinson's disease. Increased serotonergic innervation might contribute to clinical differences in LRRK2 Parkinson's disease, including the emergence of non-motor symptoms and, potentially, differences in the long-term response to levodopa. Canada Research Chairs, Michael J Fox Foundation, National Institutes of Health, Pacific Alzheimer Research Foundation, Pacific Parkinson's Research Institute, National Research Council of Canada. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Parkinson Disease Phenotype in Ashkenazi Jews with and without LRRK2 G2019S mutations
Alcalay, Roy N.; Mirelman, Anat; Saunders-Pullman, Rachel; Tang, Ming-X; Santana, Helen Mejia; Raymond, Deborah; Roos, Ernest; Orbe-Reilly, Martha; Gurevich, Tanya; Shira, Anat Bar; Weisz, Mali Gana; Yasinovsky, Kira; Zalis, Maayan; Thaler, Avner; Deik, Andres; Barrett, Matthew James; Cabassa, Jose; Groves, Mark; Hunt, Ann L.; Lubarr, Naomi; Luciano, Marta San; Miravite, Joan; Palmese, Christina; Sachdev, Rivka; Sarva, Harini; Severt, Lawrence; Shanker, Vicki; Swan, Matthew Carrington; Soto-Valencia, Jeannie; Johannes, Brooke; Ortega, Robert; Fahn, Stanley; Cote, Lucien; Waters, Cheryl; Mazzoni, Pietro; Ford, Blair; Louis, Elan; Levy, Oren; Rosado, Llency; Ruiz, Diana; Dorovski, Tsvyatko; Pauciulo, Michael; Nichols, William; Orr-Urtreger, Avi; Ozelius, Laurie; Clark, Lorraine; Giladi, Nir; Bressman, Susan; Marder, Karen S
2013-01-01
Background The phenotype of Parkinson disease (PD) patients with and without LRRK2 G2019S mutations is reported to be similar; however large uniformly evaluated series are lacking. Objective To characterize the clinical phenotype of Ashkenazi Jewish (AJ) PD carriers of the LRRK2 G2019S mutation. Methods We studied 553 AJ PD patients, including 65 patients who were previously reported, from three sites (two in New York and one in Tel-Aviv). GBA mutation carriers were excluded. Evaluations included the Montreal Cognitive Assessment (MoCA), the Unified Parkinson's Disease Rating Scale (UPDRS), the geriatric depression scale (GDS) and the non-motor symptoms (NMS) questionnaire. Regression models were constructed to test the association between clinical and demographic features and LRRK2 status (outcome) in 488 newly recruited participants. Results LRRK2 G2019S carriers (n=97) and non-carriers (n=391) were similar in age and age-at-onset of PD. Carriers had longer disease duration (8.6years versus 6.1years, p<0.001), were more likely to be women (51.5% versus 37.9%, p=0.015) and more often reported first symptoms in lower extremities (40.0% versus 19.2%, p<0.001). In logistic models adjusted for age, disease duration, gender, education, and site, carriers were more likely to have lower extremity onset (p<0.001), postural instability gait difficulty (PIGD, p=0.043) and persistent levodopa response for>5 years (p=0.042). Performance on UPDRS, MoCA, GDS and NMS did not differ by mutation status. Conclusion PD in AJ-LRRK2 G2019S mutation carriers is similar to idiopathic PD, but characterized by more frequent lower extremity involvement at onset and PIGD without the associated cognitive impairment. PMID:24243757
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Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition
Hockey, Leanne N.; Kilpatrick, Bethan S.; Eden, Emily R.; Lin-Moshier, Yaping; Brailoiu, G. Cristina; Brailoiu, Eugen; Futter, Clare E.; Schapira, Anthony H.; Marchant, Jonathan S.; Patel, Sandip
2015-01-01
ABSTRACT Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca2+-dependent trafficking in Parkinson disease. PMID:25416817
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Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.
Hockey, Leanne N; Kilpatrick, Bethan S; Eden, Emily R; Lin-Moshier, Yaping; Brailoiu, G Cristina; Brailoiu, Eugen; Futter, Clare E; Schapira, Anthony H; Marchant, Jonathan S; Patel, Sandip
2015-01-15
Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease. © 2015. Published by The Company of Biologists Ltd.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak
Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less
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2015-01-01
We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors. PMID:25075558
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Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak; ...
2014-07-17
Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Liu, Qingjie; Batt, Douglas G.; Lippy, Jonathan S.
Four series of disubstituted carbazole-1-carboxamides were designed and synthesised as inhibitors of Bruton’s tyrosine kinase (BTK). 4,7- and 4,6-disubstituted carbazole-1-carboxamides were potent and selective inhibitors of BTK, while 3,7- and 3,6-disubstituted carbazole-1-carboxamides were potent and selective inhibitors of Janus kinase 2 (JAK2).
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Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B
2012-01-01
In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.
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Lower cognitive performance in healthy G2019S LRRK2 mutation carriers
Thaler, Avner; Mirelman, Anat; Gurevich, Tanya; Simon, Ely; Orr-Urtreger, Avi; Marder, Karen; Bressman, Susan
2012-01-01
Objective: To assess cognitive abilities of healthy first-degree relatives of Ashkenazi patients with Parkinson disease (PD), carriers of the G2019S mutation in the LRRK2 gene. Methods: In this observational study, 60 consecutive healthy first-degree relatives (aged 50.9 ± 6.2 years; 48% male; 30 G2019S carriers) were assessed using a computerized cognitive program, the Montreal Cognitive Assessment questionnaire, the Unified Parkinson's Disease Rating Scale Part III, and the Geriatric Depression Scale. Results: G2019S carriers scored significantly lower on the computerized executive function index (p = 0.04) and on specific executive function tasks (Stroop test, p = 0.007). Conclusion: Carrying the LRRK2 G2019S mutation was associated with lower executive performance in a population at risk for PD. PMID:22914834
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Lack of association between the LRRK2 A419V variant and Asian Parkinson's disease.
Gopalai, Aroma Agape; Lim, Shen Yang; Aziz, Zariah Abdul; Lim, Soo Kun; Tan, Li Ping; Chong, Yip Boon; Tan, Chong Tin; Puvanarajah, Santhi; Viswanathan, Shanti; Kuppusamy, Rishikesan; Tan, Ai Huey; Lim, Thien Thien; Eow, Gaik Bee; Norlinah, Mohamed Ibrahim; Li, Hui Hua; Zhao, Yi; Ahmad-Annuar, Azlina
2013-05-01
The G2385R and R1628P LRRK2 gene variants have been associated with an increased risk of Parkinson's disease (PD) in the Asian population. Recently, a new LRRK2 gene variant, A419V, was reported to be a third risk variant for PD in Asian patients. Our objective was to investigate this finding in our cohort of Asian subjects. Eight hundred and twenty-eight subjects (404 PD patients, and 424 age and gender-matched control subjects without neurological disorders) were recruited. Genotyping was done by Taqman® allelic discrimination assay on an Applied Biosystems 7500 Fast Real-Time PCR machine. The heterozygous A419V genotype was found in only 1 patient with PD, compared to 3 in the control group (0.4% vs 1.3%), giving an odds ratio of 0.35 (95% confidence interval (CI), 0.01 to 3.79; P = 0.624). A419V is not an important LRRK2 risk variant in our Asian cohort of patients with PD. Our data are further supported by a literature review which showed that 4 out of 6 published studies reported a negative association of this variant in PD.
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Marlowe, Timothy A.; Lenzo, Felicia L.; Figel, Sheila A.; Grapes, Abigail T.; Cance, William G.
2016-01-01
Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms which drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTKs) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK’s critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. Additionally, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: 1) the rapid phosphorylation and activation of RTK signaling pathways in RTKHigh cells and 2) the long-term acquisition of RTKs novel to the parental cell line in RTKLow cells. Finally, HER2+ cancer cells displayed resistance to FAK-kinase inhibition in 3D–growth assays using a HER2 isogenic system and HER2+ cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. PMID:27638858
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Asquith, Christopher R M; Godoi, Paulo H; Couñago, Rafael M; Laitinen, Tuomo; Scott, John W; Langendorf, Christopher G; Oakhill, Jonathan S; Drewry, David H; Zuercher, William J; Koutentis, Panayiotis A; Willson, Timothy M; Kalogirou, Andreas S
2018-05-19
We demonstrate for the first time that 4 H -1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4 H -1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.
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Ma, X H; Wang, R; Tan, C Y; Jiang, Y Y; Lu, T; Rao, H B; Li, X Y; Go, M L; Low, B C; Chen, Y Z
2010-10-04
Multitarget agents have been increasingly explored for enhancing efficacy and reducing countertarget activities and toxicities. Efficient virtual screening (VS) tools for searching selective multitarget agents are desired. Combinatorial support vector machines (C-SVM) were tested as VS tools for searching dual-inhibitors of 11 combinations of 9 anticancer kinase targets (EGFR, VEGFR, PDGFR, Src, FGFR, Lck, CDK1, CDK2, GSK3). C-SVM trained on 233-1,316 non-dual-inhibitors correctly identified 26.8%-57.3% (majority >36%) of the 56-230 intra-kinase-group dual-inhibitors (equivalent to the 50-70% yields of two independent individual target VS tools), and 12.2% of the 41 inter-kinase-group dual-inhibitors. C-SVM were fairly selective in misidentifying as dual-inhibitors 3.7%-48.1% (majority <20%) of the 233-1,316 non-dual-inhibitors of the same kinase pairs and 0.98%-4.77% of the 3,971-5,180 inhibitors of other kinases. C-SVM produced low false-hit rates in misidentifying as dual-inhibitors 1,746-4,817 (0.013%-0.036%) of the 13.56 M PubChem compounds, 12-175 (0.007%-0.104%) of the 168 K MDDR compounds, and 0-84 (0.0%-2.9%) of the 19,495-38,483 MDDR compounds similar to the known dual-inhibitors. C-SVM was compared to other VS methods Surflex-Dock, DOCK Blaster, kNN and PNN against the same sets of kinase inhibitors and the full set or subset of the 1.02 M Zinc clean-leads data set. C-SVM produced comparable dual-inhibitor yields, slightly better false-hit rates for kinase inhibitors, and significantly lower false-hit rates for the Zinc clean-leads data set. Combinatorial SVM showed promising potential for searching selective multitarget agents against intra-kinase-group kinases without explicit knowledge of multitarget agents.
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Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D
2018-02-15
Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.
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Byrne, Dominic P; Li, Yong; Ngamlert, Pawin; Ramakrishnan, Krithika; Eyers, Claire E; Wells, Carrow; Drewry, David H; Zuercher, William J; Berry, Neil G; Fernig, David G; Eyers, Patrick A
2018-06-22
Protein tyrosine sulphation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors. ©2018 The Author(s).
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Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.
Bayguinov, O; Dwyer, L; Kim, H; Marklew, A; Sanders, K M; Koh, S D
2011-06-01
The Rho-kinase pathway regulates agonist-induced contractions in several smooth muscles, including the intestine, urinary bladder and uterus, via dynamic changes in the Ca(2+) sensitivity of the contractile apparatus. However, there is evidence that Rho-kinase also modulates other cellular effectors such as ion channels. We examined the regulation of colonic smooth muscle excitability by Rho-kinase using conventional microelectrode recording, isometric force measurements and patch-clamp techniques. The Rho-kinase inhibitors, Y-27632 and H-1152, decreased nerve-evoked on- and off-contractions elicited at a range of frequencies and durations. The Rho-kinase inhibitors decreased the spontaneous contractions and the responses to carbachol and substance P independently of neuronal inputs, suggesting Y-27632 acts directly on smooth muscle. The Rho-kinase inhibitors significantly reduced the depolarization in response to carbachol, an effect that cannot be due to regulation of Ca(2+) sensitization. Patch-clamp experiments showed that Rho-kinase inhibitors reduce GTPγS-activated non-selective cation currents. The Rho-kinase inhibitors decreased contractions evoked by nerve stimulation, carbachol and substance P. These effects were not solely due to inhibition of the Ca(2+) sensitization pathway, as the Rho-kinase inhibitors also inhibited the non-selective cation conductances activated by excitatory transmitters. Thus, Rho-kinase may regulate smooth muscle excitability mechanisms by regulating non-selective cation channels as well as changing the Ca(2+) sensitivity of the contractile apparatus. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.
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Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G
2016-12-01
Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Han, Seungil; Mistry, Anil; Chang, Jeanne S.
Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptormore » tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.« less
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Protocols for the Design of Kinase-focused Compound Libraries.
Jacoby, Edgar; Wroblowski, Berthold; Buyck, Christophe; Neefs, Jean-Marc; Meyer, Christophe; Cummings, Maxwell D; van Vlijmen, Herman
2018-05-01
Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Schaenzer, Adam J.; Wlodarchak, Nathan; Drewry, David H.; Zuercher, William J.; Rose, Warren E.; Striker, Rob; Sauer, John-Demian
2017-01-01
Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial Penicillin-binding-protein And Serine/Threonine kinase-Associated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition. PMID:28821610
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Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian
2017-10-13
Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.
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Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency
DOE Office of Scientific and Technical Information (OSTI.GOV)
Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.
2010-10-01
Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.
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Disclosure of Research Results in Genetic Studies of Parkinson Disease Due to LRRK2 Mutations
Bressman, Susan; Raymond, Deborah; Glickman, Amanda; Tolosa, Eduardo
2015-01-01
With the advent of large genetic studies examining both symptomatic and asymptomatic individuals, whether and how to disclose genetic research results have become pressing questions. The need is particularly acute in the case of LRRK2 research: Movement centers worldwide are recruiting cohorts of individuals with PD and their family members, including asymptomatic carriers, clinical features and treatment are complex and evolving, and disclosure policies vary at different sites and have been modified during the course of some studies. Herein, we present the major ethical principles of autonomy, beneficence, non-maleficence, and honesty that should guide disclosure policies in studies of families with LRRK2 mutations. We make recommendations regarding: genetic counseling, policies of either active or passive disclosure, responsibilities of funders to budget for genetic counseling, clinical genetic testing where locally required for disclosure, and aspects of study design to avoid mandatory disclosure whenever feasible. PMID:25952684
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Chemical Proteomics and Structural Biology Define EPHA2 Inhibition by Clinical Kinase Drugs.
Heinzlmeir, Stephanie; Kudlinzki, Denis; Sreeramulu, Sridhar; Klaeger, Susan; Gande, Santosh Lakshmi; Linhard, Verena; Wilhelm, Mathias; Qiao, Huichao; Helm, Dominic; Ruprecht, Benjamin; Saxena, Krishna; Médard, Guillaume; Schwalbe, Harald; Kuster, Bernhard
2016-12-16
The receptor tyrosine kinase EPHA2 (Ephrin type-A receptor 2) plays important roles in oncogenesis, metastasis, and treatment resistance, yet therapeutic targeting, drug discovery, or investigation of EPHA2 biology is hampered by the lack of appropriate inhibitors and structural information. Here, we used chemical proteomics to survey 235 clinical kinase inhibitors for their kinase selectivity and identified 24 drugs with submicromolar affinities for EPHA2. NMR-based conformational dynamics together with nine new cocrystal structures delineated drug-EPHA2 interactions in full detail. The combination of selectivity profiling, structure determination, and kinome wide sequence alignment allowed the development of a classification system in which amino acids in the drug binding site of EPHA2 are categorized into key, scaffold, potency, and selectivity residues. This scheme should be generally applicable in kinase drug discovery, and we anticipate that the provided information will greatly facilitate the development of selective EPHA2 inhibitors in particular and the repurposing of clinical kinase inhibitors in general.
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Saccharomyces cerevisiae as a platform for assessing sphingolipid lipid kinase inhibitors
Agah, Sayeh; Mendelson, Anna J.; Eletu, Oluwafunmilayo T.; Barkey-Bircann, Peter; Gesualdi, James
2018-01-01
Successful medicinal chemistry campaigns to discover and optimize sphingosine kinase inhibitors require a robust assay for screening chemical libraries and for determining rank order potencies. Existing assays for these enzymes are laborious, expensive and/or low throughput. The toxicity of excessive levels of phosphorylated sphingoid bases for the budding yeast, Saccharomyces cerevisiae, affords an assay wherein inhibitors added to the culture media rescue growth in a dose-dependent fashion. Herein, we describe our adaptation of a simple, inexpensive, and high throughput assay for assessing inhibitors of sphingosine kinase types 1 and 2 as well as ceramide kinase and for testing enzymatic activity of sphingosine kinase type 2 mutants. The assay was validated using recombinant enzymes and generally agrees with the rank order of potencies of existing inhibitors. PMID:29672528
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Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.
Zhu, Jin-Yi; Cuellar, Rebecca A; Berndt, Norbert; Lee, Hee Eun; Olesen, Sanne H; Martin, Mathew P; Jensen, Jeffrey T; Georg, Gunda I; Schönbrunn, Ernst
2017-09-28
Members of the Wee family of kinases negatively regulate the cell cycle via phosphorylation of CDK1 and are considered potential drug targets. Herein, we investigated the structure-function relationship of human Wee1, Wee2, and Myt1 (PKMYT1). Purified recombinant full-length proteins and kinase domain constructs differed substantially in phosphorylation states and catalytic competency, suggesting complex mechanisms of activation. A series of crystal structures reveal unique features that distinguish Wee1 and Wee2 from Myt1 and establish the structural basis of differential inhibition by the widely used Wee1 inhibitor MK-1775. Kinome profiling and cellular studies demonstrate that, in addition to Wee1 and Wee2, MK-1775 is an equally potent inhibitor of the polo-like kinase PLK1. Several previously unrecognized inhibitors of Wee kinases were discovered and characterized. Combined, the data provide a comprehensive view on the catalytic and structural properties of Wee kinases and a framework for the rational design of novel inhibitors thereof.
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Yousufzai, S Y; Gao, G; Abdel-Latif, A A
2000-10-27
The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F(2alpha)-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F(2alpha) and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca(2+) mobilization pathway.
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Greco, Rita; Li, Zhifang; Sun, Fangxian; Barberis, Claude; Tabart, Michel; Patel, Vinod; Schio, Laurent; Hurley, Raelene; Chen, Bo; Cheng, Hong; Lengauer, Christoph; Pollard, Jack; Watters, James; Garcia-Echeverria, Carlos; Wiederschain, Dmitri; Adrian, Francisco; Zhang, JingXin
2014-01-01
Inhibitors of JAK2 kinase are emerging as an important treatment modality for myeloproliferative neoplasms (MPN). However, similar to other kinase inhibitors, resistance to JAK2 inhibitors may eventually emerge through a variety of mechanisms. Effective drug combination is one way to enhance therapeutic efficacy and combat resistance against JAK2 inhibitors. To identify potential combination partners for JAK2 compounds in MPN cell lines, we performed pooled shRNA screen targeting 5,000 genes in the presence or absence of JAK2 blockade. One of the top hits identified was MYC, an oncogenic transcription factor that is difficult to inhibit directly, but could be targeted by modulation of upstream regulatory elements such as kinases. We demonstrate herein that PIM kinase inhibitors efficiently suppress MYC protein levels in MPN cell lines. Overexpression of MYC restores the viability of PIM inhibitor-treated cells, revealing causal relationship between MYC down-regulation and cell growth inhibition by PIM compounds. Combination of various PIM inhibitors with a JAK2 inhibitor results in significant synergistic growth inhibition of multiple MPN cancer cell lines and induction of apoptosis. Mechanistic studies revealed strong downregulation of phosphorylated forms of S6 and 4EBP1 by JAK2/PIM inhibitor combination treatment. Finally, such combination was effective in eradicating in vitro JAK2 inhibitor-resistant MPN clones, where MYC is consistently up-regulated. These findings demonstrate that simultaneous suppression of JAK2 and PIM kinase activity by small molecule inhibitors is more effective than either agent alone in suppressing MPN cell growth. Our data suggest that JAK2 and PIM combination might warrant further investigation for the treatment of JAK2-driven hematologic malignancies. PMID:24830942
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Xu, Shucheng
2010-09-01
The role of a calcium-dependent and calmodulin (CaM)-stimulated protein kinase in abscisic acid (ABA)-induced antioxidant defense was determined in leaves of maize (Zea mays). In-gel kinase assays showed that treatments with ABA or H(2)O(2) induced the activation of a 49-kDa protein kinase and a 52-kDa protein kinase significantly. Furthermore, we showed that the 52-kDa protein kinase has the characteristics of CaM-stimulating activity and is sensitive to calcium-CaM-dependent protein kinase II (CaMK II) inhibitor KN-93 or CaM antagonist W-7. Treatments with ABA or H(2)O(2) not only induced the activation of the 52-kDa protein kinase, but also enhanced the total activities of the antioxidant enzymes, including catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with a CaMK inhibitor and a reactive oxygen species (ROS) inhibitor or scavenger. Pretreatment with the CaMK inhibitor also substantially arrested the ABA-induced H(2)O(2) production. Kinase activity enhancements induced by ABA were attenuated by pretreatment with an ROS inhibitor or scavenger. These results suggest that the 52-kDa CaMK is involved in ABA-induced antioxidant defense and that cross-talk between CaMK and H(2)O(2) plays a pivotal role in ABA signaling. We infer that CaMK acts both upstream and downstream of H(2)O(2), but mainly acts between ABA and H(2)O(2) in ABA-induced antioxidant-defensive signaling.
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Dual kinase-bromodomain inhibitors for rationally designed polypharmacology
Ciceri, Pietro; Müller, Susanne; O’Mahony, Alison; Fedorov, Oleg; Filippakopoulos, Panagis; Hunt, Jeremy P.; Lasater, Elisabeth A.; Pallares, Gabriel; Picaud, Sarah; Wells, Christopher; Martin, Sarah; Wodicka, Lisa M.; Shah, Neil P.; Treiber, Daniel K.; Knapp, Stefan
2014-01-01
Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multi-target profile has, however, necessitated the application of combination therapies, which can pose significant clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as novel targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase/bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, clinical PLK1 and JAK2/FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a novel strategy for rational single agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase/bromodomain inhibitors. PMID:24584101
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Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.
2016-01-01
Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090
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NASA Astrophysics Data System (ADS)
Shahin, Rand; Swellmeen, Lubna; Shaheen, Omar; Aboalhaija, Nour; Habash, Maha
2016-01-01
Targeting Proviral integration-site of murine Moloney leukemia virus 1 kinase, hereafter called Pim-1 kinase, is a promising strategy for treating different kinds of human cancer. Headed for this a total list of 328 formerly reported Pim-1 kinase inhibitors has been explored and divided based on the pharmacophoric features of the most active molecules into 10 subsets projected to represent potential active binding manners accessible to ligands within the binding pocket of Pim-1 kinase. Discovery Studio 4.1 (DS 4.1) was employed to detect potential pharmacophoric active binding manners anticipated by Pim-1 Kinase inhibitors. The pharmacophoric models were then allowed to compete within Quantitative Structure Activity Relationship (QSAR) framework with other 2D descriptors. Accordingly Genetic algorithm and multiple linear regression investigation were engaged to find the finest QSAR equation that has the best predictive power r 262 2 = 0.70, F = 119.14, r LOO 2 = 0.693, r PRESS 2 against 66 external test inhibitors = 0.71 q2 = 0.55. Three different pharmacophores appeared in the successful QSAR equation this represents three different binding modes for inhibitors within the Pim-1 kinase binding pocket. Pharmacophoric models were later used to screen compounds within the National Cancer Institute database. Several low micromolar Pim-1 Kinase inhibitors were captured. The most potent hits show IC50 values of 0.77 and 1.03 µM. Also, upon analyzing the successful QSAR Equation we found that some polycyclic aromatic electron-rich structures namely 6-Chloro-2-methoxy-acridine can be considered as putative hits for Pim-1 kinase inhibition.
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Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies
Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles
2015-01-01
Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586
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Fiore, Mario; Forli, Stefano; Manetti, Fabrizio
2015-01-01
The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway, but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases, to prevent tumor metastasis, and to increase tumor sensitivity to chemotherapeutics. PMID:26502061
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Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi
2016-07-01
Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.
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Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi
2016-01-01
Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334
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The selectivity of protein kinase inhibitors: a further update
Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip
2007-01-01
The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214
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Disclosure of research results in genetic studies of Parkinson's disease caused by LRRK2 mutations.
Pont-Sunyer, Claustre; Bressman, Susan; Raymond, Deborah; Glickman, Amanda; Tolosa, Eduardo; Saunders-Pullman, Rachel
2015-06-01
With the advent of large genetic studies examining both symptomatic and asymptomatic individuals, whether and how to disclose genetic research results have become pressing questions. The need is particularly acute in the case of LRRK2 research: Movement centers worldwide are recruiting cohorts of individuals with Parkinson's disease (PD) and their family members, including asymptomatic carriers. Clinical features and treatment are complex and evolving, and disclosure policies vary at different sites and have been modified during the course of some studies. We present the major ethical principles of autonomy, beneficence, nonmaleficence, and honesty that should guide disclosure policies in studies of families with LRRK2 mutations. We make recommendations regarding genetic counseling, policies of either active or passive disclosure, responsibilities of funders to budget for genetic counseling, clinical genetic testing where locally required for disclosure, and aspects of study design to avoid mandatory disclosure whenever feasible. © 2015 International Parkinson and Movement Disorder Society. © 2015 International Parkinson and Movement Disorder Society.
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Prodromal Parkinsonism and Neurodegenerative Risk Stratification in REM Sleep Behavior Disorder
Lawton, Michael; Rolinski, Michal; Evetts, Samuel; Baig, Fahd; Ruffmann, Claudio; Gornall, Aimie; Klein, Johannes C; Lo, Christine; Dennis, Gary; Bandmann, Oliver; Quinnell, Timothy; Zaiwalla, Zenobia; Ben-Shlomo, Yoav; Hu, Michele TM
2017-01-01
Abstract Objectives Rapid eye movement (REM) sleep behavior disorder (RBD) is the most specific marker of prodromal alpha-synucleinopathies. We sought to delineate the baseline clinical characteristics of RBD and evaluate risk stratification models. Methods Clinical assessments were performed in 171 RBD, 296 control, and 119 untreated Parkinson’s (PD) participants. Putative risk measures were assessed as predictors of prodromal neurodegeneration, and Movement Disorders Society (MDS) criteria for prodromal PD were applied. Participants were screened for common leucine-rich repeat kinase 2 (LRRK2)/glucocerebrosidase gene (GBA) gene mutations. Results Compared to controls, participants with RBD had higher rates of solvent exposure, head injury, smoking, obesity, and antidepressant use. GBA mutations were more common in RBD, but no LRRK2 mutations were found. RBD participants performed significantly worse than controls on Unified Parkinson’s Disease Rating Scale (UPDRS)-III, timed “get-up-and-go”, Flamingo test, Sniffin Sticks, and cognitive tests and had worse measures of constipation, quality of life (QOL), and orthostatic hypotension. For all these measures except UPDRS-III, RBD and PD participants were equally impaired. Depression, anxiety, and apathy were worse in RBD compared to PD participants. Stratification of people with RBD according to antidepressant use, obesity, and age altered the odds ratio (OR) of hyposmia compared to controls from 3.4 to 45.5. 74% (95% confidence interval [CI] 66%, 80%) of RBD participants met the MDS criteria for probable prodromal Parkinson’s compared to 0.3% (95% CI 0.009%, 2%) of controls. Conclusions RBD are impaired across a range of clinical measures consistent with prodromal PD and suggestive of a more severe nonmotor subtype. Clinical risk stratification has the potential to select higher risk patients for neuroprotective interventions. PMID:28472425
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MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE
ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.
MAP Kinase signal transduction is associated with a variety ... -
BIM expression in treatment naïve cancers predicts responsiveness to kinase inhibitors
Faber, Anthony; Corcoran, Ryan B.; Ebi, Hiromichi; Sequist, Lecia V.; Waltman, Belinda A.; Chung, Euiheon; Incio, Joao; Digumarthy, Subba R.; Pollack, Sarah F.; Song, Youngchul; Muzikansky, Alona; Lifshits, Eugene; Roberge, Sylvie; Coffman, Erik J.; Benes, Cyril; Gómez, Henry; Baselga, Jose; Arteaga, Carlos L.; Rivera, Miguel N.; Dias-Santagata, Dora; Jain, Rakesh K.; Engelman, Jeffrey A.
2011-01-01
Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pre-treatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR mutant, HER2 amplified, and PIK3CA mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms. PMID:22145099
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Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon
2013-01-01
Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279
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The Pim kinases: new targets for drug development.
Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis
2011-12-01
The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.
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Byrne, Dominic P; Li, Yong; Ramakrishnan, Krithika; Barsukov, Igor L; Yates, Edwin A; Eyers, Claire E; Papy-Garcia, Dulcé; Chantepie, Sandrine; Pagadala, Vijayakanth; Lu, Jian; Wells, Carrow; Drewry, David H; Zuercher, William J; Berry, Neil G; Fernig, David G; Eyers, Patrick A
2018-06-22
Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2- O -sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3'-phosphoadenosine 5'-phosphosulphate) to the 2- O position of a-L-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro , including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST. ©2018 The Author(s).
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Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian
2012-07-11
G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complexmore » in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.« less
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Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.
2010-07-19
G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.
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Han, Mei; Li, Shan; Ai, Jing; Sheng, Rong; Hu, Yongzhou; Hu, Youhong; Geng, Meiyu
2016-12-01
A series of novel 4-chloro-benzamides derivatives containing substituted five-membered heteroaryl ring were designed, synthesized and evaluated as RET kinase inhibitors for cancer therapy. Most of compounds exhibited moderate to high potency in ELISA-based kinase assay. In particular, compound I-8 containing 1,2,4-oxadiazole strongly inhibited RET kinase activity both in molecular and cellular level. In turn, I-8 inhibited cell proliferation driven by RET wildtype and gatekeeper mutation. The results implied that 4-chloro-3-(5-(pyridin-3-yl)-1,2,4-oxadiazole-3-yl)benzamides are promising lead compounds as novel RET kinase inhibitor for further investigation. Copyright © 2016 Elsevier Ltd. All rights reserved.
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The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*
Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut
2015-01-01
Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157
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Oakley, Fiona; Meso, Muriel; Iredale, John P; Green, Karen; Marek, Carylyn J; Zhou, Xiaoying; May, Michael J; Millward-Sadler, Harry; Wright, Matthew C; Mann, Derek A
2005-01-01
Resolution of liver fibrosis is associated with clearance of hepatic myofibroblasts by apoptosis; development of strategies that promote this process in a selective way is therefore important. The aim of this study was to determine whether the inhibitor of kappaB kinase suppressor sulfasalazine stimulates hepatic myofibroblast apoptosis and recovery from fibrosis. Hepatic myofibroblasts were generated by culture activation of rat and human hepatic stellate cells. Fibrosis was established in rat livers by chronic injury with carbon tetrachloride followed by recovery with or without sulfasalazine (150 mg/kg) treatment. Treatment of hepatic stellate cells with sulfasalazine (0.5-2.0 mmol/L) induced apoptosis of activated rat and human hepatic stellate cells. A single in vivo administration of sulfasalazine promoted accelerated recovery from fibrosis as assessed by improved fibrosis score, selective clearance of smooth muscle alpha-actin-positive myofibroblasts, reduced hepatic procollagen I and tissue inhibitor of metalloproteinase 1 messenger RNA expression, and increased matrix metalloproteinase 2 activity. Mechanistic studies showed that sulfasalazine selectively blocks nuclear factor-kappaB-dependent gene transcription, inhibits hepatic stellate cell expression of Gadd45beta, stimulates phosphorylation of Jun N-terminal kinase 2, and promotes apoptosis by a mechanism that is prevented by the Jun N-terminal kinase inhibitor SP600125. As further evidence for a survival role for the inhibitor of kappaB kinase/nuclear factor-kappaB pathway in activated hepatic stellate cells, a highly selective cell-permeable peptide inhibitor of kappaB kinase activation also stimulated hepatic stellate cell apoptosis via a Jun N-terminal kinase-dependent mechanism. Inhibition of the inhibitor of kappaB kinase/nuclear factor-kappaB pathway is sufficient to increase the rate at which activated hepatic stellate cells undergo apoptosis both in vitro and in vivo, and drugs that selectively target inhibitor of kappaB kinase have potential as antifibrotics.
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A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2.
Wang, Hong; Brautigan, David L
2002-12-20
Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.
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An, Xiao-De; Liu, Hongyan; Xu, Zhong-Liang; Jin, Yi; Peng, Xia; Yao, Ying-Ming; Geng, Meiyu; Long, Ya-Qiu
2015-02-01
Starting from our previously identified novel c-Met kinase inhibitors bearing 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one scaffold, a global structural exploration was conducted to furnish an optimal binding motif for further development, directed by the enzyme inhibitory mechanism. First round SAR study picked two imidazonaphthyridinone frameworks with 1,8- and 3,5-disubstitution pattern as class I and class II c-Met kinase inhibitors, respectively. Further structural optimization on type II inhibitors by truncation of the imidazonaphthyridinone core and incorporation of an N-phenyl cyclopropane-1,1-dicarboxamide pharmacophore led to the discovery of novel imidazopyridine-based c-Met kinase inhibitors, displaying nanomolar enzyme inhibitory activity and improved Met kinase selectivity. More significantly, the new chemotype c-Met kinase inhibitors effectively inhibited Met phosphorylation and its downstream signaling as well as the proliferation of Met-dependent EBC-1 human lung cancer cells at submicromolar concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Ripple, Maureen O.; Kim, Namjoon; Springett, Roger
2013-01-01
The Ras-MEK1/2-ERK1/2 kinase signaling pathway regulates proliferation, survival, and differentiation and, because it is often aberrant in tumors, is a popular target for small molecule inhibition. A novel metabolic analysis that measures the real-time oxidation state of NAD(H) and the hemes of the electron transport chain and oxygen consumption within intact, living cells found that structurally distinct MEK1/2 inhibitors had an immediate, dose-dependent effect on mitochondrial metabolism. The inhibitors U0126, MIIC and PD98059 caused NAD(H) reduction, heme oxidation, and decreased oxygen consumption, characteristic of complex I inhibition. PD198306, an orally active MEK1/2 inhibitor, acted as an uncoupler. Each MEK1/2 inhibitor depleted phosphorylated ERK1/2 and inhibited proliferation, but the most robust antiproliferative effects always correlated with the metabolic failure which followed mitochondrial inhibition rather than inhibition of MEK1/2. This warrants rethinking the role of ERK1/2 in proliferation and emphasizes the importance of mitochondrial function in this process. PMID:23235157
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Kuhlmann, Naila; Kadgien, Chelsie A; Tatarnikov, Igor; Fox, Jesse; Khinda, Jaskaran; Mitchell, Emma; Bergeron, Sabrina; Melrose, Heather
2017-01-01
LRRK2 mutations produce end-stage Parkinson’s disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD. PMID:28930069
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Volta, Mattia; Beccano-Kelly, Dayne A; Paschall, Sarah A; Cataldi, Stefano; MacIsaac, Sarah E; Kuhlmann, Naila; Kadgien, Chelsie A; Tatarnikov, Igor; Fox, Jesse; Khinda, Jaskaran; Mitchell, Emma; Bergeron, Sabrina; Melrose, Heather; Farrer, Matthew J; Milnerwood, Austen J
2017-09-20
LRRK2 mutations produce end-stage Parkinson's disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD.
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Structure of the β-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090
Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Matsumoto, Yoshiyuki; Takimoto-Kamimura, Midori
2013-01-01
Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2), a serine/threonine kinase from the p38 mitogen-activated protein kinase signalling pathway, plays an important role in the production of TNF-α and other cytokines. In a previous report, it was shown that MK2 in complex with the selective inhibitor TEI-I01800 adopts an α-helical glycine-rich loop that is induced by the stable nonplanar conformer of TEI-I01800. To understand the mechanism of the structural change, the structure of MK2 bound to TEI-L03090, which lacks the key substituent found in TEI-I01800, was determined. MK2–TEI-L03090 has a β-sheet glycine-rich loop in common with other kinases, as predicted. This result suggests that a small compound can induce a drastic conformational change in the target protein structure and can be used to design potent and selective inhibitors. PMID:24316826
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Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors
Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.
2003-06-03
The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.
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The Future of Janus Kinase Inhibitors in Inflammatory Bowel Disease
De Vries, L.C.S.; Wildenberg, M.E.; De Jonge, W.J.
2017-01-01
Abstract Inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease, are disabling conditions characterised by chronic, relapsing inflammation of the gastrointestinal tract. Current treatments are not universally effective or, in the case of therapeutic antibodies, are hampered by immune responses. Janus kinase inhibitors are orally delivered small molecules that target cytokine signalling by preventing phosphorylation of Janus kinases associated with the cytokine receptor. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling and transcription of cytokines in the nucleus will be diminished. Key cytokines in the pathogenesis of inflammatory bowel diseases are targeted by Janus kinase inhibitors. Several Janus kinase inhibitors are in development for the treatment of inflammatory bowel diseases. Tofacitinib, inhibiting signalling via all Janus kinase family members, was effective in phase 2 and 3 trials in moderate-severe ulcerative colitis. GSK2586184, a Janus kinase 1 selective inhibitor, induced clinical and endoscopic response in ulcerative colitis; however, the study was discontinued at an early stage due to liver toxicity observed in systemic lupus patients receiving the drug. Filgotinib, a Janus kinase 1 selective inhibitor investigated in treatment of Crohn’s disease, was superior to placebo. As adverse events associated with the broad immunological effect of these agents have been reported, the future application of these drugs is potentially limited. We will discuss the treatment efficacy of Janus kinase inhibition in inflammatory bowel diseases, how current Janus kinase inhibitors available target immune responses relevant in inflammatory bowel disease, and whether more specific kinase inhibition could be effective. PMID:28158411
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The Future of Janus Kinase Inhibitors in Inflammatory Bowel Disease.
De Vries, L C S; Wildenberg, M E; De Jonge, W J; D'Haens, G R
2017-07-01
Inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease, are disabling conditions characterised by chronic, relapsing inflammation of the gastrointestinal tract. Current treatments are not universally effective or, in the case of therapeutic antibodies, are hampered by immune responses. Janus kinase inhibitors are orally delivered small molecules that target cytokine signalling by preventing phosphorylation of Janus kinases associated with the cytokine receptor. Subsequently, phosphorylation of signal transducers and activators of transcription that relay Janus kinase signalling and transcription of cytokines in the nucleus will be diminished. Key cytokines in the pathogenesis of inflammatory bowel diseases are targeted by Janus kinase inhibitors. Several Janus kinase inhibitors are in development for the treatment of inflammatory bowel diseases. Tofacitinib, inhibiting signalling via all Janus kinase family members, was effective in phase 2 and 3 trials in moderate-severe ulcerative colitis. GSK2586184, a Janus kinase 1 selective inhibitor, induced clinical and endoscopic response in ulcerative colitis; however, the study was discontinued at an early stage due to liver toxicity observed in systemic lupus patients receiving the drug. Filgotinib, a Janus kinase 1 selective inhibitor investigated in treatment of Crohn's disease, was superior to placebo. As adverse events associated with the broad immunological effect of these agents have been reported, the future application of these drugs is potentially limited. We will discuss the treatment efficacy of Janus kinase inhibition in inflammatory bowel diseases, how current Janus kinase inhibitors available target immune responses relevant in inflammatory bowel disease, and whether more specific kinase inhibition could be effective. © European Crohn’s and Colitis Organisation (ECCO) 2017.
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2-Aminobenzimidazoles as potent Aurora kinase inhibitors.
Zhong, Min; Bui, Minna; Shen, Wang; Baskaran, Subramanian; Allen, Darin A; Elling, Robert A; Flanagan, W Michael; Fung, Amy D; Hanan, Emily J; Harris, Shannon O; Heumann, Stacey A; Hoch, Ute; Ivy, Sheryl N; Jacobs, Jeffrey W; Lam, Stuart; Lee, Heman; McDowell, Robert S; Oslob, Johan D; Purkey, Hans E; Romanowski, Michael J; Silverman, Jeffrey A; Tangonan, Bradley T; Taverna, Pietro; Yang, Wenjin; Yoburn, Josh C; Yu, Chul H; Zimmerman, Kristin M; O'Brien, Tom; Lew, Willard
2009-09-01
This Letter describes the discovery and key structure-activity relationship (SAR) of a series of 2-aminobenzimidazoles as potent Aurora kinase inhibitors. 2-Aminobenzimidazole serves as a bioisostere of the biaryl urea residue of SNS-314 (1c), which is a potent Aurora kinase inhibitor and entered clinical testing in patients with solid tumors. Compared to SNS-314, this series of compounds offers better aqueous solubility while retaining comparable in vitro potency in biochemical and cell-based assays; in particular, 6m has also demonstrated a comparable mouse iv PK profile to SNS-314.
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Levinson, Nicholas M.; Boxer, Steven G.
2012-01-01
Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name “bosutinib”, and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity. PMID:22493660
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Kaieda, Akira; Takahashi, Masashi; Takai, Takafumi; Goto, Masayuki; Miyazaki, Takahiro; Hori, Yuri; Unno, Satoko; Kawamoto, Tomohiro; Tanaka, Toshimasa; Itono, Sachiko; Takagi, Terufumi; Hamada, Teruki; Shirasaki, Mikio; Okada, Kengo; Snell, Gyorgy; Bragstad, Ken; Sang, Bi-Ching; Uchikawa, Osamu; Miwatashi, Seiji
2018-02-01
We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Fan, Ya-Ping; Puri, Rajinder N; Rattan, Satish
2002-03-01
Effect of ANG II was investigated in in vitro smooth muscle strips and in isolated smooth muscle cells (SMC). Among different species, rat internal and sphincter (IAS) smooth muscle showed significant and reproducible contraction that remained unmodified by different neurohumoral inhibitors. The AT(1) antagonist losartan but not AT(2) antagonist PD-123319 antagonized ANG II-induced contraction of the IAS smooth muscle and SMC. ANG II-induced contraction of rat IAS smooth muscle and SMC was attenuated by tyrosine kinase inhibitors genistein and tyrphostin, protein kinase C (PKC) inhibitor H-7, Ca(2+) channel blocker nicardipine, Rho kinase inhibitor Y-27632 or p(44/42) mitogen-activating protein kinase (MAPK(44/42)) inhibitor PD-98059. Combinations of nicardipine and H-7, Y-27632, and PD-98059 caused further attenuation of the ANG II effects. Western blot analyses revealed the presence of both AT(1) and AT(2) receptors. We conclude that ANG II causes contraction of rat IAS smooth muscle by the activation of AT(1) receptors at the SMC and involves multiple intracellular pathways, influx of Ca(2+), and activation of PKC, Rho kinase, and MAPK(44/42).
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Grant, Karen M.; Dunion, Morag H.; Yardley, Vanessa; Skaltsounis, Alexios-Leandros; Marko, Doris; Eisenbrand, Gerhard; Croft, Simon L.; Meijer, Laurent; Mottram, Jeremy C.
2004-01-01
The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation. We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target. A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors. Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro. Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 μM. These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine. The paullones and staurosporine derivatives were toxic to macrophages. The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations. The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 μM, respectively. In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control. Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development. PMID:15273118
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G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives.
Guccione, Manuela; Ettari, Roberta; Taliani, Sabrina; Da Settimo, Federico; Zappalà, Maria; Grasso, Silvana
2016-10-27
G-protein-coupled receptor kinase 2 (GRK2) is a G-protein-coupled receptor kinase that is ubiquitously expressed in many tissues and regulates various intracellular mechanisms. The up- or down-regulation of GRK2 correlates with several pathological disorders. GRK2 plays an important role in the maintenance of heart structure and function; thus, this kinase is involved in many cardiovascular diseases. GRK2 up-regulation can worsen cardiac ischemia; furthermore, increased kinase levels occur during the early stages of heart failure and in hypertensive subjects. GRK2 up-regulation can lead to changes in the insulin signaling cascade, which can translate to insulin resistance. Increased GRK2 levels also correlate with the degree of cognitive impairment that is typically observed in Alzheimer's disease. This article reviews the most potent and selective GRK2 inhibitors that have been developed. We focus on their mechanism of action, inhibition profile, and structure-activity relationships to provide insight into the further development of GRK2 inhibitors as drug candidates.
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Koch, Heiner; Busto, M Estela Del Castillo; Kramer, Karl; Médard, Guillaume; Kuster, Bernhard
2015-06-05
Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR), is in clinical use for treating non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high initial response rates, many patients develop resistance to gefitinib. The molecular mechanisms of TKI resistance often remain unclear. Here, we describe a chemical proteomic approach comprising kinase affinity purification (kinobeads) and quantitative mass spectrometry for the identification of kinase inhibitor resistance mechanisms in cancer cells. We identified the previously described amplification of MET and found EPHA2 to be more than 10-fold overexpressed (p < 0.001) in gefitinib-resistant HCC827 cells suggesting a potential role in developing resistance. siRNA-mediated EPHA2 knock-down or treating cells with the multikinase inhibitor dasatinib restored sensitivity to gefitinib. Of all dasatinib targets, EPHA2 exhibited the most drastic effect (p < 0.001). In addition, EPHA2 knockdown or ephrin-A1 treatment of resistant cells decreased FAK phosphorylation and cell migration. These findings confirm EPHA2 as an actionable drug target, provide a rational basis for drug combination approaches, and indicate that chemical proteomics is broadly applicable for the discovery of kinase inhibitor resistance.
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Anti-inflammatory properties of Gö 6850: a selective inhibitor of protein kinase C.
Jacobson, P B; Kuchera, S L; Metz, A; Schächtele, C; Imre, K; Schrier, D J
1995-11-01
Protein kinase C (PKC) regulates a variety of signal transduction events implicated in the pathogenesis of inflammation, including the biosynthesis of inflammatory cytokines and superoxide and the activation of phospholipase A2. Because of the significant role of PKC in these inflammatory processes, we evaluated a specific and potent inhibitor of C kinase for efficacy in several in vitro and in vivo murine models of inflammation. Unlike the relatively nonspecific kinase inhibitor staurosporine, the bisindolylmaleimide 3-[1-[-3-(dimethylaminopropyl]-1H-indol-3-yl]- 4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (Gö 6850) demonstrated increased selectivity for C kinase in purified enzyme assays (respective IC50 values (microM) for Gö 6850 and staurosporine: protein kinase C (0.032, 0.009); myosin light-chain kinase (0.6, 0.01); protein kinase G (4.6, 0.018); protein kinase A (33, 0.04); tyrosine kinase1 (94, 0.4); tyrosine kinase2 (> 100, > 1)). Topically applied Gö 6850 inhibited phorbol myristate acetate-induced edema, neutrophil influx and vascular permeability in murine epidermis in a dose- and time-dependent manner at levels comparable to indomethacin. In a murine model of delayed type hypersensitivity, Gö 6850 inhibited dinitrofluorobenzene-induced contact dermatitis with and ID50 value of 150 micrograms/ear. Cellular studies in mouse peritoneal macrophages demonstrated that Gö 6850 was a potent inhibitor of phorbol myristate acetate-induced prostaglandin E2 production. Superoxide production in phorbol myristate acetate-stimulated murine neutrophils was also inhibited by Gö 6850 (IC50 = 88 nM).(ABSTRACT TRUNCATED AT 250 WORDS)
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Structure of the Human Protein Kinase ZAK in Complex with Vemurafenib
Mathea, Sebastian; Abdul Azeez, Kamal R.; Salah, Eidarus; Tallant, Cynthia; Wolfreys, Finn; Konietzny, Rebecca; Fischer, Roman; Lou, Hua Jane; Brennan, Paul E.; Schnapp, Gisela; Pautsch, Alexander; Kessler, Benedikt M.; Turk, Benjamin E.; Knapp, Stefan
2017-01-01
The mixed lineage kinase ZAK is a key regulator of the MAPK pathway mediating cell survival and inflammatory response. ZAK is targeted by several clinically approved kinase inhibitors, and inhibition of ZAK has been reported to protect from doxorubicin-induced cardiomyopathy. On the other hand, unintended targeting of ZAK has been linked to severe adverse effects such as the development of cutaneous squamous cell carcinoma. Therefore, both specific inhibitors of ZAK, as well as anticancer drugs lacking off-target activity against ZAK, may provide therapeutic benefit. Here we report the first crystal structure of ZAK in complex with the B-RAF inhibitor vemurafenib. The co-crystal structure displayed a number of ZAK-specific features including a highly distorted P loop conformation enabling rational inhibitor design. Positional scanning peptide library analysis revealed a unique substrate specificity of the ZAK kinase including unprecedented preferences for histidine residues at positions −1 and +2 relative to the phosphoacceptor site. In addition, we screened a library of clinical kinase inhibitors identifying several inhibitors that potently inhibit ZAK, demonstrating that this kinase is commonly mistargeted by currently used anticancer drugs. PMID:26999302
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Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity
Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.
2011-01-01
Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377
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Loaëc, Nadège; Attanasio, Eletta; Villiers, Benoît; Durieu, Emilie; Tahtouh, Tania; Cam, Morgane; Alencar, Aline; Roué, Mélanie; Bourguet-Kondracki, Marie-Lise; Proksch, Peter; Limanton, Emmanuelle; Guiheneuf, Solène; Carreaux, François; Bazureau, Jean-Pierre; Klautau, Michelle
2017-01-01
A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets. PMID:29039762
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Loaëc, Nadège; Attanasio, Eletta; Villiers, Benoît; Durieu, Emilie; Tahtouh, Tania; Cam, Morgane; Davis, Rohan A; Alencar, Aline; Roué, Mélanie; Bourguet-Kondracki, Marie-Lise; Proksch, Peter; Limanton, Emmanuelle; Guiheneuf, Solène; Carreaux, François; Bazureau, Jean-Pierre; Klautau, Michelle; Meijer, Laurent
2017-10-17
A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina . The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.
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2012-01-01
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A Kd = 7.5 nM, Aurora-B Kd = 48 nM), FLT3 kinase (Kd = 6.2 nM), and FLT3 mutants including FLT3-ITD (Kd = 38 nM) and FLT3(D835Y) (Kd = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20–35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4–11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children. PMID:23043539
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Stuhlmiller, Timothy J; Zawistowski, Jon S; Chen, Xin; Sciaky, Noah; Angus, Steven P; Hicks, Sean T; Parry, Traci L; Huang, Wei; Beak, Ju Youn; Willis, Monte S; Johnson, Gary L; Jensen, Brian C
2017-10-19
Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.
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Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms
Atallah, Ehab; Verstovsek, Srdan
2016-01-01
The discovery of the Janus kinase (JAK)2 V617F mutation in patients with myeloproliferative neoplasms was a major milestone in understanding the biology of those disorders. Several groups simultaneously reported on the high incidence of this mutation in patients with myeloproliferative neoplasms: almost all patients with polycythemia vera harbor the mutation and about 50% of patients with essential thrombocythemia and primary myelofibrosis have the mutation, making the development of JAK2 tyrosine kinase inhibitors an attractive therapeutic goal. In addition, inhibition of JAK2 kinase may have a therapeutic role in other hematologic malignancies, such as chronic myeloid leukemia or lymphoma. A number of molecules that inhibit JAK2 kinase have been described in the literature, and several are being evaluated in a clinical setting. Here, we summarize current clinical experience with JAK2 inhibitors. PMID:19445582
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A Tangled Web – Tau and Sporadic Parkinson's Disease
Wray, Selina; Lewis, Patrick A.
2010-01-01
Parkinson's disease (PD) represents a major challenge for health care systems around the world: it is the most common degenerative movement disorder of old age, affecting over 100,000 people in the UK alone (Schrag et al., 2000). Despite the remarkable success of treatments directed at potentiating or replacing dopamine within the brain, which can relieve symptoms for over a decade, PD remains an incurable and invariably fatal disorder. As such, efforts to understand the processes that lead to cell death in the brains of patients with PD are a priority for neurodegenerative researchers. A great deal of progress has been made in this regard by taking advantage of advances in genetics, initially by the identification of genes responsible for rare Mendelian forms of PD (outlined in Table 1), and more recently by applying genome wide association studies (GWAS) to the sporadic form of the disease (Hardy et al., 2009). Several such GWAS have now been carried out, with a meta-analysis currently under way. Using over 6000 cases and 10,000 controls, two of these studies have identified variation at a number of loci as being associated with an increased risk of disease (Satake et al., 2009; Simon-Sanchez et al., 2009). Three genes stand out as candidates from these studies – the SNCA gene, coding for α-synuclein, the LRRK2 gene, coding for leucine rich repeat kinase 2, and MAPT, coding for the microtubule-associated protein tau. Mutations at all three of these loci have been associated with Mendelian forms of disease presenting with the clinical syndrome of Parkinsonism, however only SNCA and LRRK2 have been previously associated with pathologically defined PD (Hardy et al., 2009). Point mutations in α-synuclein, along with gene multiplication events, result in autosomal dominant PD, often with a significant dementia component. In addition to this, α-synuclein is the principle component of the main pathological hallmark of idiopathic PD, the Lewy body, making it an unsurprising hit in the GWAS (Spillantini et al., 1997). Mutations in LRRK2 are the most common genetic cause of PD, and so again made this gene a likely candidate as a susceptibility locus for the sporadic form of disease (Kumari and Tan, 2009). More surprising, perhaps, was the identification of tau as a susceptibility factor for Parkinson's. In this review we will outline the role of tau in neurodegeneration and in different forms of Parkinsonism, and speculate as to what the functional basis of the association between MAPT and PD might be. PMID:21423457
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A tangled web - tau and sporadic Parkinson's disease.
Wray, Selina; Lewis, Patrick A
2010-01-01
Parkinson's disease (PD) represents a major challenge for health care systems around the world: it is the most common degenerative movement disorder of old age, affecting over 100,000 people in the UK alone (Schrag et al., 2000). Despite the remarkable success of treatments directed at potentiating or replacing dopamine within the brain, which can relieve symptoms for over a decade, PD remains an incurable and invariably fatal disorder. As such, efforts to understand the processes that lead to cell death in the brains of patients with PD are a priority for neurodegenerative researchers. A great deal of progress has been made in this regard by taking advantage of advances in genetics, initially by the identification of genes responsible for rare Mendelian forms of PD (outlined in Table 1), and more recently by applying genome wide association studies (GWAS) to the sporadic form of the disease (Hardy et al., 2009). Several such GWAS have now been carried out, with a meta-analysis currently under way. Using over 6000 cases and 10,000 controls, two of these studies have identified variation at a number of loci as being associated with an increased risk of disease (Satake et al., 2009; Simon-Sanchez et al., 2009). Three genes stand out as candidates from these studies - the SNCA gene, coding for α-synuclein, the LRRK2 gene, coding for leucine rich repeat kinase 2, and MAPT, coding for the microtubule-associated protein tau. Mutations at all three of these loci have been associated with Mendelian forms of disease presenting with the clinical syndrome of Parkinsonism, however only SNCA and LRRK2 have been previously associated with pathologically defined PD (Hardy et al., 2009). Point mutations in α-synuclein, along with gene multiplication events, result in autosomal dominant PD, often with a significant dementia component. In addition to this, α-synuclein is the principle component of the main pathological hallmark of idiopathic PD, the Lewy body, making it an unsurprising hit in the GWAS (Spillantini et al., 1997). Mutations in LRRK2 are the most common genetic cause of PD, and so again made this gene a likely candidate as a susceptibility locus for the sporadic form of disease (Kumari and Tan, 2009). More surprising, perhaps, was the identification of tau as a susceptibility factor for Parkinson's. In this review we will outline the role of tau in neurodegeneration and in different forms of Parkinsonism, and speculate as to what the functional basis of the association between MAPT and PD might be.
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Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors
Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L.; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E.; Cuny, Gregory D.; Uhlig, Holm H.; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N.
2015-01-01
Summary RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. PMID:26320862
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Sakai, Hiroyasu; Watanabe, Yu; Honda, Mai; Tsuiki, Rika; Ueda, Yusuke; Nagai, Yuki; Narita, Minoru; Misawa, Miwa; Chiba, Yoshihiko
2013-05-01
Tyrosine (Tyr) kinases and mitogen-activated protein kinases have been thought to participate in the contractile response in various smooth muscles. The aim of the current study was to investigate the involvement of the Tyr kinase pathway in the contraction of bronchial smooth muscle. Ring preparations of bronchi isolated from rats were suspended in an organ bath. Isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine the phosphorylation of c-Jun N-terminal kinasess (JNKs) in bronchial smooth muscle. To examine the role of mitogen-activated protein kinase(s) in bronchial smooth muscle contraction, the effects of MPAK inhibitors were investigated in this study. The contraction induced by carbachol (CCh) was significantly inhibited by pretreatment with selective Tyr kinase inhibitors (genistein and ST638, n = 6, respectively), and a JNK inhibitor (SP600125, n = 6). The contractions induced by high K depolarization (n = 4), orthovanadate (a potent Tyr phosphatase inhibitor) and sodium fluoride (a G protein activator; NaF) were also significantly inhibited by selective Tyr kinase inhibitors and a JNK inhibitor (n = 4, respectively). However, the contraction induced by calyculin-A was not affected by SP600125. On the other hand, JNKs were phosphorylated by CCh (2.2 ± 0,4 [mean±SEM] fold increase). The JNK phosphorylation induced by CCh was significantly inhibited by SP600125 (n = 4). These findings suggest that the Tyr kinase/JNK pathway may play a role in bronchial smooth muscle contraction. Strategies to inhibit JNK activation may represent a novel therapeutic approach for diseases involving airway obstruction, such as asthma and chronic obstructive pulmonary disease.
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Heterodimeric JAK-STAT Activation as a Mechanism of Persistence to JAK2 Inhibitor Therapy
Koppikar, Priya; Bhagwat, Neha; Kilpivaara, Outi; Manshouri, Taghi; Adli, Mazhar; Hricik, Todd; Liu, Fan; Saunders, Lindsay M.; Mullally, Ann; Abdel-Wahab, Omar; Leung, Laura; Weinstein, Abby; Marubayashi, Sachie; Goel, Aviva; Gönen, Mithat; Estrov, Zeev; Ebert, Benjamin L.; Chiosis, Gabriela; Nimer, Stephen D.; Bernstein, Bradley E.; Verstovsek, Srdan; Levine, Ross L.
2012-01-01
The identification of somatic activating mutations in JAK21–4 and in the thrombopoietin receptor (MPL)5 in the majority of myeloproliferative neoplasm (MPN) patients led to the clinical development of JAK2 kinase inhibitors6,7. JAK2 inhibitor therapy improves MPN-associated splenomegaly and systemic symptoms, but does not significantly reduce or eliminate the MPN clone in most MPN patients. We therefore sought to characterize mechanisms by which MPN cells persist despite chronic JAK2 inhibition. Here we show that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. Further, this phenomenon is reversible, such that JAK2 inhibitor withdrawal is associated with resensitization to JAK2 kinase inhibitors and with reversible changes in JAK2 expression. We saw increased JAK2 heterodimerization and sustained JAK2 activation in cell lines, murine models, and patients treated with JAK2 inhibitors. RNA interference and pharmacologic studies demonstrate that JAK2 inhibitor persistent cells remain dependent on JAK2 protein expression. Consequently, therapies that result in JAK2 degradation retain efficacy in persistent cells and may provide additional benefit to patients with JAK2-dependent malignancies treated with JAK2 inhibitors. PMID:22820254
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Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition
Wu, Wen-I; Voegtli, Walter C.; Sturgis, Hillary L.; Dizon, Faith P.; Vigers, Guy P. A.; Brandhuber, Barbara J.
2010-01-01
AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones. PMID:20886116
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Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E
2015-08-01
The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.
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Neuropsychological performance in LRRK2 G2019S carriers with Parkinson’s disease
Alcalay, Roy N.; Mejia-Santana, Helen; Mirelman, Anat; Saunders-Pullman, Rachel; Raymond, Deborah; Palmese, Christina; Caccappolo, Elise; Ozelius, Laurie; Orr-Urtreger, Avi; Clark, Lorraine; Giladi, Nir; Bressman, Susan; Marder, Karen
2014-01-01
Background Ashkenazi Jewish (AJ) LRRK2 carriers are more likely to manifest the postural instability gait difficulty (PIGD) motor phenotype than non-carriers but perform similarly to non-carriers on cognitive screening tests. Objective To compare the cognitive profiles of AJ with Parkinson’s disease (PD) with and without LRRK2 G2019S mutations using a comprehensive neuropsychological battery. Methods We administered a neuropsychological battery to PD participants in the Michael J. Fox Foundation AJ consortium. Participants (n=236) from Beth Israel Medical Center, NY, Columbia University Medical Center, NY and Tel Aviv Medical Center, Israel included 116 LRRK2 G2019S carriers and 120 non-carriers. Glucocerbrosidase mutation carriers were excluded. We compared performance on each neuropsychological test between carriers and non-carriers. Participants in New York (n=112) were evaluated with the entire battery. Tel Aviv participants (n=124) were evaluated on attention, executive function and psychomotor speed tasks. The association between G2019S mutation status (predictor) and each neuropsychological test (outcome) was assessed using linear regression models adjusted for PIGD motor phenotype, site, sex, age, disease duration, education, Unified Parkinson’s Disease Rating Scale (UPDRS) Part III, levodopa equivalent dose, and Geriatric Depression Score (GDS). Results Carriers had longer disease duration (p<0.001) and were more likely to manifest the PIGD phenotype (p=0.024). In adjusted regression models, carriers performed better than non-carriers in Stroop Word Reading (p<0.001), Stroop Interference (p=0.011) and Category Fluency (p=0.026). Conclusion In AJ-PD, G2019S mutation status is associated with better attention (Stroop Word Reading), executive function (Stroop Interference) and language (Category Fluency) after adjustment for PIGD motor phenotype. PMID:25434972
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Angiotensin II increases Pax-2 expression in fetal kidney cells via the AT2 receptor.
Zhang, Shao-Ling; Moini, Babak; Ingelfinger, Julie R
2004-06-01
Although both the renin angiotensin system (RAS) and the paired homeobox 2 gene (Pax-2) seem critically important in renal organogenesis, whether and how they might interact has not been addressed. The present study asked whether a link between the RAS and Pax-2 exists in fetal renal cells, speculating that such an interaction, if present, might influence renal development. Embryonic kidney explants and embryonic renal cells (mouse late embryonic mesenchymal epithelial cells [MK4] and mouse early embryonic mesenchymal fibroblasts [MK3]) were used. Pax-2 protein and Pax-2 mRNA were detected by immunofluorescence, Western blot, reverse transcription-PCR, and real-time PCR. Angiotensin II (AngII) upregulated Pax-2 protein and Pax-2 mRNA expression via the AngII type 2 (AT(2)) receptor in MK4 but not in MK3 cells. The stimulatory effect of AngII on Pax-2 gene expression could be blocked by PD123319 (AT(2) inhibitor), AG 490 (a specific Janus kinase 2 inhibitor), and genistein (a tyrosine kinase inhibitor) but not by losartan (AT(1) inhibitor), SB203580 (specific p38 mitogen-activated protein kinase inhibitor), PD98059 (specific MEK inhibitor), SP600125 (JNK inhibitor), and diphenyleneiodonium chloride (an NADPH oxidase inhibitor). Moreover, embryonic kidney explants in culture confirmed that AngII upregulates Pax-2 gene expression via the AT(2) receptor. These studies demonstrate that the stimulatory effect of AngII on Pax-2 gene expression is mediated, at least in part, via the Janus kinase 2/signal transducers and activators of transcription signaling transduction pathway, suggesting that RAS and Pax-2 interactions may be important in renal development.
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Li, Bei; Cociorva, Oana M; Nomanbhoy, Tyzoon; Weissig, Helge; Li, Qiang; Nakamura, Kai; Liyanage, Marek; Zhang, Melissa C; Shih, Ann Y; Aban, Arwin; Hu, Yi; Cajica, Julia; Pham, Lan; Kozarich, John W; Shreder, Kevin R
2013-09-15
As the result of a rhJNK1 HTS, the imidazo[1,2-a]quinoxaline 1 was identified as a 1.6 μM rhJNK1 inhibitor. Optimization of this compound lead to AX13587 (rhJNK1 IC50=160 nM) which was co-crystallized with JNK1 to identify key molecular interactions. Kinase profiling against 125+ kinases revealed AX13587 was an inhibitor of JNK, MAST3, and MAST4 whereas its methylene homolog AX14373 (native JNK1 IC50=47 nM) was a highly specific JNK inhibitor. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Bagley, Mark C; Dwyer, Jessica E; Molina, Maria D Beltran; Rand, Alexander W; Rand, Hayley L; Tomkinson, Nicholas C O
2015-06-28
Microwave irradiation of 2-halobenzonitriles and methyl thioglycolate in the presence of triethylamine in DMSO at 130 °C provides rapid access to 3-aminobenzo[b]thiophenes in 58-96% yield. This transformation has been applied in the synthesis of the thieno[2,3-b]pyridine core motif of LIMK1 inhibitors, the benzo[4,5]thieno[3,2-e][1,4]diazepin-5(2H)-one scaffold of MK2 inhibitors and a benzo[4,5]thieno[3,2-d]pyrimidin-4-one inhibitor of the PIM kinases.
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Wang, Sheng; Xu, Lei; Lu, Yu-Ting; Liu, Yu-Fei; Han, Bing; Liu, Ting; Tang, Jie; Li, Jia; Wu, Jiangping; Li, Jing-Ya; Yu, Li-Fang; Yang, Fan
2017-04-21
Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is a serine/threonine kinase that plays a key role in a wide variety of cell death signaling pathways. Inhibition of DRAK2 was found to efficiently protect islet β-cells from apoptosis and hence DRAK2 inhibitors represent a promising therapeutic strategy for the treatment of diabetes. Only very few chemical entities targeting DRAK2 are currently known. We carried out a high throughput screening and identified compound 4 as a moderate DRAK2 inhibitor with an IC 50 value of 3.15 μM. Subsequent SAR studies of hit compound 4 led to the development of novel benzofuran-3(2H)-one series of DRAK2 inhibitors with improved potency and favorable selectivity profiles against 26 selected kinases. Importantly, most potent compounds 40 (IC 50 = 0.33 μM) and 41 (IC 50 = 0.25 μM) were found to protect islet β-cells from apoptosis in dose-dependent manners. These data support the notion that small molecule inhibitors of DRAK2 represents a promising strategy for the treatment of diabetes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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NASA Astrophysics Data System (ADS)
Cao, Shandong
2012-08-01
The purpose of the present study was to develop in silico models allowing for a reliable prediction of polo-like kinase inhibitors based on a large diverse dataset of 136 compounds. As an effective method, quantitative structure activity relationship (QSAR) was applied using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The proposed QSAR models showed reasonable predictivity of thiophene analogs (Rcv2=0.533, Rpred2=0.845) and included four molecular descriptors, namely IC3, RDF075m, Mor02m and R4e+. The optimal model for imidazopyridine derivatives (Rcv2=0.776, Rpred2=0.876) was shown to perform good in prediction accuracy, using GATS2m and BEHe1 descriptors. Analysis of the contour maps helped to identify structural requirements for the inhibitors and served as a basis for the design of the next generation of the inhibitor analogues. Docking studies were also employed to position the inhibitors into the polo-like kinase active site to determine the most probable binding mode. These studies may help to understand the factors influencing the binding affinity of chemicals and to develop alternative methods for prescreening and designing of polo-like kinase inhibitors.
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Hit to lead optimization of pyrazolo[1,5-a]pyrimidines as B-Raf kinase inhibitors.
Gopalsamy, Ariamala; Ciszewski, Greg; Shi, Mengxiao; Berger, Dan; Hu, Yongbo; Lee, Frederick; Feldberg, Larry; Frommer, Eileen; Kim, Steven; Collins, Karen; Wojciechowicz, Donald; Mallon, Robert
2009-12-15
Our continued effort towards optimization of the pyrazolo[1,5-a]pyrimidine scaffold as B-Raf kinase inhibitors is described. Structure guided design was utilized to introduce kinase hinge region interacting groups in the 2-position of the scaffold. This strategy led to the identification of lead compound 9 with enhanced enzyme and cellular potency, while maintaining good selectivity over a number of kinases.
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Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun
2015-05-01
Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
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2015-01-01
Drug-resistance acquisition through kinase gate-keeper mutations is a major hurdle in the clinic. Here, we determined the first crystal structures of the human FGFR4 kinase domain (FGFR4K) alone and complexed with ponatinib, a promiscuous type-2 (DFG-out) kinase inhibitor, and an oncogenic FGFR4K harboring the V550L gate-keeper mutation bound to FIIN-2, a new type-1 irreversible inhibitor. Remarkably, like ponatinib, FIIN-2 also binds in the DFG-out mode despite lacking a functional group necessary to occupy the pocket vacated upon the DFG-out flip. Structural analysis reveals that the covalent bond between FIIN-2 and a cysteine, uniquely present in the glycine-rich loop of FGFR kinases, facilitates the DFG-out conformation, which together with the internal flexibility of FIIN-2 enables FIIN-2 to avoid the steric clash with the gate-keeper mutation that causes the ponatinib resistance. The structural data provide a blueprint for the development of next generation anticancer inhibitors through combining the salient inhibitory mechanisms of ponatinib and FIIN-2. PMID:25317566
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Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C
2016-05-01
The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents. Copyright © 2016 Elsevier Inc. All rights reserved.
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Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko
2014-01-01
It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095
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Hunt, J T; Floyd, D M; Lee, V G; Little, D K; Moreland, S
1989-01-01
Although the amino acid residues that are important for peptide substrates of myosin light-chain kinase have been reported, those that are important for peptide inhibitors of this enzyme have not previously been investigated. Synthetic peptides based on the sequence Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -Asn-Val21-Phe22-Ala of the chicken gizzard myosin light chain were tested as inhibitors of pig carotid-artery myosin light-chain kinase. The basic amino acid residues of the known myosin light-chain kinase inhibitor Lys-Lys-Arg-Ala-Ala-Arg-Ala-Thr-Ser-NH2 (IC50 = 14 microM) [Pearson, Misconi & Kemp (1986) J. Biol. Chem. 261, 25-27] were shown to be the important residues that contribute to inhibitor potency, as evidence by the finding that the hexapeptide Lys-Lys-Arg-Ala-Ala-Arg-NH2 had an IC50 value of 22 microM. This indicates that binding of the phosphorylatable serine residue to myosin light-chain kinase, which is of obvious importance for a substrate, does not enhance the potency of an inhibitor. With the aim of preparing more potent inhibitors, peptides Lys-Lys-Arg-Ala-Ala-Arg-Ala-Ala-Xaa-NH2 were prepared with a variety of amino acids substituted for the phosphorylatable serine residue. None of these peptides was a more potent inhibitor than the serine peptide. PMID:2920029
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Liu, Qingsong; Kirubakaran, Sivapriya; Hur, Wooyoung; Niepel, Mario; Westover, Kenneth; Thoreen, Carson C; Wang, Jinhua; Ni, Jing; Patricelli, Matthew P; Vogel, Kurt; Riddle, Steve; Waller, David L; Traynor, Ryan; Sanda, Takaomi; Zhao, Zheng; Kang, Seong A; Zhao, Jean; Look, A Thomas; Sorger, Peter K; Sabatini, David M; Gray, Nathanael S
2012-03-23
An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.
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Genetic Testing and Parkinson Disease: Assessment of Patient Knowledge, Attitudes, and Interest
Wood, Elisabeth McCarty; Xie, Sharon X.; Siderowf, Andrew; Van Deerlin, Vivianna M.
2012-01-01
The most common genetic contributor to late-onset Parkinson disease (PD) is the LRRK2 gene. In order to effectively integrate LRRK2 genetic testing into clinical practice, a strategy tailored to the PD population must be developed. We assessed 168 individuals with PD for baseline knowledge of genetics, perceived risk, and interest and opinions regarding genetic counseling and testing. Most participants felt that they were familiar with general genetics terms but overall knowledge levels were low, with an average score of 55%. The majority of participants thought it was likely they inherited a PD gene (72%), believed genetic testing for PD would be useful (86%), and were interested in genetic testing (59%) and genetic counseling (56%). However, only a few participants had heard of any genetic tests for PD (29%) or LRRK2 (10%). There appears to be a significant level of interest in genetics and genetic testing within the PD population, but a considerable deficit in genetics knowledge and an over-estimation of risk. Genetic education and counseling tools to address these needs were developed to provide patients with the ability to make informed and knowledgeable genetic testing decisions. PMID:21476119
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Sun, Jian; Li, Jing-Ran; Fang, Fei; Du, Qian-Ru; Qian, Yong; Gong, Hai-Bin; Zhu, Hai-Liang
2013-01-01
4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC50 values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor. PMID:23936329
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Effect of TGFβ on calcium signaling in megakaryocytes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yan, Jing; Schmid, Evi; Department of Pediatric Surgery and Pediatric Urology, University Children's Hospital Tübingen, Tübingen
2015-05-22
TGFβ is a powerful regulator of megakaryocyte maturation and platelet formation. As previously shown for other cell types, TGFβ may up-regulate the expression of the serum & glucocorticoid inducible kinase SGK1, an effect requiring p38 kinase. SGK1 has in turn recently been shown to participate in the regulation of cytosolic Ca{sup 2+} activity ([Ca{sup 2+}]{sub i}) in megakaryocytes and platelets. SGK1 phosphorylates the IκB kinase (IKKα/β), which in turn phosphorylates the inhibitor protein IκBα resulting in nuclear translocation of nuclear factor NFκB. Genes up-regulated by NFκB include Orai1, the pore forming ion channel subunit accomplishing store operated Ca{sup 2+} entrymore » (SOCE). The present study explored whether TGFβ influences Ca{sup 2+} signaling in megakaryocytes. [Ca{sup 2+}]{sub i} was determined by Fura-2 fluorescence and SOCE from the increase of [Ca{sup 2+}]{sub i} following re-addition of extracellular Ca{sup 2+} after store depletion by removal of extracellular Ca{sup 2+} and inhibition of the sarcoendoplasmatic Ca{sup 2+} ATPase (SERCA) with thapsigargin (1 μM). As a result, TGFβ (60 ng, 24 h) increased SOCE, an effect significantly blunted by p38 kinase inhibitor Skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) and NFκB inhibitor wogonin (100 μM). In conclusion, TGFβ is a powerful regulator of store operated Ca{sup 2+} entry into megakaryocytes, an effect mediated by a signaling cascade involving p38 kinase, SGK1 and NFκB. - Highlights: • TGFβ up-regulates store operated Ca{sup 2+} entry (SOCE) in megakaryocytes. • The effect of TGFβ on SOCE is blunted by p38 kinase inhibitor Skepinone-L. • The effect of TGFβ on SOCE is virtually abrogated by SGK1 inhibitor EMD638683. • The effect of TGFβ on SOCE is almost abolished by NFκB inhibitor wogonin. • The effect of TGFβ is expected to enhance sensitivity of platelets to activation.« less
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LRRK2 Mediated Changes in TAU Phosphorylation
2012-10-01
DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE 01 October 2012 2. REPORT TYPE Final 3. DATES COVERED 4 April 2011 – 3 September...2012 4 . TITLE AND SUBTITLE 5a. CONTRACT NUMBER LRRK2 Mediated Changes in TAU Phosphorylation 5b. GRANT NUMBER W81XWH-11-1-0338 5c. PROGRAM...Page Introduction…………………………………………………………….………..….. 4 Body………………………………………………………………………………….. 4 Key Research
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Lu, Dong; Shen, Aijun; Liu, Yang; Peng, Xia; Xing, Weiqiang; Ai, Jing; Geng, Meiyu; Hu, Youhong
2016-06-10
Analysis of the results of studies of docking 1 and 7a with c-Met kinase led to the identification of benzo[d]oxazol-2(3H)-one-quinolone derivatives as potential inhibitors of this enzyme. A molecular hybrid strategy, using a 4-ethoxy-7-substituted-quinoline core and a benzo[d]oxazol-2(3H)-one scaffold, was employed to design members of this family for study as inhibitors of the kinase and proliferation of EBC-1 cells. Most of the substances were found to display good to excellent c-Met kinase inhibitory activities. The results of a structure-activity relationship (SAR) study led to the discovery of benzo[d]oxazol-2(3H)-one-quinolone 13, which has IC50 values of 1 nM against c-Met kinase and 5 nM against proliferation of the EBC-1 cell line. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
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HALOACETIC ACIDS AND KINASE INHIBITORS PERTURB MOUSE NEURAL CREST CELLS IN VITRO
HUNTER, E.S.1, J. SMITH2, J. ANDREWS1. 1 Reproductive Toxicology Division, NHEERL, US EPA, Research Triangle Park and 2 Department of Cell and Developmental Biology, UNC-CH, Chapel Hill, North Carolina. Haloacetic acids and kinase inhibitors perturb mouse neural crest cells in vi...
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JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors
Gao, Sizhi P.; Chang, Qing; Mao, Ninghui; Daly, Laura A.; Vogel, Robert; Chan, Tyler; Liu, Shu Hui; Bournazou, Eirini; Schori, Erez; Zhang, Haiying; Brewer, Monica Red; Pao, William; Morris, Luc; Ladanyi, Marc; Arcila, Maria; Manova-Todorova, Katia; de Stanchina, Elisa; Norton, Larry; Levine, Ross L.; Altan-Bonnet, Gregoire; Solit, David; Zinda, Michael; Huszar, Dennis; Lyden, David; Bromberg, Jacqueline F.
2016-01-01
Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non–small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells’ dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC. PMID:27025877
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Lusková, Petra; Dráber, Petr
2004-01-01
Mast cells and basophils are major effector cells in the immunoglobulin E (IgE)-dependent allergic reactions as well as in the innate immunity. They are distributed throughout the body and, upon allergen exposure, are stimulated via the high affinity IgE receptor (FcepsilonRI) to release several pro-inflammatory mediators such as leukotrienes, immunoregulatory cytokines and histamine. FcepsilonRI-mediated signaling is initiated by tyrosine phosphorylation of FcepsilonRI subunits by Src family kinase Lyn, which is followed by an activation of Syk/Zap family kinase Syk. The activated kinases then in turn phosphorylate and activate other enzymes [phospholipase Cgamma (PLCgamma) isoforms, phosphatidylinositol-3 kinase (PI3K) isoforms, protein kinase C (PKC) isoforms, Bruton's tyrosine kinase (Btk) and others], adaptors [linker for activation of T cells (LAT), Cbl, Grb2 and others] and GTP exchange factors/GTPases (Vav, Ras, Rho, and others), and subsequently induce the mobilization of stored and extracellular Ca(2+). These and other biochemical events lead within seconds and minutes to the secretory response and later to the production of chemokines. This review is focused on the use of tyrosine kinase inhibitors specific for Src family kinases (PP1/PP2, SU6656 and CT5269), Syk kinase (piceatannol, ER-27319 and BAY 61-3606) and Btk (terreic acid and LFM-A13) for a modulation of FcepsilonRI-mediated signaling in mast cells. Potential use of the inhibitors in the treatment of inflammatory and allergy diseases as well as future directions in the development of highly specific tyrosine kinases inhibitors of new generations and their use in an intended modulation of mast cell signaling are discussed.
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Gabryel, Bozena; Pudelko, Anna; Malecki, Andrzej
2004-06-28
The present study focused on the mechanism of cytoprotective effect of aniracetam on the primary rat astrocyte cultures exposed to simulated ischemia conditions in vitro. To study these mechanisms, the aniracetam-mediated modulation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/Akt kinase pathways was determined. Simulated in vitro ischemia caused death of approximately 35% of astrocytes via apoptosis and decreased cell viability about 50% at 8 h. Exposure to aniracetam at concentrations of 0.1-10 microM in these conditions significantly decreased the number of apoptotic cells. Moreover, the intensification of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolinum bromide (MTT) conversion and the decrease of lactate dehydrogenase (LDH) release after 1 and 10 microM aniracetam treatment were observed indicating a significant increase in cell viability. When cultured astrocytes were incubated during 8 h simulated ischemia with [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] (U0126), an extracellular regulated kinase 1 and 2 (Erk1/2) inhibitor or wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase)/Akt inhibitor, the cell apoptosis was accelerated. These effects of used kinase inhibitors (both U0126 and wortmannin) were antagonized by adding 1 and 10 microM aniracetam to the culture medium. In addition, aniracetam significantly stimulated of phospho-Erk1/2 kinase and phospho-Akt expression. Maximum levels of Erk1/2 and Akt activation were observed as a result of treatment with 10 microM aniracetam. U0126 and wortmannin markedly attenuated the effects of aniracetam on expression of activated kinases. Results of the present study indicate that both Erk1/2 and PI 3-K/Akt kinase pathways are vital for cytoprotective effect of aniracetam.
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Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase Inhibitors.
Canning, Peter; Ruan, Qui; Schwerd, Tobias; Hrdinka, Matous; Maki, Jenny L; Saleh, Danish; Suebsuwong, Chalada; Ray, Soumya; Brennan, Paul E; Cuny, Gregory D; Uhlig, Holm H; Gyrd-Hansen, Mads; Degterev, Alexei; Bullock, Alex N
2015-09-17
RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
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NASA Astrophysics Data System (ADS)
Poulsen, Anders; William, Anthony; Blanchard, Stéphanie; Lee, Angeline; Nagaraj, Harish; Wang, Haishan; Teo, Eeling; Tan, Evelyn; Goh, Kee Chuan; Dymock, Brian
2012-04-01
Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.
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Small molecule inhibitors reveal PTK6 kinase is not an oncogenic driver in breast cancers
Gajiwala, Ketan S.; Cronin, Ciarán N.; Nagata, Asako; Johnson, Eric; Kraus, Michelle; Tatlock, John; Kania, Robert; Foley, Timothy
2018-01-01
Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment. PMID:29879184
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Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru
2013-01-01
Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693
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Taguchi, Yoshimitsu; Kondo, Tadakazu; Watanabe, Mitsumasa; Miyaji, Michihiko; Umehara, Hisanori; Kozutsumi, Yasunori; Okazaki, Toshiro
2004-11-15
Interleukin 2 (IL-2) rescued human natural killer (NK) KHYG-1 cells from apoptosis along with a reduction of ceramide. Conversely, an increase of ceramide inhibited IL-2-rescued survival. IL-2 deprivation-induced activation of acid sphingomyelinase (SMase) and inhibition of glucosylceramide synthase (GCS) and sphingomyelin synthase (SMS) were normalized by IL-2 supplementation. A phosphatidyl inositol-3 (PI-3) kinase inhibitor, LY294002, inhibited IL-2-rescued survival, but a mitogen-activated protein kinase inhibitor, PD98059, and an inhibitor of Janus tyrosine kinase/signal transducer and activator of transcription pathway, AG490, did not. LY294002 inhibited IL-2-induced reduction of ceramide through activation of acid SMase and inhibition of GCS and SMS, suggesting the positive involvement of PI-3 kinase in ceramide reduction through enzymatic regulation. Indeed, a constitutively active PI-3 kinase enhanced growth rate and ceramide reduction through inhibition of acid SMase and activation of GCS and SMS. Further, LY294002 inhibited IL-2-induced changes of transcriptional level as well as mRNA and protein levels in acid SMase and GCS but did not affect the stability of the mRNAs. These results suggest that PI-3 kinase-dependent reduction of ceramide through regulation of acid SMase, GCS, and SMS plays a role in IL-2-rescued survival of NK cells.
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Meirson, Tomer; Samson, Abraham O; Gil-Henn, Hava
2017-01-01
The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK) was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. PMID:28572720
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Zhu, L; Yang, Y; Lu, X
2016-01-01
The Rho-associated kinases (ROCKs) have long been recognized as an attractive therapeutic target for various neurological diseases; selective inhibition of ROCK1 and ROCK2 isoforms would result in distinct biological effects on neurogenesis, neuroplasticity and neuroregeneration after brain surgery and traumatic brain injury. However, the discovery and design of isoform-selective inhibitors remain a great challenge due to the high conservation and similarity between the kinase domains of ROCK1 and ROCK2. Here, a structure-based quantitative structure-selectivity relationship (SB-QSSR) approach was used to correlate experimentally measured selectivity with the difference in inhibitor binding to the two kinase isoforms. The resulting regression models were examined rigorously through both internal cross-validation and external blind validation; a nonlinear predictor was found to have high fitting stability and strong generalization ability, which was then employed to perform virtual screening against a structurally diverse, drug-like compound library. Consequently, five and seven hits were identified as promising candidates of 1-o-2 and 2-o-1 selective inhibitors, respectively, from which seven purchasable compounds were tested in vitro using a standard kinase assay protocol to determine their inhibitory activity against and selectivity between ROCK1 and ROCK2. The structural basis, energetic property and biological implication underlying inhibitor selectivity and promiscuity were also investigated systematically using a hybrid quantum mechanics/molecular mechanics (QM/MM) scheme.
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Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases
Patterson, H; Nibbs, R; McInnes, I; Siebert, S
2014-01-01
Protein kinases mediate protein phosphorylation, which is a fundamental component of cell signalling, with crucial roles in most signal transduction cascades: from controlling cell growth and proliferation to the initiation and regulation of immunological responses. Aberrant kinase activity is implicated in an increasing number of diseases, with more than 400 human diseases now linked either directly or indirectly to protein kinases. Protein kinases are therefore regarded as highly important drug targets, and are the subject of intensive research activity. The success of small molecule kinase inhibitors in the treatment of cancer, coupled with a greater understanding of inflammatory signalling cascades, has led to kinase inhibitors taking centre stage in the pursuit for new anti-inflammatory agents for the treatment of immune-mediated diseases. Herein we discuss the main classes of kinase inhibitors; namely Janus kinase (JAK), mitogen-activated protein kinase (MAPK) and spleen tyrosine kinase (Syk) inhibitors. We provide a mechanistic insight into how these inhibitors interfere with kinase signalling pathways and discuss the clinical successes and failures in the implementation of kinase-directed therapeutics in the context of inflammatory and autoimmune disorders. PMID:24313320
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Dombroski, Beth A; Galasko, Douglas R; Mata, Ignacio F; Zabetian, Cyrus P; Craig, Ulla-Katrina; Garruto, Ralph M; Oyanagi, Kiyomitsu; Schellenberg, Gerard D
2013-06-01
High-prevalence foci of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) exist in Japanese on the Kii Peninsula of Japan and in the Chamorros of Guam. Clinical and neuropathologic similarities suggest that the disease in these 2 populations may be related. Recent findings showed that some of the Kii Peninsula ALS cases had pathogenic C9orf72 repeat expansions, a genotype that causes ALS in Western populations. To perform genotyping among Guam residents to determine if the C9orf72 expanded repeat allele contributes to ALS-PDC in this population and to evaluate LRRK2 for mutations in the same population. Case-control series from neurodegenerative disease research programs on Guam that screened residents for ALS, PDC, and dementia. Study participants included 24 with ALS and 22 with PDC and 43 older control subjects with normal cognition ascertained between 1956 and 2006. All but one participant were Chamorro, the indigenous people of Guam. A single individual of white race/ethnicity with ALS was ascertained on Guam during the study. Participants were screened for C9orf72 hexanucleotide repeat length. Participants with repeat numbers in great excess of 30 were considered to have pathogenic repeat expansions. LRRK2 was screened for point mutations by DNA sequencing. We found a single individual with an expanded pathogenic hexanucleotide repeat. This individual of white race/ethnicity with ALS was living on Guam at the time of ascertainment but had been born in the United States. All Chamorro participants with ALS and PDC and control subjects had normal repeats, ranging from 2 to 17 copies. No pathogenic LRRK2 mutations were found. Unlike participants with ALS from the Kii Peninsula, C9orf72 expansions do not cause ALS-PDC in Chamorros. Likewise, LRRK2 mutations do not cause Guam ALS-PDC.
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Fox, T.; Coll, J. T.; Xie, X.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.; Fleming, M. A.; Galullo, V.; Su, M. S.; Wilson, K. P.
1998-01-01
Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation. PMID:9827991
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Koo, Junghui; Yue, Ping; Gal, Anthony A.; Khuri, Fadlo R.; Sun, Shi-Yong
2014-01-01
mTOR kinase inhibitors which target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacological inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. PMID:24626091
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Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong
2014-05-01
mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.
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Molecular structures of cdc2-like kinases in complex with a new inhibitor chemotype
Helmer, Renate; Loaëc, Nadège; Preu, Lutz; Ott, Ingo; Knapp, Stefan; Meijer, Laurent
2018-01-01
Cdc2-like kinases (CLKs) represent a family of serine-threonine kinases involved in the regulation of splicing by phosphorylation of SR-proteins and other splicing factors. Although compounds acting against CLKs have been described, only a few show selectivity against dual-specificity tyrosine phosphorylation regulated-kinases (DYRKs). We here report a novel CLK inhibitor family based on a 6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one core scaffold. Within the series, 3-(3-chlorophenyl)-6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one (KuWal151) was identified as inhibitor of CLK1, CLK2 and CLK4 with a high selectivity margin towards DYRK kinases. The compound displayed a potent antiproliferative activity in an array of cultured cancer cell lines. The X-ray structure analyses of three members of the new compound class co-crystallized with CLK proteins corroborated a molecular binding mode predicted by docking studies. PMID:29723265
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Approved and Experimental Small-Molecule Oncology Kinase Inhibitor Drugs: A Mid-2016 Overview.
Fischer, Peter M
2017-03-01
Kinase inhibitor research is a comparatively recent branch of medicinal chemistry and pharmacology and the first small-molecule kinase inhibitor, imatinib, was approved for clinical use only 15 years ago. Since then, 33 more kinase inhibitor drugs have received regulatory approval for the treatment of a variety of cancers and the volume of reports on the discovery and development of kinase inhibitors has increased to an extent where it is now difficult-even for those working in the field-easily to keep an overview of the compounds that are being developed, as currently there are 231 such compounds, targeting 38 different protein and lipid kinases (not counting isoforms), in clinical use or under clinical investigation. The purpose of this review is thus to provide an overview of the biomedical rationales for the kinases being targeted on the one hand, and the design principles, as well as chemical, pharmacological, pharmaceutical, and toxicological kinase inhibitor properties, on the other hand. Two issues that are especially important in kinase inhibitor research, target selectivity and drug resistance, as well as the underlying structural concepts, are discussed in general terms and in the context of relevant kinases and their inhibitors. © 2016 Wiley Periodicals, Inc.
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Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge
2004-08-01
In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.
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Guarnieri, Michael T.; Blagg, Brian S. J.
2011-01-01
Abstract Bacterial histidine kinases (HK) are members of the GHKL superfamily, which share a unique adenosine triphosphate (ATP)-binding Bergerat fold. Our previous studies have shown that Gyrase, Hsp90, MutL (GHL) inhibitors bind to the ATP-binding pocket of HK and may provide lead compounds for the design of novel antibiotics targeting these kinases. In this article, we developed a competition assay using the fluorescent ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate. The method can be used for high-throughput screening of compound libraries targeting HKs or other ATP-binding proteins. We utilized the assay to screen a library of GHL inhibitors targeting the bacterial HK PhoQ, and discuss the applications of the 2′,3′-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate competition assay beyond GHKL inhibitor screening. PMID:21050069
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Macroporous hydrogel micropillars for quantifying Met kinase activity in cancer cell lysates.
Powers, Alicia D; Liu, Bi; Lee, Andrew G; Palecek, Sean P
2012-09-07
Overactive and overexpressed kinases have been implicated in the cause and progression of many cancers. Kinase inhibitors offer a targeted approach for treating cancers associated with increased or deregulated kinase activity. Often, however, cancer cells exhibit initial resistance to these inhibitors or evolve to develop resistance during treatment. Additionally, cancers of any one tissue type are typically heterogeneous in their oncogenesis mechanisms, and thus diagnosis of a particular type of cancer does not necessarily provide insight into what kinase therapies may be effective. For example, while some lung cancer cells that overexpress the epidermal growth factor receptor (EFGR) respond to treatment with EGFR kinase inhibitors, overexpression or hyperactivity of Met kinase correlates with resistance to EGFR kinase inhibitors. Here we describe a microfluidic-based assay for quantifying Met kinase activity in cancer cell lysates with the eventual goals of predicting cancer cell responsiveness to kinase inhibitors and monitoring development of resistance to these inhibitors. In this assay, we immobilized a phosphorylation substrate for Met kinase into macroporous hydrogel micropillars. We then exposed the micropillars to a cancer cell lysate and detected substrate phosphorylation using a fluorescently conjugated antibody. This assay is able to quantify Met kinase activity in whole cell lysate from as few as 150 cancer cells. It can also detect cells expressing overactive Met kinase in a background of up to 75% non-cancerous cells. Additionally, the assay can quantify kinase inhibition by the Met-specific kinase inhibitors SU11274 and PHA665752, suggesting predictive capability for cellular response to kinase inhibitors.
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NASA Technical Reports Server (NTRS)
Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.
1999-01-01
The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.
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Anti-hyperglycemic action of apigenin-6-C-β-fucopyranoside from Averrhoa carambola.
Cazarolli, Luisa Helena; Kappel, Virgínia Demarchi; Pereira, Danielle Fontana; Moresco, Henrique Hunger; Brighente, Inês Maria Costa; Pizzolatti, Moacir Geraldo; Silva, Fátima Regina Mena Barreto
2012-10-01
A stimulatory effect of apigenin-6-C-β-fucopyranoside (1) on glucose uptake was observed when rat soleus muscle was incubated with 1, 10 and 100 μM of this flavonoid glycoside. The presence of specific insulin signaling inhibitors, such as wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C (PKC), PD98059, an inhibitor of mitogen-activated protein kinase (MEK), and HNMPA(AM)₃, an insulin receptor tyrosine kinase activity inhibitor showed that apigenin-6-C-β-fucopyranoside triggers different metabolic pathways in skeletal muscle. The oral administration of crude extract, fractions and isolated flavonoids (apigenin-6-C-β-fucopyranoside (1) and apigenin-6-C-(2″-O-α-rhamnopyranosyl)-β-fucopyranoside (2)) from Averrhoa carambola leaves exhibited a potential hypoglycemic activity in hyperglycemic normal rats. Additionally, both flavonoids significantly increased the muscle and liver glycogen content after an acute treatment. The results indicate that A. carambola can be regarded as a potent antihyperglycemic agent with insulin secretagogue and insulin mimetic properties. Copyright © 2012 Elsevier B.V. All rights reserved.
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Liu, Ling-Ling; Long, Zi-Jie; Wang, Le-Xun; Zheng, Fei-Meng; Fang, Zhi-Gang; Yan, Min; Xu, Dong-Fan; Chen, Jia-Jie; Wang, Shao-Wu; Lin, Dong-Jun; Liu, Quentin
2013-11-01
Aurora kinases are overexpressed in large numbers of tumors and considered as potential therapeutic targets. In this study, we found that the Aurora kinases inhibitors MK-0457 (MK) and ZM447439 (ZM) induced polyploidization in acute myeloid leukemia (AML) cell lines. The level of glycolytic metabolism was significantly increased in the polyploidy cells, which were sensitive to glycolysis inhibitor 2-deoxy-D-glucose (2DG), suggesting that polyploidy cells might be eliminated by metabolism deprivation. Indeed, inhibition of mTOR pathway by mTOR inhibitors (rapamycin and PP242) or 2DG promoted not only apoptosis but also autophagy in the polyploidy cells induced by Aurora inhibitors. Mechanically, PP242 or2DGdecreased the level of glucose uptake and lactate production in polyploidy cells as well as the expression of p62/SQSTM1. Moreover, knockdown of p62/SQSTM1 sensitized cells to the Aurora inhibitor whereas overexpression of p62/SQSTM1 reduced drug efficacy. Thus, our results revealed that inhibition of mTOR pathway decreased the glycolytic metabolism of the polyploidy cells, and increased the efficacy of Aurora kinases inhibitors, providing a novel approach of combination treatment in AML. ©2013 AACR.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tamm, Christoffer, E-mail: christoffer.tamm@imbim.uu.se; Galito, Sara Pijuan, E-mail: sara.pijuan@imbim.uu.se; Anneren, Cecilia, E-mail: cecilia.anneren@imbim.uu.se
2012-02-15
The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt inmore » proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.« less
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Harling, John D.; Deakin, Angela M.; Campos, Sébastien; Grimley, Rachel; Chaudry, Laiq; Nye, Catherine; Polyakova, Oxana; Bessant, Christina M.; Barton, Nick; Somers, Don; Barrett, John; Graves, Rebecca H.; Hanns, Laura; Kerr, William J.; Solari, Roberto
2013-01-01
IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both TH1 and TH2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models. PMID:23935099
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p21-activated kinase inhibitors.
Rudolph, Joachim; Crawford, James J; Hoeflich, Klaus P; Chernoff, Jonathan
2013-01-01
The p21-activated kinases (PAKs) are Ser/Thr kinases in the STE20 kinase family with important roles in regulating cytoskeletal organization, cell migration, and signaling. The PAK enzyme family comprises six members subdivided into two groups: Group I, represented by PAK1, 2, and 3, and Group II, represented by PAK 4, 5, and 6, based on sequence and structural homology. Individual PAK isoforms were found to be overexpressed and amplified in a variety of human cancers, and in vitro and in vivo studies using genetically engineered systems as well as small-molecule tool compounds have suggested therapeutic utility of PAKs as oncology targets. The identification of potent and kinome-selective ATP-competitive PAK inhibitors has proven challenging, likely caused by the openness and unique plasticity of the ATP-binding site of PAK enzymes. Progress in achieving increased kinase selectivity has been achieved with certain inhibitors but at the expense of increased molecular weight. Allosteric inhibitors, such as IPA-3, leverage the unique Group I PAK autoregulatory domain for selective inhibition, and this approach might provide an outlet to evade the kinase selectivity challenges observed with ATP-competitive PAK inhibitors. © 2013 Elsevier Inc. All rights reserved.
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Tran, Hai B; Barnawi, Jameel; Ween, Miranda; Hamon, Rhys; Roscioli, Eugene; Hodge, Greg; Reynolds, Paul N; Pitson, Stuart M; Davies, Lorena T; Haberberger, Rainer; Hodge, Sandra
2016-07-01
Alveolar macrophages from chronic obstructive pulmonary disease patients and cigarette smokers are deficient in their ability to phagocytose apoptotic bronchial epithelial cells (efferocytosis). We hypothesized that the defect is mediated via inhibition of sphingosine kinases and/or their subcellular mislocalization in response to cigarette smoke and can be normalized with exogenous sphingosine-1-phosphate or FTY720 (fingolimod), a modulator of sphingosine-1-phosphate signaling, which has been shown to be clinically useful in multiple sclerosis. Measurement of sphingosine kinase 1/2 activities by [(32)P]-labeled sphingosine-1-phosphate revealed a 30% reduction of sphingosine kinase 1 (P < 0.05) and a nonsignificant decrease of sphingosine kinase 2 in THP-1 macrophages after 1 h cigarette smoke extract exposure. By confocal analysis macrophage sphingosine kinase 1 protein was normally localized to the plasma membrane and cytoplasm and sphingosine kinase 2 to the nucleus and cytoplasm but absent at the cell surface. Cigarette smoke extract exposure (24 h) led to a retraction of sphingosine kinase 1 from the plasma membrane and sphingosine kinase 1/2 clumping in the Golgi domain. Selective inhibition of sphingosine kinase 2 with 25 µM ABC294640 led to 36% inhibition of efferocytosis (P < 0.05); 10 µM sphingosine kinase inhibitor/5C (sphingosine kinase 1-selective inhibitor) induced a nonsignificant inhibition of efferocytosis, but its combination with ABC294640 led to 56% inhibition (P < 0.01 vs. control and < 0.05 vs. single inhibitors). Cigarette smoke-inhibited efferocytosis was significantly (P < 0.05) reversed to near-control levels in the presence of 10-100 nM exogenous sphingosine-1-phosphate or FTY720, and FTY720 reduced cigarette smoke-induced clumping of sphingosine kinase 1/2 in the Golgi domain. These data strongly support a role of sphingosine kinase 1/2 in efferocytosis and as novel therapeutic targets in chronic obstructive pulmonary disease. © Society for Leukocyte Biology.
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Malami, Ibrahim; Abdul, Ahmad Bustamam; Abdullah, Rasedee; Bt Kassim, Nur Kartinee; Waziri, Peter; Christopher Etti, Imaobong
2016-04-08
Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.
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Sadras, Teresa; Heatley, Susan L; Kok, Chung H; McClure, Barbara J; Yeung, David; Hughes, Timothy P; Sutton, Rosemary; Ziegler, David S; White, Deborah L
2017-10-01
We report a novel somatic mutation in the kinase domain of JAK2 (R938Q) in a high-risk pediatric case of B-cell acute lymphoblastic leukemia (ALL). The patient developed on-therapy relapse at 12 months, and interestingly, the JAK2 locus acquired loss of heterozygosity during treatment resulting in 100% mutation load. Furthermore, we show that primary ALL mononuclear cells harboring the JAK2 R938Q mutation display reduced sensitivity to the JAK1/2 ATP-competitive inhibitor ruxolitinib in vitro, compared to ALL cells that carry a more common JAK2 pseudokinase domain mutation. Our findings are in line with previous reports that demonstrate that mutations within the kinase domain of JAK2 are associated with resistance to type I JAK inhibitors. Importantly, given the recent inclusion of ruxolitinib in trial protocols for children with JAK pathway alterations, we predict that inter-patient genetic variability may result in suboptimal responses to JAK inhibitor therapy in a subset of cases. The need for alternate targeted and/or combination therapies for patients who display inherent or developed resistance to JAK inhibitor therapy will be warranted, and we propose that kinase-mutants less sensitive to type I JAK inhibitors may present a currently unexplored platform for investigation of improved therapies. Copyright © 2017. Published by Elsevier Inc.
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Hsu, Chia-Jen; Hsu, Wen-Chi; Lee, Der-Jay; Liu, An-Lun; Chang, Chia-Ming; Shih, Huei-Jhen; Huang, Wun-Han; Lee-Chen, Guey-Jen; Hsieh-Li, Hsiu Mei; Lee, Guan-Chiun; Sun, Ying-Chieh
2017-08-01
GSK3β kinase is a noteworthy target for discovery of the drugs that will be used to treat several diseases. In the effort to identify a new inhibitor lead compound, we utilized thermodynamic integration (TI)-molecular dynamics (MD) simulation and kinase assay to investigate the bindings between GSK3β kinase and five compounds that were analogous to a known inhibitor with an available crystal structure. TI-MD simulations of the first two compounds (analogs 1 and 2) were used for calibration. The computed binding affinities of analogs 1 and 2 agreed well with the experimental results. The rest three compounds (analogs 3-5) were newly obtained from a database search, and their affinity data were newly measured in our labs. TI-MD simulations predicted the binding modes and the computed ΔΔG values have a reasonably good correlation with the experimental affinity data. These newly identified inhibitors appear to be new leads according to our survey of GSK3β inhibitors listed in recent review articles. The predicted binding modes of these compounds should aid in designing new derivatives of these compounds in the future. © 2017 John Wiley & Sons A/S.
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Bettayeb, Karima; Tirado, Oscar M; Marionneau-Lambot, Séverine; Ferandin, Yoan; Lozach, Olivier; Morris, Jonathan C; Mateo-Lozano, Silvia; Drueckes, Peter; Schächtele, Christoph; Kubbutat, Michael H G; Liger, François; Marquet, Bernard; Joseph, Benoît; Echalier, Aude; Endicott, Jane A; Notario, Vicente; Meijer, Laurent
2007-09-01
Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.
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Yousufzai, S Y; Abdel-Latif, A A
1998-11-06
We investigated the effects of the protein tyrosine kinase inhibitors, genistein, tyrphostin 47, and herbimycin on prostaglandin F2alpha- and carbachol-induced inositol-1,4,5-trisphosphate (IP3) production, [Ca2+]i mobilization and contraction in cat iris sphincter smooth muscle. Prostaglandin F2alpha and carbachol induced contraction in a concentration-dependent manner with EC50 values of 0.92 x 10(-9) and 1.75 x 10(-8) M, respectively. The protein tyrosine kinase inhibitors blocked the stimulatory effects of prostaglandin F2alpha, but not those evoked by carbachol, on IP3 accumulation, [Ca2+]i mobilization and contraction, suggesting involvement of protein tyrosine kinase activity in the physiological actions of the prostaglandin. Daidzein and tyrphostin A, inactive negative control compounds for genistein and tyrphostin 47, respectively, were without effect. Latanoprost, a prostaglandin F2alpha analog used as an antiglaucoma drug, induced contraction and this effect was blocked by genistein. Genistein (10 microM) markedly reduced (by 67%) prostaglandin F2alpha-stimulated increase in [Ca2+]i but had little effect on that of carbachol in cat iris sphincter smooth muscle cells. Vanadate, a potent inhibitor of protein tyrosine phosphatase, induced a slow gradual muscle contraction in a concentration-dependent manner with an EC50 of 82 microM and increased IP3 generation in a concentration-dependent manner with an EC50 of 90 microM. The effects of vanadate were abolished by genistein (10 microM). Wortmannin, a myosin light chain kinase inhibitor, reduced prostaglandin F2alpha- and carbachol-induced contraction, suggesting that the involvement of protein tyrosine kinase activity may lie upstream of the increases in [Ca2+]i evoked by prostaglandin F2alpha. Further studies aimed at elucidating the role of protein tyrosine kinase activity in the coupling mechanism between prostaglandin F2alpha receptor activation and increases in intracellular Ca2+ mobilization and identifying the tyrosine-phosphorylated substrates will provide important information about the role of protein tyrosine kinase in the mechanism of smooth muscle contraction, as well as about the mechanism of the intraocular pressure lowering effect of the prostaglandin in glaucoma patients.
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Chang, Alice Y W
2012-11-17
Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun.
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PREDICT-PD: An online approach to prospectively identify risk indicators of Parkinson's disease.
Noyce, Alastair J; R'Bibo, Lea; Peress, Luisa; Bestwick, Jonathan P; Adams-Carr, Kerala L; Mencacci, Niccolo E; Hawkes, Christopher H; Masters, Joseph M; Wood, Nicholas; Hardy, John; Giovannoni, Gavin; Lees, Andrew J; Schrag, Anette
2017-02-01
A number of early features can precede the diagnosis of Parkinson's disease (PD). To test an online, evidence-based algorithm to identify risk indicators of PD in the UK population. Participants aged 60 to 80 years without PD completed an online survey and keyboard-tapping task annually over 3 years, and underwent smell tests and genotyping for glucocerebrosidase (GBA) and leucine-rich repeat kinase 2 (LRRK2) mutations. Risk scores were calculated based on the results of a systematic review of risk factors and early features of PD, and individuals were grouped into higher (above 15th centile), medium, and lower risk groups (below 85th centile). Previously defined indicators of increased risk of PD ("intermediate markers"), including smell loss, rapid eye movement-sleep behavior disorder, and finger-tapping speed, and incident PD were used as outcomes. The correlation of risk scores with intermediate markers and movement of individuals between risk groups was assessed each year and prospectively. Exploratory Cox regression analyses with incident PD as the dependent variable were performed. A total of 1323 participants were recruited at baseline and >79% completed assessments each year. Annual risk scores were correlated with intermediate markers of PD each year and baseline scores were correlated with intermediate markers during follow-up (all P values < 0.001). Incident PD diagnoses during follow-up were significantly associated with baseline risk score (hazard ratio = 4.39, P = .045). GBA variants or G2019S LRRK2 mutations were found in 47 participants, and the predictive power for incident PD was improved by the addition of genetic variants to risk scores. The online PREDICT-PD algorithm is a unique and simple method to identify indicators of PD risk. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. © 2016 International Parkinson and Movement Disorder Society.
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Dynamic regulation of a metabolic multi-enzyme complex by protein kinase CK2.
An, Songon; Kyoung, Minjoung; Allen, Jasmina J; Shokat, Kevan M; Benkovic, Stephen J
2010-04-09
The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, tetrabromocinammic acid, 4,4',5,5',6,6'-hexahydroxydiphenic acid 2,2',6,6'-dilactone (ellagic acid) as well as by silencing the endogenous human CK2alpha catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole, another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which 4,5,6,7-tetrabromobenzotriazole affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly, and thus the purinosome may be one of the ultimate targets of kinase inhibitors.
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Targeting cancer with kinase inhibitors
Gross, Stefan; Rahal, Rami; Stransky, Nicolas; Lengauer, Christoph; Hoeflich, Klaus P.
2015-01-01
Kinase inhibitors have played an increasingly prominent role in the treatment of cancer and other diseases. Currently, more than 25 oncology drugs that target kinases have been approved, and numerous additional therapeutics are in various stages of clinical evaluation. In this Review, we provide an in-depth analysis of activation mechanisms for kinases in cancer, highlight recent successes in drug discovery, and demonstrate the clinical impact of selective kinase inhibitors. We also describe the substantial progress that has been made in designing next-generation inhibitors to circumvent on-target resistance mechanisms, as well as ongoing strategies for combining kinase inhibitors in the clinic. Last, there are numerous prospects for the discovery of novel kinase targets, and we explore cancer immunotherapy as a new and promising research area for studying kinase biology. PMID:25932675
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Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening.
Li, Siyuan; Sun, Xianqiang; Zhao, Hongli; Tang, Yun; Lan, Minbo
2012-06-15
By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Vaidhyanathan, Shruthi; Wilken-Resman, Brynna; Ma, Daniel J.; Parrish, Karen E.; Mittapalli, Rajendar K.; Carlson, Brett L.; Sarkaria, Jann N.
2016-01-01
Small molecule inhibitors targeting the mitogen-activated protein kinase pathway (Braf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) have had success in extending survival for patients with metastatic melanoma. Unfortunately, resistance may occur via cross-activation of alternate signaling pathways. One approach to overcome resistance is to simultaneously target the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway. Recent reports have shown that GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl) benzenesulfonamide], a dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor, can overcome acquired resistance to Braf and mitogen-activated protein kinase kinase inhibitors in vitro. These resistance mechanisms may be especially important in melanoma brain metastases because of limited drug delivery across the blood–brain barrier. The purpose of this study was to investigate factors that influence the brain distribution of GSK2126458 and to examine the efficacy of GSK2126458 in a novel patient-derived melanoma xenograft (PDX) model. Both in vitro and in vivo studies indicate that GSK2126458 is a substrate for P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), two dominant active efflux transporters in the blood–brain barrier. The steady-state brain distribution of GSK2126458 was 8-fold higher in the P-gp/Bcrp knockout mice compared with the wild type. We also observed that when simultaneously infused to steady state, GSK212658, dabrafenib, and trametinib, a rational combination to overcome mitogen-activated protein kinase inhibitor resistance, all had limited brain distribution. Coadministration of elacridar, a P-gp/Bcrp inhibitor, increased the brain distribution of GSK2126458 by approximately 7-fold in wild-type mice. In the PDX model, GSK2126458 showed efficacy in flank tumors but was ineffective in intracranial melanoma. These results show that P-gp and Bcrp are involved in limiting the brain distribution of GSK2126458 and provide a rationale for the lack of efficacy of GSK2126458 in the orthotopic PDX model. PMID:26604245
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Genetics in Parkinson disease: Mendelian versus non-Mendelian inheritance.
Hernandez, Dena G; Reed, Xylena; Singleton, Andrew B
2016-10-01
Parkinson's disease is a common, progressive neurodegenerative disorder, affecting 3% of those older than 75 years of age. Clinically, Parkinson's disease (PD) is associated with resting tremor, postural instability, rigidity, bradykinesia, and a good response to levodopa therapy. Over the last 15 years, numerous studies have confirmed that genetic factors contribute to the complex pathogenesis of PD. Highly penetrant mutations producing rare, monogenic forms of the disease have been discovered in singular genes such as SNCA, Parkin, DJ-1, PINK 1, LRRK2, and VPS35. Unique variants with incomplete penetrance in LRRK2 and GBA have been shown to be strong risk factors for PD in certain populations. Additionally, over 20 common variants with small effect sizes are now recognized to modulate the risk for PD. Investigating Mendelian forms of PD has provided precious insight into the pathophysiology that underlies the more common idiopathic form of disease; however, no treatment methodologies have developed. Furthermore, for identified common risk alleles, the functional basis underlying risk principally remains unknown. The challenge over the next decade will be to strengthen the findings delivered through genetic discovery by assessing the direct, biological consequences of risk variants in tandem with additional high-content, integrated datasets. This review discusses monogenic risk factors and mechanisms of Mendelian inheritance of Parkinson disease. Highly penetrant mutations in SNCA, Parkin, DJ-1, PINK 1, LRRK2 and VPS35 produce rare, monogenic forms of the disease, while unique variants within LRRK2 and GBA show incomplete penetrance and are strong risk factors for PD. Additionally, over 20 common variants with small effect sizes modulate disease risk. The challenge over the next decade is to strengthen genetic findings by assessing direct, biological consequences of risk variants in tandem with high-content, integrated datasets. This article is part of a special issue on Parkinson disease. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.
2015-02-13
Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can bemore » clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.« less
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Guanidinium-based derivatives: searching for new kinase inhibitors.
Diez-Cecilia, Elena; Kelly, Brendan; Perez, Concepcion; Zisterer, Daniela M; Nevin, Daniel K; Lloyd, David G; Rozas, Isabel
2014-06-23
Considering the structural similarities between the kinase inhibitor sorafenib and 4,4'-bis-guanidinium derivatives previously prepared by Rozas and co., which display interesting cytotoxicity in cancer cells, we have studied whether this activity could result from kinase inhibition. Five new families have been prepared consisting of unsubstituted and aryl-substituted 3,4'-bis-guanidiniums, 3,4'-bis-2-aminoimidazolinium and 3-acetamide-4'-(4-chloro-3-trifluoromethylphenyl)guanidinium derivatives. Cytotoxicity (measuring the IC50 values) and apoptosis studies in human HL-60 promyelocytic leukemia cells were carried out for these compounds. Additionally, their potential inhibitory effect was explored on a panel of kinases known to be involved in apoptotic pathways. The previously prepared cytotoxic 4,4'-bis-guanidiniums did not inhibit any of these kinases; however, some of the novel 3,4'-substituted derivatives showed a high percentage inhibition of RAF-1/MEK-1, for which the potential mode of binding was evaluated by docking studies. The interesting antitumour properties showed by these compounds open up new exciting lines of investigation for kinase inhibitors as anticancer agents and also highlights the relevance of the guanidinium moiety for protein kinase inhibitors chemical design. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
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Szczepankiewicz, Bruce G; Kosogof, Christi; Nelson, Lissa T J; Liu, Gang; Liu, Bo; Zhao, Hongyu; Serby, Michael D; Xin, Zhili; Liu, Mei; Gum, Rebecca J; Haasch, Deanna L; Wang, Sanyi; Clampit, Jill E; Johnson, Eric F; Lubben, Thomas H; Stashko, Michael A; Olejniczak, Edward T; Sun, Chaohong; Dorwin, Sarah A; Haskins, Kristi; Abad-Zapatero, Cele; Fry, Elizabeth H; Hutchins, Charles W; Sham, Hing L; Rondinone, Cristina M; Trevillyan, James M
2006-06-15
The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.
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Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.
Cannon, C M; Pozniak, J; Scott, M C; Ito, D; Gorden, B H; Graef, A J; Modiano, J F
2015-03-01
We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted. © 2013 Blackwell Publishing Ltd.
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Bailey, Tameka A.; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L.; Natarajan, Amarnath; Raja, Srikumar M.; Band, Vimla; Band, Hamid
2014-01-01
ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca2+-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling. PMID:25225290
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Tang, Bor Luen
2017-12-01
Sec16 is classically perceived to be a scaffolding protein localized to the transitional endoplasmic reticulum (tER) or the ER exit sites (ERES), and has a conserved function in facilitating coat protein II (COPII) complex-mediated ER exit. Recent findings have, however, pointed toward a role for Sec16 in unconventional exocytosis of certain membrane proteins, such as the Cystic fibrosis transmembrane conductance regulator (CFTR) in mammalian cells, and possibly also α-integrin in certain contexts of Drosophila development. In this regard, Sec16 interacts with components of a recently deciphered pathway of stress-induced unconventional exocytosis, which is dependent on the tether protein Golgi reassembly stacking proteins (GRASPs) and the autophagy pathway. Intriguingly, Sec16 also appears to be post-translationally modified by autophagy-related signaling processes. Sec16 is known to be phosphorylated by the atypical extracellular signal regulated kinase 7 (Erk7) upon serum and amino acid starvation, both represent conditions that trigger autophagy. Recent work has also shown that Sec16 is phosphorylated, and thus regulated by the prominent autophagy-initiating Unc-51-like autophagy activating kinase 1 (Ulk1), as well as another autophagy modulator Leucine-rich repeat kinase 2 (Lrrk2). The picture emerging from Sec16's network of physical and functional interactors allows the speculation that Sec16 is situated (and may in yet undefined ways function) at the interface between COPII-mediated exocytosis of conventional vesicular traffic and the GRASP/autophagy-dependent mode of unconventional exocytosis. © 2017 Wiley Periodicals, Inc.
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Anastasaki, Corina; Estep, Anne L; Marais, Richard; Rauen, Katherine A; Patton, E Elizabeth
2009-07-15
The Ras/MAPK pathway is critical for human development and plays a central role in the formation and progression of most cancers. Children born with germ-line mutations in BRAF, MEK1 or MEK2 develop cardio-facio-cutaneous (CFC) syndrome, an autosomal dominant syndrome characterized by a distinctive facial appearance, heart defects, skin and hair abnormalities and mental retardation. CFC syndrome mutations in BRAF promote both kinase-activating and kinase-impaired variants. CFC syndrome has a progressive phenotype, and the availability of clinically active inhibitors of the MAPK pathway prompts the important question as to whether such inhibitors might be therapeutically effective in the treatment of CFC syndrome. To study the developmental effects of CFC mutant alleles in vivo, we have expressed a panel of 28 BRAF and MEK alleles in zebrafish embryos to assess the function of human disease alleles and available chemical inhibitors of this pathway. We find that both kinase-activating and kinase-impaired CFC mutant alleles promote the equivalent developmental outcome when expressed during early development and that treatment of CFC-zebrafish embryos with inhibitors of the FGF-MAPK pathway can restore normal early development. Importantly, we find a developmental window in which treatment with a MEK inhibitor can restore the normal early development of the embryo, without the additional, unwanted developmental effects of the drug.
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The anthraquinone emodin inhibits the non-exported FIKK kinase from Plasmodium falciparum.
Lin, Benjamin C; Harris, Darcy R; Kirkman, Lucy M D; Perez, Astrid M; Qian, Yiwen; Schermerhorn, Janse T; Hong, Min Y; Winston, Dennis S; Xu, Lingyin; Lieber, Alexander M; Hamilton, Matthew; Brandt, Gabriel S
2017-12-01
The FIKK family of kinases is unique to parasites of the Apicomplexan order, which includes all malaria parasites. Plasmodium falciparum, the most virulent form of human malaria, has a family of 19 FIKK kinases, most of which are exported into the host red blood cell during malaria infection. Here, we confirm that FIKK 8 is a non-exported member of the FIKK kinase family. Through expression and purification of the recombinant kinase domain, we establish that emodin is a relatively high-affinity (IC 50 =2μM) inhibitor of PfFk8. Closely related anthraquinones do not inhibit PfFk8, suggesting that the particular substitution pattern of emodin is critical to the inhibitory pharmacophore. This first report of a P. falciparum FIKK kinase inhibitor lays the groundwork for developing specific inhibitors of the various members of the FIKK kinase family in order to probe their physiological function. Copyright © 2017 Elsevier Inc. All rights reserved.
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Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei
2011-06-01
Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
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ERK pathway inhibitors: how low should we go?
Nissan, Moriah H; Rosen, Neal; Solit, David B
2013-07-01
Resistance to RAF inhibitors is generally accompanied by reactivation of extracellular signal-regulated kinase (ERK) signaling. SCH772984, a selective, ATP-competitive inhibitor of ERK1 and ERK2, is effective in BRAF-mutant models in which resistance is the result of ERK reactivation. SCH772984 may also have a role in the treatment of tumors in which ERK is dysregulated by mutant RAS, NF1, or activated receptor tyrosine kinases, settings in which current RAF inhibitors are ineffective. ©2013 AACR.
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[The specific enzyme inhibitors for potential therapeutic use].
Bretner, Maria
2015-01-01
Therapy for hepatitis C virus (HCV) initially consisted on administering ribavirin - having a broad spectrum of action - and pegylated interferon, and was only effective in 40-50% of patients. Appropriate was to find effective inhibitors of viral replication e.g. by inhibition of a viral enzyme, NTPase/helicase required in the process of translation and RNA replication of the HCV. We developed methods of synthesis of many compounds belonging to different groups - derivatives of nucleosides, benzotriazole, benzimidazole, tropolone and epirubicine. Some of the derivatives inhibit HCV helicase activity at low concentrations and reduces replication of the viral RNA in subgenomic replicon system. In the process of HCV replication casein kinase CK2 plays an important role. It regulates the level of phosphorylation of HCV protein NS5A, which affects the production of infectious virions of HCV. Effective and selective inhibitors of kinase CK2 could be of use in the treatment of HCV in combination with other drugs. CK2 kinase phosphorylates approximately 300 proteins that affect the growth, differentiation, proliferation or apoptosis. Elevated CK2 kinase activity has been observed in several types of cancer and other diseases, therefore, inhibitors of this enzyme are potential therapeutic importance, particularly for anti-cancer treatment. Research carried out in collaboration with prof. Shugar led to the synthesis of one of the most selective inhibitors of this enzyme which is 4,5,6,7-tetrabromo-1H-benzotriazole, used for the study of the role of kinase CK2 in a number of metabolic processes in tumor cells.
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Ryter, Stefan W; Xi, Sichuan; Hartsfield, Cynthia L; Choi, Augustine M K
2002-08-01
Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.
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Tyner, Jeffrey W.; Bumm, Thomas G.; Deininger, Jutta; Wood, Lisa; Aichberger, Karl J.; Loriaux, Marc M.; Druker, Brian J.; Burns, Christopher J.; Fantino, Emmanuelle
2010-01-01
Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells. PMID:20385788
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NASA Astrophysics Data System (ADS)
Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis
1991-02-01
CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.
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Kurasawa, Osamu; Homma, Misaki; Oguro, Yuya; Miyazaki, Tohru; Mori, Kouji; Uchiyama, Noriko; Iwai, Kenichi; Ohashi, Akihiro; Hara, Hideto; Yoshida, Sei; Cho, Nobuo
2017-07-15
In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC 50 =0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Willemsen-Seegers, Nicole; Uitdehaag, Joost C M; Prinsen, Martine B W; de Vetter, Judith R F; de Man, Jos; Sawa, Masaaki; Kawase, Yusuke; Buijsman, Rogier C; Zaman, Guido J R
2017-02-17
Target residence time (τ) has been suggested to be a better predictor of the biological activity of kinase inhibitors than inhibitory potency (IC 50 ) in enzyme assays. Surface plasmon resonance binding assays for 46 human protein and lipid kinases were developed. The association and dissociation constants of 80 kinase inhibitor interactions were determined. τ and equilibrium affinity constants (K D ) were calculated to determine kinetic selectivity. Comparison of τ and K D or IC 50 values revealed a strikingly different view on the selectivity of several kinase inhibitors, including the multi-kinase inhibitor ponatinib, which was tested on 10 different kinases. In addition, known pan-Aurora inhibitors resided much longer on Aurora B than on Aurora A, despite having comparable affinity for Aurora A and B. Furthermore, the γ/δ-selective PI3K inhibitor duvelisib and the δ-selective drug idelalisib had similar 20-fold selectivity for δ- over γ-isoform but duvelisib resided much longer on both targets. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Bidirectional signaling between TM4SF5 and IGF1R promotes resistance to EGFR kinase inhibitors.
Choi, Jungeun; Kang, Minkyung; Nam, Seo Hee; Lee, Gyu-Ho; Kim, Hye-Jin; Ryu, Jihye; Cheong, Jin Gyu; Jung, Jae Woo; Kim, Tai Young; Lee, Ho-Young; Lee, Jung Weon
2015-10-01
The membrane glycoprotein TM4SF5 (transmembrane 4 L6 family member 5), which is similar to the tetraspanins, is highly expressed in different cancers and causes epithelial-mesenchymal transition (EMT). TM4SF5 interacts with other membrane proteins during its pro-tumorigenic roles, presumably at tetraspanin-enriched microdomains (TEMs/TERMs). Here, we explored TM4SF5-mediated resistance against the clinically important EGFR kinase inhibitors, with regards to cooperation with other membrane proteins, particularly the insulin-like growth factor 1 receptor (IGF1R). Using cancer cells including NSCLC with TM4SF5 overexpression or IGF1R suppression in either normal 2 dimensional (2D), 3D aqueous spheroids, or 3D collagen I gels systems, the sensitivity to tyrosine kinase inhibitors (TKIs) were evaluated. We found that TM4SF5 and IGF1R transcriptionally modulated one another, with each protein promoting the expressions of the other. Expression of TM4SF5 in gefitinib-sensitive HCC827 cells caused resistance to erlotinib and gefitinib, but not to sorafenib [a platelet derived growth factor receptor (PDGFR) inhibitor]; whereas suppression of IGF1R from gefitinib-resistant NCI-H1299 cells caused enhanced sensitization to the inhibitors. Expression of TM4SF5 and IGF1R in the drug-sensitive cells promoted signaling activities of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and S6 kinase (S6K), and resulted in a higher residual EGFR activity, even after EGFR kinase inhibitor treatment. Complex formation between TM4SF5 and IGF1R was observed, and also included EGFR, dependent on TM4SF5 expression. The TM4SF5-mediated drug resistance was further confirmed in an aqueous 3D spheroid system or upon being embedded in 3D extracellular matrix (ECM)-surrounded gel systems. Collectively, these data suggest that anti-TM4SF5 reagents may be combined with the EGFR kinase inhibitors to enhance the efficacy of chemotherapies against NSCLC. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
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Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase.
Qin, Qining; Downey, James M; Cohen, Michael V
2003-02-01
Adenosine and acetylcholine (ACh) trigger preconditioning by different signaling pathways. The involvement of phosphatidylinositol 3-kinase (PI3-kinase), a protein tyrosine kinase, and Src family tyrosine kinase in preconditioning was evaluated in isolated rabbit hearts. Either wortmannin (PI3-kinase blocker), genistein (tyrosine kinase blocker), lavendustin A (tyrosine kinase blocker), or 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4-d]pyrimidine (PP2; Src family tyrosine kinase blocker) was given for 15 min to bracket a 5-min infusion of either adenosine or ACh (trigger phase). The hearts then underwent 30 min of regional ischemia. Infarct size for ACh alone was 9.3 +/- 3.5% of the risk zone versus 34.3 +/- 4.1% in controls. All four inhibitors blocked ACh-induced protection. When wortmannin or PP2 was infused only during the 30-min ischemic period (mediator phase), ACh-induced protection was not affected (7.4 +/- 2.1% and 9.7 +/- 1.7% infarction, respectively). Adenosine-triggered protection was not blocked by any of the inhibitors. Therefore, PI3-kinase and at least one protein tyrosine kinase, probably Src kinase, are involved in the trigger phase of ACh-induced, but not adenosine-induced, preconditioning. Neither PI3-kinase nor Src kinase is a mediator of the protection of ACh.
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Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent
Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas
2016-01-01
JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457
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Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J
2004-03-01
Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.
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Martin, Ludovic; Magnaudeix, Amandine; Wilson, Cornelia M; Yardin, Catherine; Terro, Faraj
2011-11-01
Alterations in glycogen synthase kinase-3β (GSK3β) and protein phosphatase-2A (PP2A) have been proposed to be involved in the abnormal tau phosphorylation and aggregation linked to Alzheimer's disease (AD). Interconnections between GSK3β and PP2A signaling pathways are well established. Targeting tau kinases was proposed to represent a therapeutic strategy for AD. However, which tau kinases should be blocked and to what extent, keeping in mind that kinases have physiological roles? Because most kinase inhibitors are relatively specific and many of them interfere with the cell cycle, it is necessary to develop more specific tau kinase inhibitors devoid of cell toxicity. Here, we used the PP2A inhibition by okadaic acid (OKA) in primary cultured cortical neurons as an in vitro model of increased tau phosphorylation and apoptosis. We tested the effects of two newly characterized indirubin derivative inhibitors of GSK3, 6-BIDECO (6-bromoindirubin-3'-[O-(N,N-diethylcarbamyl)-oxime] and 6-BIMYEO (6-bromoindirubin-3'-[O-(2-morpholin-1-ylethyl)-oxime] hydrochloride) on OKA-induced tau phosphorylation and neuronal apoptosis. Both compounds exhibit higher selectivity toward GSK3 compared with other tau kinases (for 6-BIDECO, IC50 is 0.03 μM for GSK3, >10 μM for CDK1, and 10 μM for CDK5; for 6-BIMYEO, IC50 is 0.11 μM for GSK3, 1.8 μM for CDK1, and 0.9 μM for CDK5). We show that 6-BIDECO and 6-BIMYEO used at micromolar concentrations are not neurotoxic and potently reversed tau phosphorylation and apoptosis induced by OKA. The neuroprotection by these compounds should be further validated in animal models of AD. Copyright © 2011 Wiley-Liss, Inc.
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Sheremet, Ya A; Yemets, A I; Azmi, A; Vissenberg, K; Verbelen, J P; Blume, Ya B
2012-01-01
To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G2/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possibly through regulation of microtubule dynamics in plant cells.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Thal, David M.; Homan, Kristoff T.; Chen, Jun
2012-08-10
G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice withmore » paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.« less
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Luke, Trevor; Maylor, Julie; Undem, Clark; Sylvester, J T; Shimoda, Larissa A
2012-05-15
Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.
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Lee, Jeong Goo
2012-01-01
Purpose. To determine the role of nuclear factor-κB (NF-κB) during FGF-2–mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs). Methods. Expression and/or activation of IL-1 receptor–associated protein kinase (IRAK), TNF receptor–associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. Results. Brief stimulation of CECs with IL-1β upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. Conclusions. These data suggest that IL-1β signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene. PMID:22323467
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Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.
Roskoski, Robert
2016-09-01
The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Tan, Nguyen; Wong, Maureen; Nannini, Michelle A; Hong, Rebecca; Lee, Leslie B; Price, Stephen; Williams, Karen; Savy, Pierre Pascal; Yue, Peng; Sampath, Deepak; Settleman, Jeffrey; Fairbrother, Wayne J; Belmont, Lisa D
2013-06-01
Although mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibition is predicted to cause cell death by stabilization of the proapoptotic BH3-only protein BIM, the induction of apoptosis is often modest. To determine if addition of a Bcl-2 family inhibitor could increase the efficacy of a MEK inhibitor, we evaluated a panel of 53 non-small cell lung cancer and pancreatic cancer cell lines with the combination of navitoclax (ABT-263), a Bcl-2/Bcl-xL (BCL2/BCL2L1) antagonist, and a novel MAP kinase (MEK) inhibitor, G-963. The combination is synergistic in the majority of lines, with an enrichment of cell lines harboring KRAS mutations in the high synergy group. Cells exposed to G-963 arrest in G1 and a small fraction undergo apoptosis. The addition of navitoclax to G-963 does not alter the kinetics of cell-cycle arrest, but greatly increases the percentage of cells that undergo apoptosis. The G-963/navitoclax combination was more effective than either single agent in the KRAS mutant H2122 xenograft model; BIM stabilization and PARP cleavage were observed in tumors, consistent with the mechanism of action observed in cell culture. Addition of the phosphatidylinositol 3-kinase (PI3K, PIK3CA) inhibitor GDC-0941 to this treatment combination increases cell killing compared with double- or single-agent treatment. Taken together, these data suggest the efficacy of agents that target the MAPK and PI3K pathways can be improved by combination with a Bcl-2 family inhibitor. ©2013 AACR
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hanan, Emily J.; Eigenbrot, Charles; Bryan, Marian C.
Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. Here in this paper, wemore » describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.« less
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Discovery of wt RET and V804M RET Inhibitors: From Hit to Lead.
Mologni, Luca; Dalla Via, Martina; Chilin, Adriana; Palumbo, Manlio; Marzaro, Giovanni
2017-08-22
Oncogenic activation of RET kinase has been found in several neoplastic diseases, like medullary thyroid carcinoma, multiple endocrine neoplasia, papillary thyroid carcinoma, and non-small-cell lung cancer. Currently approved RET inhibitors were not originally designed to be RET inhibitors, and their potency against RET kinase has not been optimized. Hence, novel compounds able to inhibit both wild-type RET ( wt RET) and its mutants (e.g., V804M RET) are needed. Herein we present the development and the preliminary evaluation of a new sub-micromolar wt RET/ V804M RET inhibitor, N-(2-fluoro-5-trifluoromethylphenyl)-N'-{4'-[(2''-benzamido)pyridin-4''-ylamino]phenyl}urea (69), endowed with a 4-anilinopyridine structure, starting from our previously identified 4-anilinopyrimidine hit compound. Profiling against a panel of kinases indicated 69 as a multi cKIT/ wt RET/ V804M RET inhibitor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu
2017-01-01
Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.
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PIM kinases as therapeutic targets against advanced melanoma
Shannan, Batool; Watters, Andrea; Chen, Quan; Mollin, Stefan; Dörr, Markus; Meggers, Eric; Xu, Xiaowei; Gimotty, Phyllis A.; Perego, Michela; Li, Ling; Benci, Joseph; Krepler, Clemens; Brafford, Patricia; Zhang, Jie; Wei, Zhi; Zhang, Gao; Liu, Qin; Yin, Xiangfan; Nathanson, Katherine L.; Herlyn, Meenhard; Vultur, Adina
2016-01-01
Therapeutic strategies for the treatment of metastatic melanoma show encouraging results in the clinic; however, not all patients respond equally and tumor resistance still poses a challenge. To identify novel therapeutic targets for melanoma, we screened a panel of structurally diverse organometallic inhibitors against human-derived normal and melanoma cells. We observed that a compound that targets PIM kinases (a family of Ser/Thr kinases) preferentially inhibited melanoma cell proliferation, invasion, and viability in adherent and three-dimensional (3D) melanoma models. Assessment of tumor tissue from melanoma patients showed that PIM kinases are expressed in pre- and post-treatment tumors, suggesting PIM kinases as promising targets in the clinic. Using knockdown studies, we showed that PIM1 contributes to melanoma cell proliferation and tumor growth in vivo; however, the presence of PIM2 and PIM3 could also influence the outcome. The inhibition of all PIM isoforms using SGI-1776 (a clinically-available PIM inhibitor) reduced melanoma proliferation and survival in preclinical models of melanoma. This was potentiated in the presence of the BRAF inhibitor PLX4720 and in the presence of PI3K inhibitors. Our findings suggest that PIM inhibitors provide promising additions to the targeted therapies available to melanoma patients. PMID:27448973
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2012-01-01
Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Conclusions Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun. PMID:23157661
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Trapp, Johannes; Jochum, Anne; Meier, Rene; Saunders, Laura; Marshall, Brett; Kunick, Conrad; Verdin, Eric; Goekjian, Peter; Sippl, Wolfgang; Jung, Manfred
2006-12-14
NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.
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Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan
2017-05-02
Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Small-molecule kinase inhibitors provide insight into Mps1 cell cycle function.
Kwiatkowski, Nicholas; Jelluma, Nannette; Filippakopoulos, Panagis; Soundararajan, Meera; Manak, Michael S; Kwon, Mijung; Choi, Hwan Geun; Sim, Taebo; Deveraux, Quinn L; Rottmann, Sabine; Pellman, David; Shah, Jagesh V; Kops, Geert J P L; Knapp, Stefan; Gray, Nathanael S
2010-05-01
Mps1, a dual-specificity kinase, is required for the proper functioning of the spindle assembly checkpoint and for the maintenance of chromosomal stability. As Mps1 function has been implicated in numerous phases of the cell cycle, the development of a potent, selective small-molecule inhibitor of Mps1 should facilitate dissection of Mps1-related biology. We describe the cellular effects and Mps1 cocrystal structures of new, selective small-molecule inhibitors of Mps1. Consistent with RNAi studies, chemical inhibition of Mps1 leads to defects in Mad1 and Mad2 establishment at unattached kinetochores, decreased Aurora B kinase activity, premature mitotic exit and gross aneuploidy, without any evidence of centrosome duplication defects. However, in U2OS cells having extra centrosomes (an abnormality found in some cancers), Mps1 inhibition increases the frequency of multipolar mitoses. Lastly, Mps1 inhibitor treatment resulted in a decrease in cancer cell viability.
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Xiao, Guo-Sheng; Zhang, Yan-Hui; Wu, Wei; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong
2017-03-01
The ultra-rapidly activating delayed rectifier K + current I Kur (encoded by K v 1.5 or KCNA5) plays an important role in human atrial repolarization. The present study investigates the regulation of this current by protein tyrosine kinases (PTKs). Whole-cell patch voltage clamp technique and immunoprecipitation and Western blotting analysis were used to investigate whether the PTK inhibitors genistein, tyrphostin AG556 (AG556) and PP2 regulate human atrial I Kur and hKv1.5 channels stably expressed in HEK 293 cells. Human atrial I Kur was decreased by genistein (a broad-spectrum PTK inhibitor) and AG556 (a highly selective EGFR TK inhibitor) in a concentration-dependent manner. Inhibition of I Kur induced by 30 μM genistein or 10 μM AG556 was significantly reversed by 1 mM orthovanadate (a protein tyrosine phosphatase inhibitor). Similar results were observed in HEK 293 cells stably expressing hK v 1.5 channels. On the other hand, the Src family kinase inhibitor PP2 (1 μM) slightly enhanced I Kur and hK v 1.5 current, and the current increase was also reversed by orthovanadate. Immunoprecipitation and Western blotting analysis showed that genistein, AG556, and PP2 decreased tyrosine phosphorylation of hK v 1.5 channels and that the decrease was countered by orthovanadate. The PTK inhibitors genistein and AG556 decrease human atrial I Kur and cloned hK v 1.5 channels by inhibiting EGFR TK, whereas the Src kinase inhibitor PP2 increases I Kur and hK v 1.5 current. These results imply that EGFR TK and the soluble Src kinases may have opposite effects on human atrial I Kur . © 2017 The British Pharmacological Society.
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2015-01-01
Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties. PMID:25383627
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Reinwald, M; Silva, J T; Mueller, N J; Fortún, J; Garzoni, C; de Fijter, J W; Fernández-Ruiz, M; Grossi, P; Aguado, J M
2018-06-01
The present review is part of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biologic therapies. To review, from an infectious diseases perspective, the safety profile of therapies targeting different intracellular signaling pathways and to suggest preventive recommendations. Computer-based Medline searches with MeSH terms pertaining to each agent or therapeutic family. Although BCR-ABL tyrosine kinase inhibitors modestly increase the overall risk of infection, dasatinib has been associated with cytomegalovirus and hepatitis B virus reactivation. BRAF/MEK kinase inhibitors do not significantly affect infection susceptibility. The effect of Bruton tyrosine kinase inhibitors (ibrutinib) among patients with B-cell malignancies is difficult to distinguish from that of previous immunosuppression. However, cases of Pneumocystis jirovecii pneumonia (PCP), invasive fungal infection and progressive multifocal leukoencephalopathy have been occasionally reported. Because phosphatidylinositol-3-kinase inhibitors (idelalisib) may predispose to opportunistic infections, anti-Pneumocystis prophylaxis and prevention strategies for cytomegalovirus are recommended. No increased rates of infection have been observed with venetoclax (antiapoptotic protein Bcl-2 inhibitor). Therapy with Janus kinase inhibitors markedly increases the incidence of infection. Pretreatment screening for chronic hepatitis B virus and latent tuberculosis infection must be performed, and anti-Pneumocystis prophylaxis should be considered for patients with additional risk factors. Cancer patients receiving mTOR inhibitors face an increased incidence of overall infection, especially those with additional risk factors (prior therapies or delayed wound healing). Specific preventive approaches are warranted in view of the increased risk of infection associated with some of the reviewed agents. Copyright © 2018. Published by Elsevier Ltd.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Patnaik, Samarjit; Stevens, Kirk L.; Gerding, Roseanne
2009-07-23
Exploration of the SAR around a series of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines led to the discovery of novel pyrrolopyridine inhibitors of the IGF-1R tyrosine kinase. Several compounds demonstrated nanomolar potency in enzyme and cellular mechanistic assays.
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2016-01-01
Drug discovery programs frequently target members of the human kinome and try to identify small molecule protein kinase inhibitors, primarily for cancer treatment, additional indications being increasingly investigated. One of the challenges is controlling the inhibitors degree of selectivity, assessed by in vitro profiling against panels of protein kinases. We manually extracted, compiled, and standardized such profiles published in the literature: we collected 356 908 data points corresponding to 482 protein kinases, 2106 inhibitors, and 661 patents. We then analyzed this data set in terms of kinome coverage, results reproducibility, popularity, and degree of selectivity of both kinases and inhibitors. We used the data set to create robust proteochemometric models capable of predicting kinase activity (the ligand–target space was modeled with an externally validated RMSE of 0.41 ± 0.02 log units and R02 0.74 ± 0.03), in order to account for missing or unreliable measurements. The influence on the prediction quality of parameters such as number of measurements, Murcko scaffold frequency or inhibitor type was assessed. Interpretation of the models enabled to highlight inhibitors and kinases properties correlated with higher affinities, and an analysis in the context of kinases crystal structures was performed. Overall, the models quality allows the accurate prediction of kinase-inhibitor activities and their structural interpretation, thus paving the way for the rational design of compounds with a targeted selectivity profile. PMID:27482722
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Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
Kinehara, Masaki; Kawamura, Suguru; Tateyama, Daiki; Suga, Mika; Matsumura, Hiroko; Mimura, Sumiyo; Hirayama, Noriko; Hirata, Mitsuhi; Uchio-Yamada, Kozue; Kohara, Arihiro; Yanagihara, Kana; Furue, Miho K.
2013-01-01
Background The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. Methodology/Principal Findings In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Conclusions/Significance Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells. PMID:23349801
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2015-01-01
The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior. PMID:25676389
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sueyoshi, Noriyuki; Takao, Toshihiko; Nimura, Takaki
2007-11-23
Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca{sup 2+}/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP familymore » phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro.« less
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Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 2.
Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard
2017-02-01
Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 2 covering J through Z. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.
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Punkvang, Auradee; Kamsri, Pharit; Saparpakorn, Patchreenart; Hannongbua, Supa; Wolschann, Peter; Irle, Stephan; Pungpo, Pornpan
2015-07-01
Substituted aminopyrimidine inhibitors have recently been introduced as antituberculosis agents. These inhibitors show impressive activity against protein kinase B, a Ser/Thr protein kinase that is essential for cell growth of M. tuberculosis. However, up to now, X-ray structures of the protein kinase B enzyme complexes with the substituted aminopyrimidine inhibitors are currently unavailable. Consequently, structural details of their binding modes are questionable, prohibiting the structural-based design of more potent protein kinase B inhibitors in the future. Here, molecular dynamics simulations, in conjunction with molecular mechanics/Poisson-Boltzmann surface area binding free-energy analysis, were employed to gain insight into the complex structures of the protein kinase B inhibitors and their binding energetics. The complex structures obtained by the molecular dynamics simulations show binding free energies in good agreement with experiment. The detailed analysis of molecular dynamics results shows that Glu93, Val95, and Leu17 are key residues responsible to the binding of the protein kinase B inhibitors. The aminopyrazole group and the pyrimidine core are the crucial moieties of substituted aminopyrimidine inhibitors for interaction with the key residues. Our results provide a structural concept that can be used as a guide for the future design of protein kinase B inhibitors with highly increased antagonistic activity. © 2014 John Wiley & Sons A/S.
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The noni anthraquinone damnacanthal is a multi-kinase inhibitor with potent anti-angiogenic effects.
García-Vilas, Javier A; Pino-Ángeles, Almudena; Martínez-Poveda, Beatriz; Quesada, Ana R; Medina, Miguel Ángel
2017-01-28
The natural bioactive compound damnacanthal inhibits several tyrosine kinases. Herein, we show that -in fact- damancanthal is a multi kinase inhibitor. A docking and molecular dynamics simulation approach allows getting further insight on the inhibitory effect of damnacanthal on three different kinases: vascular endothelial growth factor receptor-2, c-Met and focal adhesion kinase. Several of the kinases targeted and inhibited by damnacanthal are involved in angiogenesis. Ex vivo and in vivo experiments clearly demonstrate that, indeed, damnacanthal is a very potent inhibitor of angiogenesis. A number of in vitro assays contribute to determine the specific effects of damnacanthal on each of the steps of the angiogenic process, including inhibition of tubulogenesis, endothelial cell proliferation, survival, migration and production of extracellular matrix remodeling enzyme. Taken altogether, these results suggest that damancanthal could have potential interest for the treatment of cancer and other angiogenesis-dependent diseases. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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The Development of CK2 Inhibitors: From Traditional Pharmacology to in Silico Rational Drug Design
Cozza, Giorgio
2017-01-01
Casein kinase II (CK2) is an ubiquitous and pleiotropic serine/threonine protein kinase able to phosphorylate hundreds of substrates. Being implicated in several human diseases, from neurodegeneration to cancer, the biological roles of CK2 have been intensively studied. Upregulation of CK2 has been shown to be critical to tumor progression, making this kinase an attractive target for cancer therapy. Several CK2 inhibitors have been developed so far, the first being discovered by “trial and error testing”. In the last decade, the development of in silico rational drug design has prompted the discovery, de novo design and optimization of several CK2 inhibitors, active in the low nanomolar range. The screening of big chemical libraries and the optimization of hit compounds by Structure Based Drug Design (SBDD) provide telling examples of a fruitful application of rational drug design to the development of CK2 inhibitors. Ligand Based Drug Design (LBDD) models have been also applied to CK2 drug discovery, however they were mainly focused on methodology improvements rather than being critical for de novo design and optimization. This manuscript provides detailed description of in silico methodologies whose applications to the design and development of CK2 inhibitors proved successful and promising. PMID:28230762
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Wang, Meining; Shen, Aijun; Zhang, Chi; Song, Zilan; Ai, Jing; Liu, Hongchun; Sun, Liping; Ding, Jian; Geng, Meiyu; Zhang, Ao
2016-06-23
Heat shock protein 90 (Hsp90) is a ubiquitous chaperone of all of the oncogenic tyrosine kinases. Many Hsp90 inhibitors, alone or in combination, have shown significant antitumor efficacy against the kinase-positive naïve and mutant models. However, clinical trials of these inhibitors are unsuccessful due to insufficient clinical benefits and nonoptimal safety profiles. Recently, much progress has been reported on the Hsp90-cochaperone-client complex, which will undoubtedly assist in the understanding of the interactions between Hsp90 and its clients. Meanwhile, Hsp90 inhibitors have shown promise against patients' resistance caused by early generation tyrosine kinase inhibitors (TKIs), and at least 13 Hsp90 inhibitors are being reevaluated in the clinic. In this regard, the objectives of the current perspective are to summarize the structure and function of the Hsp90-cochaperone-client complex, to analyze the structural and functional insights into the Hsp90-client interactions to address several existing unresolved problems with Hsp90 inhibitors, and to highlight the preclinical and clinical studies of Hsp90 inhibitors as an effective treatment against resistance to tyrosine kinase inhibitors.
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Jin, Cheng Hua; Krishnaiah, Maddeboina; Sreenu, Domalapally; Subrahmanyam, Vura B; Rao, Kota S; Lee, Hwa Jeong; Park, So-Jung; Park, Hyun-Ju; Lee, Kiho; Sheen, Yhun Yhong; Kim, Dae-Kee
2014-05-22
A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-β type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.
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Kim, Jong-Eun; Son, Joe Eun; Jang, Young Jin; Lee, Dong Eun; Kang, Nam Joo; Jung, Sung Keun; Heo, Yong-Seok; Lee, Ki Won; Lee, Hyong Joo
2011-09-01
Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a promising strategy for developing chemopreventive agents, because TNF-α-mediated cyclooxygenase (COX)-2 expression plays a key role in inflammation and carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] exerts anticarcinogenic effects, although little is known about the underlying molecular mechanisms and specific targets of this compound. In the present study, we found that luteolin inhibited TNF-α-induced COX-2 expression by down-regulating the transactivation of nuclear factor-κB and activator protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/ERK/p90(RSK), mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase/c-Jun, and Akt/p70(S6K). However, it had no effect on the phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling pathways and COX-2 expression are similar to those achieved by blocking tumor progression locus 2 serine/threonine kinase (TPL2) using pharmacologic inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays by binding directly in an ATP-competitive manner. Overall, these results indicate that luteolin exerts potent chemopreventive activities, which primarily target TPL2.
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Identification of signalling cascades involved in red blood cell shrinkage and vesiculation.
Kostova, Elena B; Beuger, Boukje M; Klei, Thomas R L; Halonen, Pasi; Lieftink, Cor; Beijersbergen, Roderick; van den Berg, Timo K; van Bruggen, Robin
2015-04-16
Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca(2+) ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)-Akt (protein kinase B) pathway, the Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway and the Raf-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation.
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Folkes, Adrian J; Ahmadi, Khatereh; Alderton, Wendy K; Alix, Sonia; Baker, Stewart J; Box, Gary; Chuckowree, Irina S; Clarke, Paul A; Depledge, Paul; Eccles, Suzanne A; Friedman, Lori S; Hayes, Angela; Hancox, Timothy C; Kugendradas, Arumugam; Lensun, Letitia; Moore, Pauline; Olivero, Alan G; Pang, Jodie; Patel, Sonal; Pergl-Wilson, Giles H; Raynaud, Florence I; Robson, Anthony; Saghir, Nahid; Salphati, Laurent; Sohal, Sukhjit; Ultsch, Mark H; Valenti, Melanie; Wallweber, Heidi J A; Wan, Nan Chi; Wiesmann, Christian; Workman, Paul; Zhyvoloup, Alexander; Zvelebil, Marketa J; Shuttleworth, Stephen J
2008-09-25
Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.
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Huang, Yongqi; Yoon, Mi-Kyung; Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W
2015-01-30
The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21(Cip1), p27(Kip1) and p57(Kip2). Their kinase inhibitory activities are mediated by a homologous N-terminal kinase inhibitory domain. The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the kinase inhibitory domain and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, as well as its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D
1995-12-01
The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.
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Verkhivker, G M
2016-10-20
Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.
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Bellik, Lydia; Vinci, Maria Cristina; Filippi, Sandra; Ledda, Fabrizio; Parenti, Astrid
2005-10-01
We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (PlGF) through the induction of functional fms-like tyrosine kinase (Flt-1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the PlGF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, PlGF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia-treated VSMCs in response to PlGF was significantly impaired by the p38 and the phosphatidylinositol 3-kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of PlGF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt-1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen-activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to PlGF, while the p38 inhibitor SB202190 markedly impaired the PlGF-induced contractile response. These effects were selectively mediated by Flt-1 without any involvement of foetal liver kinase-1 receptors. These data are the first evidence that different intracellular pathways activated by Flt-1 receptor in VSMCs are involved in diverse biological effects of PlGF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase(1/2) and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of PlGF in vascular pathology.
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Novel mutant-selective EGFR kinase inhibitors against EGFR T790M
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhou, Wenjun; Ercan, Dalia; Chen, Liang
2010-01-12
The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potentmore » against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.« less
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Molecular Imaging of Phosphorylation Events for Drug Development
Chan, C. T.; Paulmurugan, R.; Reeves, R. E.; Solow-Cordero, D.; Gambhir, S. S.
2014-01-01
Purpose Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging. Procedures An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA). Results The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity. Conclusion This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events. PMID:19048345
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Chua, Jing Yi; Lim, Thien Thien; Mohamed Ibrahim, Norlinah; Tan, Ai Huey; Eow, Gaik Bee; Abdul Aziz, Zariah; Puvanarajah, Santhi Datuk; Viswanathan, Shanthi; Lim, Soo Kun; Tan, Li Ping; Chong, Yip Boon; Tan, Chong Tin; Zhao, Yi; Tan, E. K.
2014-01-01
The LRRK2 gene has been associated with both familial and sporadic forms of Parkinson's disease (PD). The G2019S variant is commonly found in North African Arab and Caucasian PD patients, but this locus is monomorphic in Asians. The G2385R and R1628P variants are associated with a higher risk of developing PD in certain Asian populations but have not been studied in the Malaysian population. Therefore, we screened the G2385R and R1628P variants in 1,202 Malaysian subjects consisting of 695 cases and 507 controls. The G2385R and R1628P variants were associated with a 2.2-fold (P = 0.019) and 1.2-fold (P = 0.054) increased risk of PD, respectively. Our data concur with other reported findings in Chinese, Taiwanese, Singaporean, and Korean studies. PMID:25243190
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Role of Abl kinase and the Wave2 signaling complex in HIV-1 entry at a post-hemifusion step.
Harmon, Brooke; Campbell, Nancy; Ratner, Lee
2010-06-17
Entry of human immunodeficiency virus type 1 (HIV-1) commences with binding of the envelope glycoprotein (Env) to the receptor CD4, and one of two coreceptors, CXCR4 or CCR5. Env-mediated signaling through coreceptor results in Galphaq-mediated Rac activation and actin cytoskeleton rearrangements necessary for fusion. Guanine nucleotide exchange factors (GEFs) activate Rac and regulate its downstream protein effectors. In this study we show that Env-induced Rac activation is mediated by the Rac GEF Tiam-1, which associates with the adaptor protein IRSp53 to link Rac to the Wave2 complex. Rac and the tyrosine kinase Abl then activate the Wave2 complex and promote Arp2/3-dependent actin polymerization. Env-mediated cell-cell fusion, virus-cell fusion and HIV-1 infection are dependent on Tiam-1, Abl, IRSp53, Wave2, and Arp3 as shown by attenuation of fusion and infection in cells expressing siRNA targeted to these signaling components. HIV-1 Env-dependent cell-cell fusion, virus-cell fusion and infection were also inhibited by Abl kinase inhibitors, imatinib, nilotinib, and dasatinib. Treatment of cells with Abl kinase inhibitors did not affect cell viability or surface expression of CD4 and CCR5. Similar results with inhibitors and siRNAs were obtained when Env-dependent cell-cell fusion, virus-cell fusion or infection was measured, and when cell lines or primary cells were the target. Using membrane curving agents and fluorescence microscopy, we showed that inhibition of Abl kinase activity arrests fusion at the hemifusion (lipid mixing) step, suggesting a role for Abl-mediated actin remodeling in pore formation and expansion. These results suggest a potential utility of Abl kinase inhibitors to treat HIV-1 infected patients.
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Recent Progress in JAK Inhibitors for the Treatment of Rheumatoid Arthritis.
Nakayamada, Shingo; Kubo, Satoshi; Iwata, Shigeru; Tanaka, Yoshiya
2016-10-01
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial inflammation and joint destruction. Considerable advance in the treatment of RA has been made following the advent of biological disease-modifying anti-rheumatic drugs (DMARDs). However, these biologics require intravenous or subcutaneous injection and some patients fail to respond to biological DMARDs or lose their primary response. Various cytokines and cell surface molecules bind to receptors on the cell surface, resulting in the activation of various cell signaling pathways, including phosphorylation of kinase proteins. Among these kinases, the non-receptor tyrosine kinase family Janus kinase (JAK) plays a pivotal role in the pathological processes of RA. Several JAK inhibitors have been developed as new therapies for patients with RA. These are oral synthetic DMARDs that inhibit JAK1, 2, and 3. One JAK inhibitor, tofacitinib, has already been approved in many countries. Results of phase III clinical trials using a JAK1/2 inhibitor, baricitinib, have shown feasible efficacy and tolerable safety. Both drugs are effective in patients who showed inadequate response to biological DMARDs as well as synthetic DMARDs. In addition, clinical phase III trials using filgotinib and ABT-494, specific JAK1 inhibitors, are currently underway. JAK inhibitors are novel therapies for RA, but further studies are needed to determine their risk-benefit ratio and selection of the most appropriate patients for such therapy.
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Hit to lead evaluation of 1,2,3-triazolo[4,5-b]pyridines as PIM kinase inhibitors.
Pastor, Joaquín; Oyarzabal, Julen; Saluste, Gustavo; Alvarez, Rosa María; Rivero, Virginia; Ramos, Francisco; Cendón, Elena; Blanco-Aparicio, Carmen; Ajenjo, Nuria; Cebriá, Antonio; Albarrán, M I; Cebrián, David; Corrionero, Ana; Fominaya, Jesús; Montoya, Guillermo; Mazzorana, Marco
2012-02-15
PIM kinases have become targets of interest due to their association with biochemical mechanisms affecting survival, proliferation and cytokine production. 1,2,3-Triazolo[4,5-b]pyridines were identified as PIM inhibitors applying a scaffold hopping approach. Initial exploration around this scaffold and X-ray crystallographic data are hereby described. Copyright © 2012 Elsevier Ltd. All rights reserved.
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Sphingosine kinase inhibitors: a review of patent literature (2006-2015).
Lynch, Kevin R; Thorpe, S Brandon; Santos, Webster L
2016-12-01
Sphingosine kinase (SphK1 & SphK2) is the sole source of the pleiotropic lipid mediator, sphingosine-1-phosphate (S1P). S1P has been implicated in a variety of diseases such as cancer, Alzheimer's disease, sickle cell disease and fibrosis and thus the biosynthetic route to S1P is a logical target for drug discovery. Areas covered: In this review, the authors consider the SphK inhibitor patent literature from 2006-2016 Q1 with the emphasis on composition of matter utility patents. The Espacenet database was queried with the search term 'sphingosine AND kinase' to identify relevant literature. Expert opinion: Early inhibitor discovery focused on SphK1 with a bias towards oncology indications. Structurally, the reported inhibitors occupy the sphingosine 'J-shaped' binding pocket. The lack of cytotoxicity with improved SphK1 inhibitors raises doubt about the enzyme as an oncology target. SphK2 inhibitors are featured in more recent patent applications. Interestingly, both SphK1 and SphK2 inhibition and gene 'knockout' share opposing effects on circulating S1P levels: SphK1 inhibition/gene ablation decreases, while SphK2 inhibition/gene ablation increases, blood S1P. As understanding of S1P's physiological roles increases and more drug-like SphK inhibitors emerge, inhibiting one or both SphK isotypes could provide unique strategies for treating disease.
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Ojo, Kayode K; Dangoudoubiyam, Sriveny; Verma, Shiv K; Scheele, Suzanne; DeRocher, Amy E; Yeargan, Michelle; Choi, Ryan; Smith, Tess R; Rivas, Kasey L; Hulverson, Matthew A; Barrett, Lynn K; Fan, Erkang; Maly, Dustin J; Parsons, Marilyn; Dubey, Jitender P; Howe, Daniel K; Van Voorhis, Wesley C
2016-12-01
Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5μM range but interfere with intracellular division at 2.5μM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis. Copyright © 2016 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.
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LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation
Mardilovich, Katerina; Baugh, Mark; Crighton, Diane; Kowalczyk, Dominika; Gabrielsen, Mads; Munro, June; Croft, Daniel R.; Lourenco, Filipe; James, Daniel; Kalna, Gabriella; McGarry, Lynn; Rath, Oliver; Shanks, Emma; Garnett, Mathew J.; McDermott, Ultan; Brookfield, Joanna; Charles, Mark; Hammonds, Tim; Olson, Michael F.
2015-01-01
The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy. PMID:26540348
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Identification of signalling cascades involved in red blood cell shrinkage and vesiculation
Kostova, Elena B.; Beuger, Boukje M.; Klei, Thomas R.L.; Halonen, Pasi; Lieftink, Cor; Beijersbergen, Roderick; van den Berg, Timo K.; van Bruggen, Robin
2015-01-01
Even though red blood cell (RBC) vesiculation is a well-documented phenomenon, notably in the context of RBC aging and blood transfusion, the exact signalling pathways and kinases involved in this process remain largely unknown. We have established a screening method for RBC vesicle shedding using the Ca2+ ionophore ionomycin which is a rapid and efficient method to promote vesiculation. In order to identify novel pathways stimulating vesiculation in RBC, we screened two libraries: the Library of Pharmacologically Active Compounds (LOPAC) and the Selleckchem Kinase Inhibitor Library for their effects on RBC from healthy donors. We investigated compounds triggering vesiculation and compounds inhibiting vesiculation induced by ionomycin. We identified 12 LOPAC compounds, nine kinase inhibitors and one kinase activator which induced RBC shrinkage and vesiculation. Thus, we discovered several novel pathways involved in vesiculation including G protein-coupled receptor (GPCR) signalling, the phosphoinositide 3-kinase (PI3K)–Akt (protein kinase B) pathway, the Jak–STAT (Janus kinase–signal transducer and activator of transcription) pathway and the Raf–MEK (mitogen-activated protein kinase kinase)–ERK (extracellular signal-regulated kinase) pathway. Moreover, we demonstrated a link between casein kinase 2 (CK2) and RBC shrinkage via regulation of the Gardos channel activity. In addition, our data showed that inhibition of several kinases with unknown functions in mature RBC, including Alk (anaplastic lymphoma kinase) kinase and vascular endothelial growth factor receptor 2 (VEGFR-2), induced RBC shrinkage and vesiculation. PMID:25757360
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Lountos, George T; Tropea, Joseph E; Zhang, Di; Jobson, Andrew G; Pommier, Yves; Shoemaker, Robert H; Waugh, David S
2009-01-01
Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC50 = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity. PMID:19177354
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Aftab, D T; Ballas, L M; Loomis, C R; Hait, W N
1991-11-01
Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.
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Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.
Heinzlmeir, Stephanie; Lohse, Jonas; Treiber, Tobias; Kudlinzki, Denis; Linhard, Verena; Gande, Santosh Lakshmi; Sreeramulu, Sridhar; Saxena, Krishna; Liu, Xiaofeng; Wilhelm, Mathias; Schwalbe, Harald; Kuster, Bernhard; Médard, Guillaume
2017-06-21
The receptor tyrosine kinase EPHA2 has gained attention as a therapeutic drug target for cancer and infectious diseases. However, EPHA2 research and EPHA2-based therapies have been hampered by the lack of selective small-molecule inhibitors. Herein we report the synthesis and evaluation of dedicated EPHA2 inhibitors based on the clinical BCR-ABL/SRC inhibitor dasatinib as a lead structure. We designed hybrid structures of dasatinib and the previously known EPHA2 binders CHEMBL249097, PD-173955, and a known EPHB4 inhibitor in order to exploit both the ATP pocket entrance as well as the ribose pocket as binding epitopes in the kinase EPHA2. Medicinal chemistry and inhibitor design were guided by a chemical proteomics approach, allowing early selectivity profiling of the newly synthesized inhibitor candidates. Concomitant protein crystallography of 17 inhibitor co-crystals delivered detailed insight into the atomic interactions that underlie the structure-affinity relationship. Finally, the anti-proliferative effect of the inhibitor candidates was confirmed in the glioblastoma cell line SF-268. In this work, we thus discovered a novel EPHA2 inhibitor candidate that features an improved selectivity profile while maintaining potency against EPHA2 and anticancer activity in SF-268 cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Tang, Chih-Hsin; Lu, Da-Yuu; Yang, Rong-Sen; Tsai, Huei-Yann; Kao, Ming-Ching; Fu, Wen-Mei; Chen, Yuh-Fung
2007-07-15
Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-kappaB inhibitor (pyrrolidine dithiocarbamate), IkappaB protease inhibitor (L-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IkappaBalpha phosphorylation inhibitor (Bay 117082), or NF-kappaB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IkappaB kinase alpha/IkappaB kinase beta, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Leptin-mediated an increase of IkappaB kinase alpha/IkappaB kinase beta activity, kappaB-luciferase activity, and p65 and p50 binding to the NF-kappaB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-kappaB and p300 signaling pathway.
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Fong, Clifford W
2016-08-01
Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Qian, Kevin; Wang, Lian; Cywin, Charles L; Farmer, Bennett T; Hickey, Eugene; Homon, Carol; Jakes, Scott; Kashem, Mohammed A; Lee, George; Leonard, Scott; Li, Jun; Magboo, Ronald; Mao, Wang; Pack, Edward; Peng, Charlene; Prokopowicz, Anthony; Welzel, Morgan; Wolak, John; Morwick, Tina
2009-04-09
A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.
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Shah, O Jameel; Lin, Xiaoyu; Li, Leiming; Huang, Xiaoli; Li, Junling; Anderson, Mark G; Tang, Hua; Rodriguez, Luis E; Warder, Scott E; McLoughlin, Shaun; Chen, Jun; Palma, Joann; Glaser, Keith B; Donawho, Cherrie K; Fesik, Stephen W; Shen, Yu
2010-07-13
Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This "addiction" can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility.
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Basit, Sulman; Ashraf, Zaman; Lee, Kwangho; Latif, Muhammad
2017-07-07
Non-small cell lung cancers (NSCLC) harboring anaplastic lymphoma kinase (ALK) gene rearrangements invariably develop resistance to 2 nd -generation ALK inhibitors. Lorlatinib (PF-06463922) (6) is a 3 rd -generation macrocyclic ALK-TKI that demonstrates many advantages over 2 nd -generation ALK inhibitors. Lorlatinib has demonstrated decent kinase selectivity, promising pharmacokinetic profile, selective brain-penetration and strong antiproliferative activity in several ALK/ROS1-driven tumor models. The current review describes the activity spectrum, key events from discovery to clinical applications and the evidences that lorlatinib acts as an ALK/ROS1 inhibitor in clinical settings. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D
2018-01-01
Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199
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Nakano, Rei; Kitanaka, Taku; Namba, Shinichi; Kitanaka, Nanako; Sugiya, Hiroshi
2018-06-04
The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E 2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca 2+ . Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E 2 synthesis in dermal fibroblasts.
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Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells
2011-01-01
Background Peripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect. Methods Cultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting. Results Midazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB. Conclusion These results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS. PMID:21682888
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Fassihi, Afshin; Sabet, Razieh
2008-01-01
Quantitative relationships between molecular structure and p56lck protein tyrosine kinase inhibitory activity of 50 flavonoid derivatives are discovered by MLR and GA-PLS methods. Different QSAR models revealed that substituent electronic descriptors (SED) parameters have significant impact on protein tyrosine kinase inhibitory activity of the compounds. Between the two statistical methods employed, GA-PLS gave superior results. The resultant GA-PLS model had a high statistical quality (R2 = 0.74 and Q2 = 0.61) for predicting the activity of the inhibitors. The models proposed in the present work are more useful in describing QSAR of flavonoid derivatives as p56lck protein tyrosine kinase inhibitors than those provided previously. PMID:19325836
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Proliferation of the human urothelium is induced by atypical β1 -adrenoceptors.
Winder, M; Wasén, C; Aronsson, P; Giglio, D
2015-09-01
We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively. Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β1/2/3 ), dobutamine (β1 ), salbutamol (β2 ), BRL 37344 (β3 ), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β1 , propranolol; β1/2 ). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the β1 -adrenoceptor. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β1 -adrenoceptors in both urothelial cell lines. The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation. © 2016 John Wiley & Sons Ltd.
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Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells
Xu, Mei; Chen, Gang; Wang, Siying; Liao, Mingjun; Frank, Jacqueline A.; Bower, Kimberly A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia
2012-01-01
Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway. PMID:23112838
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The 'retro-design' concept for novel kinase inhibitors.
Müller, Gerhard; Sennhenn, Peter C; Woodcock, Timothy; Neumann, Lars
2010-07-01
Protein kinases are among the most attractive therapeutic targets for a broad range of diseases. This feature review highlights and classifies the main design principles employed to generate active and selective kinase inhibitors. In particular, emphasis is focused on a fragment-based lead-generation approach, which constitutes a novel design method for developing type II kinase inhibitors with distinct binding kinetic attributes. This 'retro-design' strategy relies on a customized fragment library, and contrasts the traditional approach used in the design of type II inhibitors.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhou, Tianjun; Commodore, Lois; Huang, Wei-Sheng
2010-09-30
Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by twomore » such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.« less
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Schlütke, Laura; Immer, Markus; Preu, Lutz; Totzke, Frank; Schächtele, Christoph; Kubbutat, Michael H G; Kunick, Conrad
2018-05-01
Rearrangements of anaplastic lymphoma kinase (ALK) are associated with several cancer diseases. Due to resistance development against existing ALK-inhibitors, new, structurally unrelated inhibitors are required. By a scaffold hopping strategy, 6,8-disubstituted purines were designed as analogues of similar ALK-inhibiting thieno[3,2-d]pyrimidines. While the new title compounds indeed inhibited ALK and several ALK mutants in submicromolar concentrations, they retained poor water solubility. Copyright © 2017 Elsevier B.V. All rights reserved.
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Novel ROCK inhibitors for the treatment of pulmonary arterial hypertension
DOE Office of Scientific and Technical Information (OSTI.GOV)
Shaw, Duncan; Hollingworth, Greg; Soldermann, Nicolas
A novel class of selective inhibitors of ROCK1 and ROCK2 has been identified by structural based drug design. PK/PD experiments using a set of highly selective Rho kinase inhibitors suggest that systemic Rho kinase inhibition is linked to a reversible reduction in lymphocyte counts. These results led to the consideration of topical delivery of these molecules, and to the identification of a lead molecule 7 which shows promising PK and PD in a murine model of pulmonary hypertension after intra-tracheal dosing.
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Kong, Xiaotian; Sun, Huiyong; Pan, Peichen; Tian, Sheng; Li, Dan; Li, Youyong; Hou, Tingjun
2016-01-21
Due to the high sequence identity of the binding pockets of cyclin-dependent kinases (CDKs), designing highly selective inhibitors towards a specific CDK member remains a big challenge. 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine derivatives are effective inhibitors of CDKs, among which the most promising inhibitor 12u demonstrates high binding affinity to CDK9 and attenuated binding affinity to other homologous kinases, such as CDK2. In this study, in order to rationalize the principle of the binding preference towards CDK9 over CDK2 and to explore crucial information that may aid the design of selective CDK9 inhibitors, MM/GBSA calculations based on conventional molecular dynamics (MD) simulations and enhanced sampling simulations (umbrella sampling and steered MD simulations) were carried out on two representative derivatives (12u and 4). The calculation results show that the binding specificity of 12u to CDK9 is primarily controlled by conformational change of the G-loop and variation of the van der Waals interactions. Furthermore, the enhanced sampling simulations revealed the different reaction coordinates and transient interactions of inhibitors 12u and 4 as they dissociate from the binding pockets of CDK9 and CDK2. The physical principles obtained from this study may facilitate the discovery and rational design of novel and specific inhibitors of CDK9.
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Lin, Po-Shuen; Chang, Hsiao-Hua; Yeh, Chien-Yang; Chang, Mei-Chi; Chan, Chiu-Po; Kuo, Han-Yueh; Liu, Hsin-Cheng; Liao, Wan-Chuen; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei
2017-05-01
In order to clarify the role of transforming growth factor beta 1 (TGF-β1) in pulp repair/regeneration responses, we investigated the differential signaling pathways responsible for the effects of TGF-β1 on collagen turnover, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human dental pulp cells. Pulp cells were exposed to TGF-β1 with/without pretreatment and coincubation by 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene (U0126; a mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] inhibitor) and 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H- imidazol-2-yl)-benzamide hydrate (SB431542; an activin receptor-like kinase-5/Smad signaling inhibitor). Sircol collagen assay was used to measure cellular collagen content. Culture medium procollagen I, TIMP-1, and MMP-3 levels were determined by enzyme-linked immunosorbent assay. TGF-β1 increased the collagen content, procollagen I, and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-β1-induced increase of collagen content and TIMP-1 production of dental pulp cells. These results indicate that TGF-β1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling. Copyright © 2016. Published by Elsevier B.V.
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Selectivity of ROCK inhibitors in the spontaneously tonic smooth muscle.
Rattan, Satish; Patel, Chirag A
2008-03-01
The selectivity of different Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscle has not been investigated. We examined this issue using Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarbox anecarboxamide, 2HCl], H-1152 [(S)-(+)-(2-methyl-5-isoquinolinyl) sulfonylhomopiperazine, 2HCl], HA-1077 [(5 isoquinolinesulfonyl) homopiperazine, 2HCl], and ROCK inhibitor II [N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea]. We compared these inhibitors in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). ROCK, protein kinase C (PKC), and myosin light chain kinase (MLCK) activities were determined in the IAS, before and after different ROCK inhibitors. Y-27632 and H-1152 were approximately 30-fold more potent in the IAS (IC(50): 4.4 x 10(-7) and 7.9 x 10(-8) M, respectively) vs. the phasic rectal smooth muscle (RSM) (IC(50): 1.3 x 10(-5) and 2.5 x 10(-6) M, respectively). HA-1077 and ROCK inhibitor II were equipotent in the IAS vs. RSM. In the IAS, H-1152 was the most potent whereas ROCK inhibitor II is the least. Y-27632 and H-1152 caused concentration-dependent decrease in the IAS tone that correlates directly with the decreases in ROCK activity, without significant effect in the PKC and MLCK activities. This specifically selective correlation between ROCK activity and decrease in the IAS tone was absent in the case of HA-1077 and ROCK inhibitor II, which also inhibited PKC and MLCK. We conclude that the IAS tone is critically dependent on ROCK activity, and H-1152 and Y-27632 are the most selective and potent ROCK inhibitors in the IAS.
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TRAF6 and Src kinase activity regulates Cot activation by IL-1.
Rodríguez, Cristina; Pozo, Maite; Nieto, Elvira; Fernández, Margarita; Alemany, Susana
2006-09-01
Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.
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Mauchle, Ulrike; Selvarajah, Gayathri T; Mol, Jan A; Kirpensteijn, Jolle; Verheije, Monique H
2015-08-01
Osteosarcoma is the most common primary bone tumour in dogs but various forms of therapy have not significantly improved clinical outcomes. As dysregulation of kinase activity is often present in tumours, kinases represent attractive molecular targets for cancer therapy. The purpose of this study was to identify novel compounds targeting kinases with the potential to induce cell death in a panel of canine osteosarcoma cell lines. The ability of 80 well-characterized kinase inhibitor compounds to inhibit the proliferation of four canine osteosarcoma cell lines was investigated in vitro. For those compounds with activity, the mechanism of action and capability to potentiate the activity of doxorubicin was further evaluated. The screening showed 22 different kinase inhibitors that induced significant anti-proliferative effects across the four canine osteosarcoma cell lines investigated. Four of these compounds (RO 31-8220, 5-iodotubercidin, BAY 11-7082 and an erbstatin analog) showed significant cell growth inhibitory effects across all cell lines in association with variable induction of apoptosis. RO 31-8220 and 5-iodotubercidin showed the highest ability to potentiate the effects of doxorubicin on cell viability. In conclusion, the present study identified several potent kinase inhibitors targeting the PKC, CK1, PKA, ErbB2, mTOR and NF-κB pathways, which may warrant further investigations for the treatment of osteosarcoma in dogs. Copyright © 2014 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Jones, Peter; Storer, R. Ian; Sabnis, Yogesh A.
By use of a structure-based computational method for identification of structurally novel Janus kinase (JAK) inhibitors predicted to bind beyond the ATP binding site, a potent series of indazoles was identified as selective pan-JAK inhibitors with a type 1.5 binding mode. Optimization of the series for potency and increased duration of action commensurate with inhaled or topical delivery resulted in potent pan-JAK inhibitor 2 (PF-06263276), which was advanced into clinical studies.
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Roles of Apicomplexan protein kinases at each life cycle stage.
Kato, Kentaro; Sugi, Tatsuki; Iwanaga, Tatsuya
2012-06-01
Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
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Pan, Min-Hsiung; Chen, Wei-Jen; Lin-Shiau, Shoei-Yn; Ho, Chi-Tang; Lin, Jen-Kun
2002-10-01
Tangeretin (5,6,7,8,4'-pentamethoxyflavone) is concentrated in the peel of citrus fruits. DNA flow cytometric analysis indicated that tangeretin blocked cell cycle progression at G1 phase in colorectal carcinoma COLO 205 cells. Over a 24 h exposure to tangeretin, the degree of phosphorylation of Rb was decreased after 12 h and G1 arrest developed. The protein expression of cyclins A, D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that tangeretin inhibited the activities of cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4) in a dose-dependent manner in the cell-free system. As the cells were exposed to tangeretin (50 microM) over 48 h a gradual loss of both Cdk2 and 4 kinase activities occurred. Tangeretin also increased the content of the Cdk inhibitor p21 protein and this effect correlated with the elevation in p53 levels. In addition, tangeretin also increased the level of the Cdk inhibitor p27 protein within 18 h. These results suggest that tangeretin either exerts its growth-inhibitory effects through modulation of the activities of several key G1 regulatory proteins, such as Cdk2 and Cdk4, or mediates the increase of Cdk inhibitors p21 and p27.
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Hanan, Emily J.; Eigenbrot, Charles; Bryan, Marian C.; ...
2014-11-10
Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. Here in this paper, wemore » describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.« less
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Shen, Mingyun; Zhou, Shunye; Li, Youyong; Li, Dan; Hou, Tingjun
2013-10-01
LIM kinases (LIMKs), downstream of Rho-associated protein kinases (ROCKs) and p21-activated protein kinases (PAKs), are shown to be promising targets for the treatment of cancers. In this study, the inhibition mechanism of 41 pyrrolopyrimidine derivatives as LIMK2 inhibitors was explored through a series of theoretical approaches. First, a model of LIMK2 was generated through molecular homology modeling, and the studied inhibitors were docked into the binding active site of LIMK2 by the docking protocol, taking into consideration the flexibility of the protein. The binding poses predicted by molecular docking for 17 selected inhibitors with different bioactivities complexed with LIMK2 underwent molecular dynamics (MD) simulations, and the binding free energies for the complexes were predicted by using the molecular mechanics/generalized born surface area (MM/GBSA) method. The predicted binding free energies correlated well with the experimental bioactivities (r(2) = 0.63 or 0.62). Next, the free energy decomposition analysis was utilized to highlight the following key structural features related to biological activity: (1) the important H-bond between Ile408 and pyrrolopyrimidine, (2) the H-bonds between the inhibitors and Asp469 and Gly471 which maintain the stability of the DFG-out conformation, and (3) the hydrophobic interactions between the inhibitors and several key residues (Leu337, Phe342, Ala345, Val358, Lys360, Leu389, Ile408, Leu458 and Leu472). Finally, a variety of LIMK2 inhibitors with a pyrrolopyrimidine scaffold were designed, some of which showed improved potency according to the predictions. Our studies suggest that the use of molecular docking with MD simulations and free energy calculations could be a powerful tool for understanding the binding mechanism of LIMK2 inhibitors and for the design of more potent LIMK2 inhibitors.
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Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity
Alsamarah, Abdelaziz; LaCuran, Alecander E.; Oelschlaeger, Peter; Hao, Jijun; Luo, Yun
2015-01-01
Abnormal alteration of bone morphogenetic protein (BMP) signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI) to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2) tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD) simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5) or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2), as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189) will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling. PMID:26133550
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Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya; Szukalska, Gabriela; Banerjee, Budhaditya; Hilinski, Michael K.; Lannigan, Deborah A.; Stukenberg, P. Todd; Derewenda, Zygmunt S.
2012-01-01
The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-L-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 Å resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors. PMID:22846040
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Bagley, Mark C; Davis, Terence; Rokicki, Michal J; Widdowson, Caroline S; Kipling, David
2010-02-01
UR-13756 is a potent and selective p38 mitogen-activated protein kinase (MAPK) inhibitor, reported to have good bioavailability and pharmacokinetic properties and, thus, is of potential use in the treatment of accelerated aging in Werner syndrome. Irradiation of 2-chloroacrylonitrile and methylhydrazine in ethanol at 100 °C gives 1-methyl-3-aminopyrazole, which reacts with 4-fluorobenzaldehyde and a ketone, obtained by Claisen condensation of 4-picoline, in a Hantzsch-type 3-component hereocyclocondensation, to give the pyrazolopyridine UR-13756. UR-13756 shows p38 MAPK inhibitory activity in human telomerase reverse transcriptase-immortalized HCA2 dermal fibroblasts, with an IC(50) of 80 nm, as shown by ELISA, is 100% efficacious for up to 24 h at 1.0 μm and displays excellent kinase selectivity over the related stress-activated c-Jun kinases. In addition, UR-13756 is an effective p38 inhibitor at 1.0 μm in Werner syndrome cells, as shown by immunoblot. The convergent synthesis of UR-13756 is realized using microwave dielectric heating and provides a highly selective inhibitor that shows excellent selectivity for p38 MAPK over c-Jun N-terminal kinase.
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Wcisło-Dziadecka, Dominika; Zbiciak-Nylec, Martyna; Brzezińska-Wcisło, Ligia; Bebenek, Katarzyna; Kaźmierczak, Agata
2017-11-01
The rapid progress of genetic engineering furthermore opens up new prospects in the therapy of this difficult-to-treat disease. IL-23 inhibitors, phosphodiesterase 4 (PDE4) inhibitors, and Janus kinase (JAK) inhibitors are currently encouraging further research. Two drugs which are IL-23 inhibitors are now in phase III of clinical trials. The aim of the action of both drugs is selective IL-23 inhibition by targeting the p19 subunit. Guselkumab is a fully human monoclonal antibody. Tildrakizumab is a humanized monoclonal antibody, which also belongs to IgG class and is targeted to subunit p19 of interleukin 23 (IL-23). Phosphodiesterase inhibitors exert an anti-inflammatory action and their most common group is the PDE4 family. PDE4 inhibits cAMP, which reduces the inflammatory response of the pathway of Th helper lymphocytes, Th17, and type 1 interferon which modulates the production of anti-inflammatory cytokines such as IL-10 interleukins. The Janus kinase (JAK) signaling pathway plays an important role in the immunopathogenesis of psoriasis. Tofacitinib suppresses the expression of IL-23, IL-17A, IL-17F, and IL-22 receptors during the stimulation of lymphocytes. Ruxolitinib is a selective inhibitor of JAK1 and JAK2 kinases and the JAK-STAT signaling pathway. This article is a review of the aforementioned drugs as described in the latest available literature. © 2017 Wiley Periodicals, Inc.
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Inhibitors of JAK-family kinases: an update on the patent literature 2013-2015, part 1.
Kettle, Jason G; Åstrand, Annika; Catley, Matthew; Grimster, Neil P; Nilsson, Magnus; Su, Qibin; Woessner, Richard
2017-02-01
Janus kinases (JAKs) are a family of four enzymes; JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) that are critical in cytokine signalling and are strongly linked to both cancer and inflammatory diseases. There are currently two launched JAK inhibitors for the treatment of human conditions: tofacitinib for Rheumatoid arthritis (RA) and ruxolitinib for myeloproliferative neoplasms including intermediate or high risk myelofibrosis and polycythemia vera. Areas covered: This review covers patents claiming activity against one or more JAK family members in the period 2013-2015 inclusive, and covers 95 patents from 42 applicants, split over two parts. The authors have ordered recent patents according to the primary applicant's name, with part 1 covering A through to I. Expert opinion: Inhibition of JAK-family kinases is an area of growing interest, catalysed by the maturity of data on marketed inhibitors ruxolitinib and tofacitinib in late stage clinical trials. Many applicants are pursuing traditional fast-follower strategies around these inhibitors, with a range of chemical strategies adopted. The challenge will be to show sufficient differentiation to the originator compounds, since dose limiting toxicities with such agents appear to be on target and mechanism-related and also considering that such agents may be available as generic compounds by the time follower agents reach market.
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Screening of Microbial Extracts for Anticancer Compounds Using Streptomyces Kinase Inhibitor Assay.
Shanbhag, Prashant; Bhave, Sarita; Vartak, Ashwini; Kulkarni-Almeida, Asha; Mahajan, Girish; Villanueva, Ivan; Davies, Julian
2015-07-01
Eukaryotic kinases are known to play an important role in signal transduction pathways by phosphorylating their respective substrates. Abnormal phosphorylations by these kinases have resulted in diseases. Hence inhibitors of kinases are of considerable pharmaceutical interest for a wide variety of disease targets, especially cancers. A number of reports have been published which indicate that eukaryotic-like kinases may complement two-component kinase systems in several bacteria. In Streptomyces sp. such kinases have been found to have a role in formation of aerial hyphae, spores, pigmentation & even in antibiotic production in some strains. Eukaryotic kinase inhibitors are seen to inhibit formation of aerial mycelia in Streptomyces without inhibiting vegetative mycelia. This property has been used to design an assay to screen for eukaryotic kinase inhibitors. The assay involves testing of compounds against Streptomyces 85E ATCC 55824 using agar well diffusion method. Inhibitors of kinases give rise to "bald" colonies where aerial mycelia and sporulation inhibition is seen. The assay has been standardized using known eukaryotic protein kinase inhibiting anticancer agents like AG-490, AG-1295, AG-1478, Flavopiridol and Imatinib as positive controls, at a concentration ranging from 10 μg/well to 100 μg/well. Anti-infective compounds which are not reported to inhibit eukaryotic protein kinases were used as negative controls. A number of microbial cultures have been screened for novel eukaryotic protein kinase inhibitors. Further these microbial extracts were tested in various cancer cell lines like Panel, HCT116, Calul, ACHN and H460 at a concentration of 10 μg/mL/ well. The anticancer data was seen correlating well with the Streptomyces kinase assay thus validating the assay.
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Trask, Oscar J; Baker, Audrey; Williams, Rhonda Gates; Nickischer, Debra; Kandasamy, Ramani; Laethem, Carmen; Johnston, Patricia A; Johnston, Paul A
2006-01-01
This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation. This chapter also presents a case history of the performance of the MK2-EGFP translocation assay, run as a "high-content" screen of a 32K kinase-biased library to identify p38 inhibitors. The assay performed very well and a number of putative p38 inhibitor hits were identified. Through the use of multiparameter data provided by the nuclear translocation algorithm and by checking images, a number of compounds were identified that were potential artifacts due to interference with the imaging format. These included fluorescent compounds, or compounds that dramatically reduced cell numbers due to cytotoxicity or by disrupting cell adherence. A total of 145 compounds produced IC(50) values <50.0 muM in the MK2-EGFP translocation assay, and a cross target query of the Lilly-RTP HTS database confirmed their inhibitory activity against in vitro kinase targets, including p38a. Compounds were confirmed structurally by LCMS analysis and profiled in cell-based imaging assays for MAPK signaling pathway selectivity. Three of the hit scaffolds identified in the MK2-EGFP translocation HCS run on the ArrayScan were selected for a p38a inhibitor hit-to-lead structure activity relationship (SAR) chemistry effort.
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Hit generation and exploration: imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases.
Bavetsias, Vassilios; Sun, Chongbo; Bouloc, Nathalie; Reynisson, Jóhannes; Workman, Paul; Linardopoulos, Spiros; McDonald, Edward
2007-12-01
A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases with IC(50) values of 0.042, 0.198 and 0.227microM, respectively. Compound 31 inhibits cell proliferation and has good microsomal stability.
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Evidence for prehistoric origins of the G2019S mutation in the North African Berber population.
Ben El Haj, Rafiqua; Salmi, Ayyoub; Regragui, Wafa; Moussa, Ahmed; Bouslam, Naima; Tibar, Houyam; Benomar, Ali; Yahyaoui, Mohamed; Bouhouche, Ahmed
2017-01-01
The most common cause of the monogenic form of Parkinson's disease known so far is the G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) gene. Its frequency varies greatly among ethnic groups and geographic regions ranging from less than 0.1% in Asia to 40% in North Africa. This mutation has three distinct haplotypes; haplotype 1 being the oldest and most common. Recent studies have dated haplotype 1 of the G2019S mutation to about 4000 years ago, but it remains controversial whether the mutation has a Near-Eastern or Moroccan-Berber ancestral origin. To decipher this evolutionary history, we genotyped 10 microsatellite markers spanning a region of 11.27 Mb in a total of 57 unrelated Moroccan PD patients carrying the G2019S mutation for which the Berber or Arab origin was established over 3 generations based on spoken language. We estimated the age of the most recent common ancestor for the 36 Arab-speaking and the 15 Berber-speaking G2019S carriers using the likelihood-based method with a mutation rate of 10-4. Data analysis suggests that the shortest haplotype originated in a patient of Berber ethnicity. The common founder was estimated to have lived 159 generations ago (95% CI 116-224) for Arab patients, and 200 generations ago (95% CI 123-348) for Berber patients. Then, 29 native North African males carrying the mutation were assessed for specific uniparental markers by sequencing the Y-chromosome (E-M81, E-M78, and M-267) and mitochondrial DNA (mtDNA) hypervariable regions (HV1 and HV2) to examine paternal and maternal contributions, respectively. Results showed that the autochthonous genetic component reached 76% for mtDNA (Eurasian and north African haplogroups) and 59% for the Y-chromosome (E-M81 and E-M78), suggesting that the G2019S mutation may have arisen in an autochthonous DNA pool. Therefore, we conclude that LRRK2 G2019S mutation most likely originated in a Berber founder who lived at least 5000 years ago (95% CI 3075-8700).
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Mielecki, Marcin; Lesyng, Bogdan
2016-01-01
Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.
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Thomas, Melodie
2003-12-01
Despite treatment advances over the past decade, long-term survival for patients with non-small cell lung cancer (NSCLC) remains poor, and treatment options available after second-line therapy are limited. Increased understanding of cancer biology has led to the identification of several potential targets for treatment. The epidermal growth factor receptor (EGFR) belongs to a family of plasma membrane receptor tyrosine kinases that controls many important cellular functions, from growth and proliferation to cell death. This receptor is a particularly promising therapeutic target because it often is overexpressed in patients with NSCLC and has been implicated in the pathogenesis as well as the proliferation, invasion, and metastasis of lung cancer and other malignancies. New agents developed to inhibit EGFR function include small-molecule tyrosine kinase inhibitors, monoclonal antibodies to EGFR, and pan-EGFR inhibitors. Completed and ongoing clinical trials have shown that EGFR inhibitors have remarkable efficacy for patients with relapsed NSCLC. Among these, two phase 2 trials have shown that ZD1839 is effective when used as monotherapy. The response rates are comparable with those for docetaxel given in the second-line setting. Another phase 2 trial has shown that OSI-774 is effective in the same setting. Data from phase 3 trials indicate that adding an EGFR tyrosine kinase inhibitor to chemotherapy does not provide an additional survival benefit, as compared with standard chemotherapy alone for first-line treatment of NSCLC. It appears that EGFR tyrosine kinase inhibitors are safe and well tolerated by patients with cancer. Further studies will elucidate how these new agents can best be used for NSCLC and other tumor types.
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Min, A; Hasuma, T; Yano, Y; Matsui-Yuasa, I; Otani, S
1995-12-01
We examined the effect of inhibitors of tyrosine kinase and tyrosine phosphatase on DNA fragmentation, protein tyrosine phosphorylation, and polyamine metabolism in the murine T-cell line CTLL-2. When cells were exposed to herbimycin A, a specific inhibitor of tyrosine kinase (Uehara et al., 1989, Biochem. Biophys. Res. Commun., 163:803-809), in the presence of interleukin 2 (IL-2), DNA was degraded into oligonucleosomal fragments in a dose-dependent fashion. Genistein, another inhibitor of tyrosine kinase (Akiyama et al., 1987, J. Biol. Chem., 262:5592-5596), had similar effects. Exposure of CTLL-2 cells to vanadate, a tyrosine phosphatase inhibitor, blocked with the DNA fragmentation induced by herbimycin A. Tyrosine phosphorylation of 55 Kd protein was inhibited by herbimycin A, and the inhibition was reduced by vanadate. Ornithine decarboxylase (ODC) activity decreased rapidly after herbimycin A was added to CTLL-2 cell cultures, while vanadate increased ODC activity. The exogenous addition of putrescine or spermine, but not that of spermidine, attenuated herbimycin A-induced DNA fragmentation. These findings suggest that phosphorylation of tyrosine residues of 55 Kd protein prevents DNA fragmentation and that polyamines are involved in regulation of apoptosis.
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Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors
NASA Astrophysics Data System (ADS)
Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten
2015-05-01
REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.
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Ocaña, Alberto; Amir, Eitan
2009-12-01
Aberrant activation of HER2 through overexpression has been shown to play an important role in some breast cancers. Therapies against this receptor including the monoclonal antibody, trastuzumab, or the small tyrosine kinase inhibitor, lapatinib have shown to improve the prognosis of such patients. Despite overexpressing HER2, some patients do not respond to these targeted treatments or progress after a short period of time. Irreversible tyrosine kinase inhibitors have been developed to bypass several pathways that could be involved in this resistance. In vitro, these agents have been shown to be more potent and to prolong target inhibition. Clinical development of these agents is ongoing and early results are promising. This review will describe the biologic rationale that justifies the development of these agents in breast cancer focusing on the current status and future directions.
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Bairy, Santhosh Kumar; Suneel Kumar, B V S; Bhalla, Joseph Uday Tej; Pramod, A B; Ravikumar, Muttineni
2009-04-01
c-Src kinase play an important role in cell growth and differentiation and its inhibitors can be useful for the treatment of various diseases, including cancer, osteoporosis, and metastatic bone disease. Three dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on quinazolin derivatives inhibiting c-Src kinase. Molecular field analysis (MFA) models with four different alignment techniques, namely, GLIDE, GOLD, LIGANDFIT and Least squares based methods were developed. glide based MFA model showed better results (Leave one out cross validation correlation coefficient r(2)(cv) = 0.923 and non-cross validation correlation coefficient r(2)= 0.958) when compared with other models. These results help us to understand the nature of descriptors required for activity of these compounds and thereby provide guidelines to design novel and potent c-Src kinase inhibitors.
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Perspective on computational and structural aspects of kinase discovery from IPK2014.
Martin, Eric; Knapp, Stefan; Engh, Richard A; Moebitz, Henrik; Varin, Thibault; Roux, Benoit; Meiler, Jens; Berdini, Valerio; Baumann, Alexander; Vieth, Michal
2015-10-01
Recent advances in understanding the activity and selectivity of kinase inhibitors and their relationships to protein structure are presented. Conformational selection in kinases is studied from empirical, data-driven and simulation approaches. Ligand binding and its affinity are, in many cases, determined by the predetermined active and inactive conformation of kinases. Binding affinity and selectivity predictions highlight the current state of the art and advances in computational chemistry as it applies to kinase inhibitor discovery. Kinome wide inhibitor profiling and cell panel profiling lead to a better understanding of selectivity and allow for target validation and patient tailoring hypotheses. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. Copyright © 2015 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Won Seok; Chang, Jai Won; Han, Nam Jeong
The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-{beta}1 (TGF-{beta}1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-{beta}1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-{kappa}B. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-{kappa}B DNA binding activity was also decreased by Syk inhibitors. Highmore » glucose increased nuclear translocation of p65 without serine phosphorylation of I{kappa}B{alpha} and without degradation of I{kappa}B{alpha}, but with an increase in tyrosine phosphorylation of I{kappa}B{alpha} that may account for the activation of NF-{kappa}B. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced I{kappa}B{alpha} tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-{beta}1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-{kappa}B pathways. This suggests that Syk might be implicated in the diabetic kidney disease.« less
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Zhang, Guowei; Wang, Huijuan; Ma, Zhiyong
2017-04-20
At present the treatment of advanced non-small cell lung cancer enters a targeted era and develops rapidly. New drugs appear constantly. Small molecular tyrosine kinase inhibitors have occupied the biggest piece of the territory, which commonly have a clear biomarker as predictor, and show remarkable effect in specific molecular classification of patients. The epidermal growth factor tyrosine kinase inhibitors such as gefitinib, erlotinib, icotinib and anaplastic lymphoma kinase tyrosine kinase inhibitors crizotinib have brought a milestone advance. In recent years new generations of tyrosine kinase inhibitors have achieved a great success in patients with acquired resistance to the above two kinds of drugs. At the same time new therapeutic targets are constantly emerging. So in this paper, we reviewed and summarized the important drugs and clinical trails on this topic, and made a prospect of the future development.
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Grande, M Teresa; López-Novoa, José M
2008-01-01
Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.
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Structural analysis of the human fibroblast growth factor receptor 4 kinase.
Lesca, E; Lammens, A; Huber, R; Augustin, M
2014-11-11
The family of fibroblast growth factor receptors (FGFRs) plays an important and well-characterized role in a variety of pathological disorders. FGFR4 is involved in myogenesis and muscle regeneration. Mutations affecting the kinase domain of FGFR4 may cause cancer, for example, breast cancer or rhabdomyosarcoma. Whereas FGFR1-FGFR3 have been structurally characterized, the structure of the FGFR4 kinase domain has not yet been reported. In this study, we present four structures of the kinase domain of FGFR4, in its apo-form and in complex with different types of small-molecule inhibitors. The two apo-FGFR4 kinase domain structures show an activation segment similar in conformation to an autoinhibitory segment observed in the hepatocyte growth factor receptor kinase but different from the known structures of other FGFR kinases. The structures of FGFR4 in complex with the type I inhibitor Dovitinib and the type II inhibitor Ponatinib reveal the molecular interactions with different types of kinase inhibitors and may assist in the design and development of FGFR4 inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.
Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar
2003-08-01
DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.
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Venkatesan, Aranapakam M; Chen, Zecheng; dos Santos, Osvaldo; Dehnhardt, Christoph; Santos, Efren Delos; Ayral-Kaloustian, Semiramis; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Chaudhary, Inder; Mansour, Tarek S
2010-10-01
A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed. Copyright © 2010. Published by Elsevier Ltd.
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Yang, Jing; Ikezoe, Takayuki; Nishioka, Chie; Tasaka, Taizo; Taniguchi, Ayuko; Kuwayama, Yoshio; Komatsu, Naoki; Bandobashi, Kentaro; Togitani, Kazuto; Koeffler, H Phillip; Taguchi, Hirokuni; Yokoyama, Akihito
2007-09-15
Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. We have recently shown that hematopoietic malignant cells including those from acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) aberrantly expressed Aurora A and B kinases, and ZM447439, a potent inhibitor of Aurora kinases, effectively induced growth arrest and apoptosis of a variety of leukemia cells. The present study explored the effect of AZD1152, a highly selective inhibitor of Aurora B kinase, on various types of human leukemia cells. AZD1152 inhibited the proliferation of AML lines (HL-60, NB4, MOLM13), ALL line (PALL-2), biphenotypic leukemia (MV4-11), acute eosinophilic leukemia (EOL-1), and the blast crisis of chronic myeloid leukemia K562 cells with an IC50 ranging from 3 nM to 40 nM, as measured by thymidine uptake on day 2 of culture. These cells had 4N/8N DNA content followed by apoptosis, as measured by cell-cycle analysis and annexin V staining, respectively. Of note, AZD1152 synergistically enhanced the antiproliferative activity of vincristine, a tubulin depolymerizing agent, and daunorubicin, a topoisomerase II inhibitor, against the MOLM13 and PALL-2 cells in vitro. Furthermore, AZD1152 potentiated the action of vincristine and daunorubicin in a MOLM13 murine xenograft model. Taken together, AZD1152 is a promising new agent for treatment of individuals with leukemia. The combined administration of AZD1152 and conventional chemotherapeutic agent to patients with leukemia warrants further investigation.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xing, Li; Shieh, Huey S.; Selness, Shaun R.
2009-07-24
PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as themore » gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.« less
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The Potential Role of Aurora Kinase Inhibitors in Haematological Malignancies
Farag, Sherif S.
2011-01-01
Summary Aurora kinases play an important role in the control of the cell cycle and have been implicated in tumourigenesis in a number of cancers. Among the haematological malignancies, overexpression of Aurora kinases has been reported in acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, multiple myeloma, aggressive non-Hodgkin lymphoma and Hodgkin lymphoma. A large number of Aurora kinase inhibitors are currently in different stages of clinical development. In addition to varying in their selectivity for the different Aurora kinases, some also have activity directed at other cellular kinases involved in important molecular pathways in cancer cells. This review summarizes the biology of Aurora kinases and discusses why they may be good therapeutic targets in different haematological cancers. We describe preclinical data that has served as the rationale for investigating Aurora kinase inhibitors in different haematological malignancies, and summarize published results from early phase clinical trials. While the anti-tumour effects of Aurora kinase inhibitors appear promising, we highlight important issues for future clinical research and suggest that the optimal use of these inhibitors is likely to be in combination with cytotoxic agents already in use for the treatment of various haematological cancers. PMID:21980926
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Skin problems and EGFR-tyrosine kinase inhibitor
Kozuki, Toshiyuki
2016-01-01
Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. PMID:26826719
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Husain, S; Abdel-Latif, A A
1999-08-15
We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.
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Husain, S; Abdel-Latif, A A
1999-01-01
We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle. PMID:10432304
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Lee, Sang Jae; Choi, Jang-Sik; Han, Byeong-Gu; Kim, Hyoun Sook; Song, Ho-Juhn; Lee, Jaekyoo; Nam, Seungyoon; Goh, Sung-Ho; Kim, Jung-Ho; Koh, Jong Sung; Lee, Byung Il
2016-10-01
Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC 50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499. The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282). © 2016 Federation of European Biochemical Societies.
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Akula, Sravani; Kamasani, Swapna; Sivan, Sree Kanth; Manga, Vijjulatha; Vudem, Dashavantha Reddy; Kancha, Rama Krishna
2018-05-01
A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.
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Jerusalem, Guy; Rorive, Andree; Collignon, Joelle
2014-01-01
Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-positive, HER2-negative advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clinically meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clinically oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-positive HER2-negative, triple-negative, and HER2-positive ER-positive/negative disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-positive, HER2-negative early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup analysis of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addition of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed in this review. Second-generation mTOR inhibitors and dual mTOR-phosphatidylinositol-3-kinase inhibitors are currently being evaluated in clinical trials. PMID:24833916
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Shah, O. Jameel; Lin, Xiaoyu; Li, Leiming; Huang, Xiaoli; Li, Junling; Anderson, Mark G.; Tang, Hua; Rodriguez, Luis E.; Warder, Scott E.; McLoughlin, Shaun; Chen, Jun; Palma, Joann; Glaser, Keith B.; Donawho, Cherrie K.; Fesik, Stephen W.; Shen, Yu
2010-01-01
Aurora kinase B inhibitors induce apoptosis secondary to polyploidization and have entered clinical trials as an emerging class of neocytotoxic chemotherapeutics. We demonstrate here that polyploidization neutralizes Mcl-1 function, rendering cancer cells exquisitely dependent on Bcl-XL/-2. This “addiction” can be exploited therapeutically by combining aurora kinase inhibitors and the orally bioavailable BH3 mimetic, ABT-263, which inhibits Bcl-XL, Bcl-2, and Bcl-w. The combination of ABT-263 with aurora B inhibitors produces a synergistic loss of viability in a range of cell lines of divergent tumor origin and exhibits more sustained tumor growth inhibition in vivo compared with aurora B inhibitor monotherapy. These data demonstrate that Bcl-XL/-2 is necessary to support viability during polyploidization in a variety of tumor models and represents a druggable molecular vulnerability with potential therapeutic utility. PMID:20616035
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Roh, Mi Ryung; Kim, Jung Min; Lee, Sang Hee; Jang, Hong Sun; Park, Kyu Hyun; Chung, Kee Yang; Rha, Sun Young
2015-09-01
Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2 μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2 μmol/L vemurafenib for 24 h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2 μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion. © 2015 Japanese Dermatological Association.
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Zhou, Zhiping; Ho, Sharon L; Singh, Ranjodh; Pisapia, David J; Souweidane, Mark M
2015-04-01
Diffuse intrinsic pontine gliomas (DIPGs) are inoperable and lethal high-grade gliomas lacking definitive therapy. Platelet-derived growth factor receptor (PDGFR) and its downstream signaling molecules are the most commonly overexpressed oncogenes in DIPG. This study tested the effective concentration of PDGFR pathway inhibitors in cell culture and then toxicity of these small-molecule kinase inhibitors delivered to the mouse brainstem via convection-enhanced delivery (CED) for potential clinical application. Effective concentrations of small-molecule kinase inhibitors were first established in cell culture from a mouse brainstem glioma model. Sixteen mice underwent CED, a local drug delivery technique, of saline or of single and multidrug combinations of dasatinib (2 M), everolimus (20 M), and perifosine (0.63 mM) in the pons. Animals were kept alive for 3 days following the completion of infusion. No animals displayed any immediate or delayed neurological deficits postoperatively. Histological analysis revealed edema, microgliosis, acute inflammation, and/or axonal injury in the experimental animals consistent with mild acute drug toxicity. Brainstem CED of small-molecule kinase inhibitors in the mouse did not cause serious acute toxicities. Future studies will be necessary to evaluate longer-term safety to prepare for potential clinical application.
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Alekseev, Oleg; Limonnik, Vladimir; Donovan, Kelly; Azizkhan-Clifford, Jane
2015-01-01
Herpes simplex virus (HSV) type I keratitis remains a leading cause of corneal morbidity, despite the availability of effective antiviral drugs. Improved understanding of virus-host interactions at the level of the host DNA damage response (DDR), a known factor in the development of HSV-1 keratitis, may shed light on potential new therapeutic targets. This report examines the role of checkpoint kinase 2 (Chk2), a DDR mediator protein, in corneal epithelial HSV-1 infection. A small-molecule inhibitor of Chk2 (Chk2 inhibitor II) was applied to HSV-1-infected cultured human corneal epithelial cells (hTCEpi and HCE) as well as to explanted and organotypically cultured human and rabbit corneas. Infection levels were assessed by plaque assay and real-time PCR. RNAi-mediated depletion of Chk2 was performed to confirm the effect of the inhibitor. Inhibition of the Chk2 kinase activity greatly suppresses the cytopathic effect, genome replication and infectious progeny production in vitro and ex vivo. This report demonstrates the critical role of Chk2 kinase in the establishment of HSV-1 corneal epithelial infection. These data contribute to our understanding of herpesvirus-host interactions and underscore the significance of DDR activation in HSV-1 keratitis.
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Liu, Mao-Hua; Chen, Shi-Bing; Yu, Juan; Liu, Cheng-Jun; Zhang, Xiao-Jing
2017-08-01
The TAM receptor tyrosine kinase family member Mer has been recognized as an attractive therapeutic target for pediatric leukemia. Beside Mer the family contains other two kinases, namely, Tyro3 and Axl, which are highly homologues with Mer and thus most existing small-molecule inhibitors show moderate or high promiscuity across the three kinases. Here, the structural basis and energetic property of selective binding of small-molecule inhibitors to the three kinases were investigated at molecular level. It is found that the selectivity is primarily determined by the size, shape and configuration of kinase's ATP-binding site; the Mer and Axl possess a small, closed active pocket as compared to the bulky, open pocket of Tyro3. The location and conformation of active-site residues of Mer and Axl are highly consistent, suggesting that small-molecule inhibitors generally have a low Mer-over-Axl selectivity and a high Mer-over-Tyro3 selectivity. We demonstrated that the difference in ATP binding potency to the three kinases is also responsible for inhibitor selectivity. We also found that the long-range interactions and allosteric effect arising from rest of the kinase's active site can indirectly influence inhibitor binding and selectivity. Copyright © 2017 Elsevier Inc. All rights reserved.
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Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C-gamma1.
Husain, Shahid; Jafri, Farahdiba
2002-10-11
In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.
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Immunoprecipitation of PDE2 phosphorylated and inactivated by an associated protein kinase.
Bentley, J Kelley
2005-01-01
A PDE2A2-associated protein kinase phosphorylates PDE2A2 in vivo and in vitro to inhibit its catalytic activity. Rat brain PDE2A2 may be solubilized using nona (ethylene glycol) mono dodecyl ether (Lubrol 12A9). PDE2A2 exists in a complex with a protein kinase regulating its activity in an adenosine triphosphate-dependent manner. When native or recombinant PDE2 is immunoprecipitated from PC12 cells using an antibody to the amino terminus in a buffer containing Lubrol 12A9, protease inhibitors, and phosphatase inhibitors, a coimmunoprecipitating nerve growth factor-stimulated protein kinase acts to phosphorylate it. PDE2A2 phosphoryla-tion occurs optimally at pH 6.5 in a sodium 2-(4-morpholino)-ethane sulfonate buffer with 5 mM MgCl2 and 1 mM Na3VO4. I describe protocols for producing an antibody to an amino-terminal bacterial fusion protein encoding amino acids 1-251 of PDE2A2 as well as the use of this antibody in immunoprecipitating a PDE2: tyrosine protein-kinase complex from rat brain or PC12 cells.
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Haan, Claude; Behrmann, Iris; Haan, Serge
2010-01-01
Abstract Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. PMID:20132407
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Structural Biology Insight for the Design of Sub-type Selective Aurora Kinase Inhibitors.
Sarvagalla, Sailu; Coumar, Mohane Selvaraj
2015-01-01
Aurora kinase A, B and C, are key regulators of mitosis and are over expressed in many of the human cancers, making them an ideal drug target for cancer chemotherapy. Currently, over a dozen of Aurora kinase inhibitors are in various phases of clinical development. The majority of the inhibitors (VX-680/MK-0457, PHA-739358, CYC116, SNS-314, AMG 900, AT-9283, SCH- 1473759, ABT-348, PF-03814735, R-763/AS-703569, KW-2449 and TAK-901) are pan-selective (isoform non-selective) and few are Aurora A (MLN8054, MLN8237, VX-689/MK5108 and ENMD 2076) and Aurora B (AZD1152 and GSK1070916) sub-type selective. Despite the intensive research efforts in the past decade, no Aurora kinase inhibitor has reached the market. Recent evidence suggests that the sub-type selective Aurora kinase A inhibitor could possess advantages over pan-selective Aurora inhibitors, by avoiding Aurora B mediated neutropenia. However, sub-type selective Aurora kinase A inhibitor design is very challenging due to the similarity in the active site among the isoforms. Structural biology and computational aspects pertaining to the design of Aurora kinase inhibitors were analyzed and found that a possible means to develop sub-type selective inhibitor is by targeting Aurora A specific residues (Leu215, Thr217 and Arg220) or Aurora B specific residues (Arg159, Glu161 and Lys164), near the solvent exposed region of the protein. Particularly, a useful strategy for the design of sub-type selective Aurora A inhibitor could be by targeting Thr217 residue as in the case of MLN8054. Further preclinical and clinical studies with the sub-type selective Aurora inhibitors could help bring them to the market for the treatment of cancer.
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Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.
2014-01-01
The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440
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Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László
2009-01-01
There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.
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A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma.
Pillai, Prajit; Desai, Shraddha; Patel, Rekha; Sajan, Mini; Farese, Robert; Ostrov, David; Acevedo-Duncan, Mildred
2011-05-01
Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma. Published by Elsevier Ltd.
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Renault, Ilana Zalcberg; Scholl, Vanesa; Hassan, Rocio; Capelleti, Paola; de Lima, Marcos; Cortes, Jorge
2011-01-01
Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging. PMID:23049363
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Stern, Bodo; Nurse, Paul
1998-01-01
The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1 arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1 arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p. PMID:9614176
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Activating MAPK1 (ERK2) mutation in an aggressive case of disseminated juvenile xanthogranuloma
Chakraborty, Rikhia; Hampton, Oliver A.; Abhyankar, Harshal; Zinn, Daniel J.; Grimes, Amanda; Skull, Brooks; Eckstein, Olive; Mahmood, Nadia; Wheeler, David A.; Lopez-Terrada, Dolores; Peters, Tricia L.; Hicks, John M.; Elghetany, Tarek; Krance, Robert; Poulikakos, Poulikos I.; Merad, Miriam; McClain, Kenneth L.; Allen, Carl E.; Parsons, Donald W.
2017-01-01
Juvenile xanthogranuloma (JXG) is a rare histiocytic disorder that is usually benign and self-limiting. We present a case of atypical, aggressive JXG harboring a novel mitogen-activated protein kinase (MAPK) pathway mutation in the MAPK1 gene, which encodes mitogen-activated protein kinase 1 or extracellular signal-regulated 2 (ERK2). Our analysis revealed that the mutation results in constitutive ERK activation that is resistant to BRAF or MEK inhibitors but susceptible to an ERK inhibitor. These data highlight the importance of identifying specific MAPK pathway alterations as part of the diagnostic workup for patients with histiocytic disorders rather than initiating empiric treatment with MEK inhibitors. PMID:28512266
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Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries
Ghisdal, Philippe; Vandenberg, Greet; Morel, Nicole
2003-01-01
The present study was aimed at investigating whether, besides its pivotal role in Ca2+-independent contraction of smooth muscle, Rho-kinase is involved in the mechanisms underlying the Ca2+ signal activated by noradrenaline in arteries. In rat aorta and mesenteric artery, the Rho-kinase inhibitor Y-27632 (10 μM) completely relaxed the contraction evoked by noradrenaline (1 μM) and simultaneously inhibited the Ca2+ signal by 54 ± 1 % (mesenteric artery) and 71 ± 15 % (aorta), and the cell membrane depolarisation by 56 ± 11 % (mesenteric artery). A similar effect was observed in arteries contracted by AlF4−, while in KCl-contracted arteries, Y-27632 decreased tension without changing cytosolic Ca2+. The same effects were observed with another inhibitor of Rho-kinase (HA1077) but not with an inhibitor of protein kinase C (Ro-31–8220). Effects of Y-27632 were not prevented by incubating the artery in 25 mM KCl, with K+ channel blockers or with the Ca2+ channel blocker nimodipine. Y-27632 did not affect either the increase in the production of inositol phosphates activated by noradrenaline, or the release of Ca2+ from non-mitochondrial stores evoked by InsP3 in permeabilised aortic cells, or the Ca2+ signals evoked by thapsigargin or caffeine. The capacitative Ca2+ entry activated by thapsigargin was not impaired by Y-27632, but the entry of Ba2+ activated by noradrenaline in the presence of nimodipine was blocked by 10 μM Y-27632. These results indicate that Rho-kinase is involved in noradrenaline activation of a Ca2+ entry distinct from voltage- or store-operated channels in rat arteries. PMID:12853654
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.
2014-10-02
Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction ofmore » phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.« less
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Rho kinase inhibitors: a patent review (2012 - 2013).
Feng, Yangbo; LoGrasso, Philip V
2014-03-01
The Rho kinase/ROCK is critical in vital signal transduction pathways central to many essential cellular activities. Since ROCK possess multiple substrates, modulation of ROCK activity is useful for treatment of many diseases. Significant progress has been made in the development of ROCK inhibitors over the past two years (Jan 2012 to Aug 2013). Patent search in this review was based on FPO IP Research and Communities and Espacenet Patent Search. In this review, patent applications will be classified into four groups for discussions. The grouping is mainly based on structures or scaffolds (groups 1 and 2) and biological functions of ROCK inhibitors (groups 3 and 4). These four groups are i) ROCK inhibitors based on classical structural elements for ROCK inhibition; ii) ROCK inhibitors based on new scaffolds; iii) bis-functional ROCK inhibitors; and iv) novel applications of ROCK inhibitors. Although currently only one ROCK inhibitor (fasudil) is used as a drug, more drugs based on ROCK inhibition are expected to be advanced into market in the near future. Several directions should be considered for future development of ROCK inhibitors, such as soft ROCK inhibitors, bis-functional ROCK inhibitors, ROCK2 isoform-selective inhibitors, and ROCK inhibitors as antiproliferation agents.
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2015-04-01
recently decoded a major conserved route that mTORC1 uses to control autophagy. These studies demonstrate that mTORC1 inactivates another kinase complex...inhibition, and 2) to further explore use of novel small molecule inhibitors of ULK1 to synergize with mTOR inhibitors to induce cell death. 15. SUBJECT...others have recently decoded a major conserved route that mTORC1 uses to control autophagy. These studies demonstrate that mTORC1 inactivates another
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Sehon, Clark A; Wang, Gren Z; Viet, Andrew Q; Goodman, Krista B; Dowdell, Sarah E; Elkins, Patricia A; Semus, Simon F; Evans, Christopher; Jolivette, Larry J; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Wright, Lois L; Smith, Gary K; Behm, David J; Bentley, Ross; Doe, Christopher P; Hu, Erding; Lee, Dennis
2008-11-13
Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.
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A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.
Kumar, Priyadarsini; Walsh, Donal A
2002-03-15
We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.
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Zhang, Chao; Wu, Xihan; Zhang, Meifang; Zhu, Liangcheng; Zhao, Rong; Xu, Danqing; Lin, Zhaohu; Liang, Chungen; Chen, Taiping; Chen, Li; Ren, Yi; Zhang, Joe; Qin, Ning; Zhang, Xiongwen
2013-01-01
Protein kinases play important roles in tumor development and progression. Lots of kinase inhibitors have entered into market and show promising clinical benefits. Here we report the discovery of a novel small molecule, well-tolerated, orally active kinase inhibitor, R1498, majorly targeting both angiogenic and mitotic pathways for the treatment of hepatocellular carcinoma (HCC) and gastric cancer (GC). A series of biochemical and cell-based assays indicated that the target kinase cluster of R1498 included Aurora kinases and VEGFR2 et al. R1498 showed moderate in vitro growth inhibition on a panel of tumor cells with IC50 of micromole range. The in vivo anti-tumor efficacy of R1498 was evaluated on a panel of GC and HCC xenografts in a parallel comparison with another multikinase inhibitor sorafenib. R1498 demonstrated superior efficacy and toxicity profile over sorafenib in all test models with >80% tumor growth inhibition and tumor regression in some xenogratfts. The therapeutic potential of R1498 was also highlighted by its efficacy on three human GC primary tumor derived xenograft models with 10-30% tumor regression rate. R1498 was shown to actively inhibit the Aurora A activity in vivo, and decrease the vascularization in tumors. Furthermore, R1498 presented good in vivo exposure and therapeutic window in the pharmacokinetic and dose range finding studies. Theses evidences indicate that R1498 is a potent, well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic and antiproliferative profile, and provide strong confidence for further development for HCC and GC therapy.
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Zhang, Chao; Wu, Xihan; Zhang, Meifang; Zhu, Liangcheng; Zhao, Rong; Xu, Danqing; Lin, Zhaohu; Liang, Chungen; Chen, Taiping; Chen, Li; Ren, Yi; Zhang, Joe; Qin, Ning; Zhang, Xiongwen
2013-01-01
Protein kinases play important roles in tumor development and progression. Lots of kinase inhibitors have entered into market and show promising clinical benefits. Here we report the discovery of a novel small molecule, well-tolerated, orally active kinase inhibitor, R1498, majorly targeting both angiogenic and mitotic pathways for the treatment of hepatocellular carcinoma (HCC) and gastric cancer (GC). A series of biochemical and cell-based assays indicated that the target kinase cluster of R1498 included Aurora kinases and VEGFR2 et al. R1498 showed moderate in vitro growth inhibition on a panel of tumor cells with IC50 of micromole range. The in vivo anti-tumor efficacy of R1498 was evaluated on a panel of GC and HCC xenografts in a parallel comparison with another multikinase inhibitor sorafenib. R1498 demonstrated superior efficacy and toxicity profile over sorafenib in all test models with >80% tumor growth inhibition and tumor regression in some xenogratfts. The therapeutic potential of R1498 was also highlighted by its efficacy on three human GC primary tumor derived xenograft models with 10–30% tumor regression rate. R1498 was shown to actively inhibit the Aurora A activity in vivo, and decrease the vascularization in tumors. Furthermore, R1498 presented good in vivo exposure and therapeutic window in the pharmacokinetic and dose range finding studies. Theses evidences indicate that R1498 is a potent, well-tolerated, orally active multitarget kinase inhibitor with a unique antiangiogenic and antiproliferative profile, and provide strong confidence for further development for HCC and GC therapy. PMID:23755206
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Semiconductor technology in protein kinase research and drug discovery: sensing a revolution.
Bhalla, Nikhil; Di Lorenzo, Mirella; Estrela, Pedro; Pula, Giordano
2017-02-01
Since the discovery of protein kinase activity in 1954, close to 600 kinases have been discovered that have crucial roles in cell physiology. In several pathological conditions, aberrant protein kinase activity leads to abnormal cell and tissue physiology. Therefore, protein kinase inhibitors are investigated as potential treatments for several diseases, including dementia, diabetes, cancer and autoimmune and cardiovascular disease. Modern semiconductor technology has recently been applied to accelerate the discovery of novel protein kinase inhibitors that could become the standard-of-care drugs of tomorrow. Here, we describe current techniques and novel applications of semiconductor technologies in protein kinase inhibitor drug discovery. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Inactivation of Mirk/Dyrk1b Kinase Targets Quiescent Pancreatic Cancer Cells *
Ewton, Daina Z.; Hu, Jing; Vilenchik, Maria; Deng, Xiaobing; Luk, Kin-chun; Polonskaia, Ann; Hoffman, Ann F.; Zipf, Karen; Boylan, John F.; Friedman, Eileen A.
2011-01-01
A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they re-enter the cell cycle. Earlier studies demonstrated that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells, that Mirk uses several mechanisms to block cell cycling, and that Mirk increases expression of antioxidant genes which lower ROS levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1 and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In prior studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. PMID:21878655
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Jobson, Andrew G; Cardellina, John H; Scudiero, Dominic; Kondapaka, Sudhir; Zhang, Hongliang; Kim, Hijoo; Shoemaker, Robert; Pommier, Yves
2007-10-01
Chk2 is a protein kinase involved in the ATM-dependent checkpoint pathway (http://discover.nci.nih.gov/mim). This pathway is activated by genomic instability and DNA damage and results in either cell cycle arrest, to allow DNA repair to occur, or cell death (apoptosis). Chk2 is activated by ATM-mediated phosphorylation and autophosphorylation and in turn phosphorylates its downstream targets (Cdc25A, Cdc25C, BRCA1, p53, Hdmx, E2F1, PP2A, and PML). Inhibition of Chk2 has been proposed to sensitize p53-deficient cells as well as protect normal tissue after exposure to DNA-damaging agents. We have developed a drug-screening program for specific Chk2 inhibitors using a fluorescence polarization assay, immobilized metal ion affinity-based fluorescence polarization (IMAP). This assay detects the degree of phosphorylation of a fluorescently linked substrate by Chk2. From a screen of over 100,000 compounds from the NCI Developmental Therapeutics Program, we identified a bis-guanylhydrazone [4,4'-diacetyldiphenylureabis(guanylhydrazone); NSC 109555] as a lead compound. In vitro data show the specific inhibition of Chk2 kinase activity by NSC 109555 using in vitro kinase assays and kinase-profiling experiments. NSC 109555 was shown to be a competitive inhibitor of Chk2 with respect to ATP, which was supported by docking of NSC 109555 into the ATP binding pocket of the Chk2 catalytic domain. The potency of NSC 109555 was comparable with that of other known Chk2 inhibitors, such as debromohymenialdisine and 2-arylbenzimidazole. These data define a novel chemotype for the development of potent and selective inhibitors of Chk2. This class of drugs may ultimately be useful in combination with current DNA-damaging agents used in the clinic.
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Jia, Zhanfeng; Jia, Yueqin; Liu, Boyi; Zhao, Zhiying; Jia, Qingzhong; Liang, Huiling; Zhang, Hailin
2008-08-01
Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.
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Roskoski, Robert
2016-01-01
Because dysregulation and mutations of protein kinases play causal roles in human disease, this family of enzymes has become one of the most important drug targets over the past two decades. The X-ray crystal structures of 21 of the 27 FDA-approved small molecule inhibitors bound to their target protein kinases are depicted in this paper. The structure of the enzyme-bound antagonist complex is used in the classification of these inhibitors. Type I inhibitors bind to the active protein kinase conformation (DFG-Asp in, αC-helix in). Type I½ inhibitors bind to a DFG-Asp in inactive conformation while Type II inhibitors bind to a DFG-Asp out inactive conformation. Type I, I½, and type II inhibitors occupy part of the adenine binding pocket and form hydrogen bonds with the hinge region connecting the small and large lobes of the enzyme. Type III inhibitors bind next to the ATP-binding pocket and type IV inhibitors do not bind to the ATP or peptide substrate binding sites. Type III and IV inhibitors are allosteric in nature. Type V inhibitors bind to two different regions of the protein kinase domain and are therefore bivalent inhibitors. The type I-V inhibitors are reversible. In contrast, type VI inhibitors bind covalently to their target enzyme. Type I, I½, and II inhibitors are divided into A and B subtypes. The type A inhibitors bind in the front cleft, the back cleft, and near the gatekeeper residue, all of which occur within the region separating the small and large lobes of the protein kinase. The type B inhibitors bind in the front cleft and gate area but do not extend into the back cleft. An analysis of the limited available data indicates that type A inhibitors have a long residence time (minutes to hours) while the type B inhibitors have a short residence time (seconds to minutes). The catalytic spine includes residues from the small and large lobes and interacts with the adenine ring of ATP. Nearly all of the approved protein kinase inhibitors occupy the adenine-binding pocket; thus it is not surprising that these inhibitors interact with nearby catalytic spine (CS) residues. Moreover, a significant number of approved drugs also interact with regulatory spine (RS) residues. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Hari, Sanjay B.; Perera, B. Gayani K.; Ranjitkar, Pratistha; Seeliger, Markus A.; Maly, Dustin J.
2013-01-01
Over the last decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely-related tyrosine kinases, like Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related kinases. PMID:24106839
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Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs inhibitors.
Liu, Yang; Peng, Xia; Guan, Xiaocong; Lu, Dong; Xi, Yong; Jin, Shiyu; Chen, Hui; Zeng, Limin; Ai, Jing; Geng, Meiyu; Hu, Youhong
2017-01-27
FGF receptors (FGFRs) are tyrosine kinases that are overexpressed in diverse tumors by genetic alterations such as gene amplifications, somatic mutations and translocations. Owing to this characteristic, FGFRs are attractive targets for cancer treatment. It has been demonstrated that most multi-targeted, ATP competitive tyrosine kinase inhibitors are active against FGFRs as well as other kinases. The design of new and more selective inhibitors of FGFRs, which might be reduced off-target and side effects, is a difficult yet significant challenge. The results of the current investigation, show that novel Ponatinib analogues are highly active as FGFR inhibitors and that they possess reduced kinase insert domain receptor (KDR) activities. Observations made in a structure and activity relationship (SAR) investigation led to the development of a promising, orally available lead compound 4, which displays a 50-100 fold in vitro selectivity for inhibition of FGFR1-3 over KDR. In addition, biological evaluation of compound 4 showed that it displays significant antitumor activities in FGFR1-amplificated H1581 and FGFR2-amplificated SNU-16 xenograft models. Copyright © 2016 Elsevier Masson SAS. All rights reserved.
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Selective JAK inhibitors in development for rheumatoid arthritis.
Norman, Peter
2014-08-01
The JAK kinases are a family of four tyrosine receptor kinases that play a pivotal role in cytokine receptor signalling pathways via their interaction with signal transducers and activators of transcription proteins. Selective inhibitors of JAK kinases are viewed as of considerable potential as disease-modifying anti-inflammatory drugs for the treatment of rheumatoid arthritis. This article provides a review of the clinical development and available clinical results for those JAK inhibitors currently under investigation. Phase II data for four JAK inhibitors (baricitinib, decernotinib, filgotinib and INCB-039110) are contrasted with that reported for the recently approved JAK inhibitor tofacitinib. The preclinical data on these, in addition to peficitinib, ABT-494, INCB-047986 and AC-410 are also discussed, as are some of the inhibitors in preclinical development. JAK inhibitors are effective in the treatment of rheumatoid arthritis as evidenced by several inhibitors enabling the majority of treated patients to achieve ACR20 responses, with baricitinib and INCB-039110 both effective when administered once daily. JAK inhibitors differ in isoform specificity profiles, with good efficacy achievable by selective inhibition of either JAK1 (filgotinib or INCB-039110) or JAK3 (decernotinib). It remains to be seen what selectivity provides the optimal side-effect profile and to what extent inhibition of JAK2 should be avoided.
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Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*
Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.
2015-01-01
The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479
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Functional kinomics identifies candidate therapeutic targets in head and neck cancer
Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M.; Gurley, Kay E.; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G.; Margolin, Adam A.; Grandori, Carla; Kemp, Christopher J.; Méndez, Eduardo
2014-01-01
Purpose To identify novel therapeutic drug targets for p53 mutant head and neck squamous cell carcinoma (HNSCC). Experimental Design RNAi kinome viability screens were performed on HNSCC cells including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19Arf. Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was utilized to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets utilizing multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition utilizing a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Results Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2/M cell cycle checkpoint, SFK, PI3K and FAK pathways. RNAi mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53 mutant HNSCC xenograft model. Conclusions WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. PMID:25125259
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Functional kinomics identifies candidate therapeutic targets in head and neck cancer.
Moser, Russell; Xu, Chang; Kao, Michael; Annis, James; Lerma, Luisa Angelica; Schaupp, Christopher M; Gurley, Kay E; Jang, In Sock; Biktasova, Asel; Yarbrough, Wendell G; Margolin, Adam A; Grandori, Carla; Kemp, Christopher J; Méndez, Eduardo
2014-08-15
To identify novel therapeutic drug targets for p53-mutant head and neck squamous cell carcinoma (HNSCC). RNAi kinome viability screens were performed on HNSCC cells, including autologous pairs from primary tumor and recurrent/metastatic lesions, and in parallel on murine squamous cell carcinoma (MSCC) cells derived from tumors of inbred mice bearing germline mutations in Trp53, and p53 regulatory genes: Atm, Prkdc, and p19(Arf). Cross-species analysis of cell lines stratified by p53 mutational status and metastatic phenotype was used to select 38 kinase targets. Both primary and secondary RNAi validation assays were performed on additional HNSCC cell lines to credential these kinase targets using multiple phenotypic endpoints. Kinase targets were also examined via chemical inhibition using a panel of kinase inhibitors. A preclinical study was conducted on the WEE1 kinase inhibitor, MK-1775. Our functional kinomics approach identified novel survival kinases in HNSCC involved in G2-M cell-cycle checkpoint, SFK, PI3K, and FAK pathways. RNAi-mediated knockdown and chemical inhibition of the WEE1 kinase with a specific inhibitor, MK-1775, had a significant effect on both viability and apoptosis. Sensitivity to the MK-1775 kinase inhibitor is in part determined by p53 mutational status, and due to unscheduled mitotic entry. MK-1775 displays single-agent activity and potentiates the efficacy of cisplatin in a p53-mutant HNSCC xenograft model. WEE1 kinase is a potential therapeutic drug target for HNSCC. This study supports the application of a functional kinomics strategy to identify novel therapeutic targets for cancer. ©2014 American Association for Cancer Research.
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Hong, S K; Matsumoto, A; Horinouchi, S; Beppu, T
1993-01-01
In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Muchir, Antoine, E-mail: a.muchir@institut-myologie.org; Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY; Wu, Wei
Highlights: • Both ACE and MEK1/2 inhibition are beneficial on cardiac function in Lmna cardiomyopathy. • MEK1/2 inhibitor has beneficial effects beyond ACE inhibition for Lmna cardiomyopathy. • These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor. - Abstract: Background: Mutations in the LMNA gene encoding A-type nuclear lamins can cause dilated cardiomyopathy with or without skeletal muscular dystrophy. Previous studies have shown abnormally increased extracellular signal-regulated kinase 1/2 activity in hearts of Lmna{sup H222P/H222P} mice, a small animal model. Inhibition of this abnormal signaling activity with a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitormore » has beneficial effects on heart function and survival in these mice. However, such treatment has not been examined relative to any standard of care intervention for dilated cardiomyopathy or heart failure. We therefore examined the effects of an angiotensin II converting enzyme (ACE) inhibitor on left ventricular function in Lmna{sup H222P/H222P} mice and assessed if adding a MEK1/2 inhibitor would provide added benefit. Methods: Male Lmna{sup H222P/H222P} mice were treated with the ACE inhibitor benazepril, the MEK1/2 inhibitor selumetinib or both. Transthoracic echocardiography was used to measure left ventricular diameters and fractional shortening was calculated. Results: Treatment of Lmna{sup H222P/H222P} mice with either benazepril or selumetinib started at 8 weeks of age, before the onset of detectable left ventricular dysfunction, lead to statistically significantly increased fractional shortening compared to placebo at 16 weeks of age. There was a trend towards a great value for fractional shortening in the selumetinib-treated mice. When treatment was started at 16 weeks of age, after the onset of left ventricular dysfunction, the addition of selumetinib treatment to benazepril lead to a statistically significant increase in left ventricular fractional shortening at 20 weeks of age. Conclusions: Both ACE inhibition and MEK1/2 inhibition have beneficial effects on left ventricular function in Lmna{sup H222P/H222P} mice and both drugs together have a synergistic benefit when initiated after the onset of left ventricular dysfunction. These results provide further preclinical rationale for a clinical trial of a MEK1/2 inhibitor in addition to standard of care in patients with dilated cardiomyopathy caused by LMNA mutations.« less
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Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy*
Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja
2011-01-01
Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors. PMID:21930714
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Janus kinase 2 inhibitors in myeloproliferative disorders.
Lucia, Eugenio; Recchia, Anna Grazia; Gentile, Massimo; Bossio, Sabrina; Vigna, Ernesto; Mazzone, Carla; Madeo, Antonio; Morabito, Lucio; Gigliotti, Vincenzo; De Stefano, Laura; Caruso, Nadia; Servillo, Pasquale; Franzese, Stefania; Bisconte, Maria Grazia; Gentile, Carlo; Morabito, Fortunato
2011-01-01
JAK2 is an obligatory kinase for the proliferation and differentiation of erythroid cells and megakaryocytes thus representing a relevant therapeutic target for agents that specifically inhibit its activity particularly in myeloproliferative disorders (MPD) harboring JAK2(V617F) mutations. We discuss the physiopathology of the JAK2 signaling pathway and review clinical trials of JAK2 inhibitors for the treatment of MPD using papers and meeting abstracts published up to September 2010. This review helps in understanding the potential role of JAK2 inhibitors in MPD clinical trials and provides a comprehensive review regarding their efficacy and safety in these disorders. JAK2 inhibitors may prove to be useful only for suppressing disease manifestations. However, unlike drugs such as IFN which are capable of eliminating the malignant clone, JAK2 inhibitors are unable to eradicate the disease. In fact, results to date indicate that although these inhibitors reduce splenomegaly and alleviate constitutional symptoms irrespective of JAK2 mutational status, most have only a modest impact on the JAK2(V617F) allele burden. Considering the relevant risk of serious complications in patients undergoing splenectomy, these drugs could find a suitable indication in patients with myelofibrosis awaiting bone marrow transplantation.
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Shi, Leilei; Zhou, Jianfeng; Wu, Jifeng; Shen, Yuemao; Li, Xun
2016-01-01
Tumor angiogenesis has always been a major gap for effective cancer therapy. Interruption of aberrant angiogenesis by specific inhibitors targeting receptor tyrosine kinases (RTKs) has been of great interests to medicinal chemists. Among the factors that are involved in tumor angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR-2) is validated as the most closely related factor which can drive angiogenesis through binding with its natural ligand VEGF. The well-validated VEGF-driven VEGFR-2 signaling pathway can stimulate many endothelial responses, including increasing vessel permeability and enhancing endothelial cell proliferation, migration and differentiation. Consequently, circumventing angiogenesis by VEGFR-2 inhibitors represents a promising strategy for counteracting various VEGFR-2-mediated disorders as well as drug resistance. Over the past decades, a considerable number of novel small molecular VEGFR-2 inhibitors have been exploited with diverse chemical scaffolds. Especially, recent frequently launched inhibitors have declared their research values and therapeutic potentials in oncology. Still, the antiangiogenesis based treatment remains an ongoing challenge. In this review, a comprehensive retrospective of newly emerged VEGFR-2 inhibitors have been summarized, with the emphasis on the structure-activity relationship (SAR) investigation, and also binding patterns of representative inhibitors with biotargets. On the basis of all of this information, varied strategies for developing potent VEGFR-2 inhibitors and the future prospect of the clinical application of antiangiogenic inhibitors are discussed hereby.
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Kovacs, Krisztina; Toth, Ambrus; Deres, Peter; Kalai, Tamas; Hideg, Kalman; Gallyas, Ferenc; Sumegi, Balazs
2006-02-14
Poly(ADP-ribose) polymerase (PARP) inhibitors protect hearts from ischemia-reperfusion (IR)-induced damages by limiting nicotinamide adenine dinucleotide (NAD+) and ATP depletion, and by other, not yet elucidated mechanisms. Our preliminary data suggested that PARP catalyzed ADP-ribosylations may affect signaling pathways in cardiomyocytes. To clarify this possibility, we studied the effect of a well-characterized (4-hydroxyquinazoline) and a novel (carboxaminobenzimidazol-derivative) PARP inhibitor on the activation of phosphatidylinositol-3-kinase (PI3-kinase)/Akt pathway in Langendorff-perfused hearts. PARP inhibitors promoted the restoration of myocardial energy metabolism (assessed by 31P nuclear magnetic resonance spectroscopy) and cardiac function compared to untreated hearts. PARP inhibitors also attenuated the infarct size and reduced the IR-induced lipid peroxidation, protein oxidation and total peroxide concentration. Moreover, PARP inhibitors facilitated Akt phosphorylation and activation, as well as the phosphorylation of its downstream target glycogen synthase kinase-3beta (GSK-3beta) in normoxia and, more robustly, during IR. Blocking PI3-kinase by wortmannin or LY294002 reduced the PARP inhibitor-elicited robust Akt and GSK-3beta phosphorylation upon ischemia-reperfusion, and significantly diminished the recovery of ATP and creatine phosphate showing the importance of Akt activation in the recovery of energy metabolism. In addition, inhibition of PI3-kinase/Akt pathway decreased the protective effect of PARP inhibitors on infarct size and the recovery of heart functions. All these data suggest that contrary to the original view, which considered preservation of NAD+ and consequently ATP pools as the exclusive underlying mechanism for the cytoprotective effect of PARP inhibitors, the activation of PI3-kinase/Akt pathway and related processes are at least equally important in the cardioprotective effects of PARP inhibitors during ischemia-reperfusion.
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NASA Astrophysics Data System (ADS)
Kumar, Vikash; Khan, Saman; Gupta, Priyanka; Rastogi, Namrata; Mishra, Durga Prasad; Ahmed, Shakil; Siddiqi, Mohammad Imran
2014-12-01
Kinases are one of the major players in cancer development and progression. Serine threonine kinases such as human checkpoint kinase-1 (Chk1), Mek1 and cyclin-dependent kinases have been identified as promising targets for cancer treatment. Chk1 is an important kinase with vital role in cell cycle arrest and many potent inhibitors targeted to Chk1 have been reported and few are currently in clinical trials. Considering the emerging importance of Chk1 inhibitors in cancer treatment there is a need to widen the chemical space of Chk1 inhibitors. In this study, we are reporting an integrated in silico approach to identify novel competitive Chk1 inhibitors. A 4-features pharmacophore model was derived from a co-crystallized structure of known potent Chk1 inhibitor and subjected to screen Maybridge compound library. Hits obtained from the screening were docked into the Chk1 active site and filtered on the basis of docking score and the number of pharmacophoric features showing conserved interaction within the active site of Chk1. Further, five compounds from the top ranking hits were subjected to in vitro evaluation as Chk1 inhibitor. After the kinase assay, four compounds were found to be active against human Chk1 (IC50 range from 4.2 to 12.5 µM). Subsequent study using the cdc25-22 mutant yeast cells revealed that one of compound (SPB07479; IC50 = 4.24 µM) promoted the formation of multinucleated cells, therefore overriding the cell cycle checkpoint. Validation studies using normal and human cancer cell lines, indicated that SPB07479 significantly inhibited proliferation of cervical cancer cells as a single agent and chemosensitized glioma and pancreatic cancer cell lines to standard chemotherapy while sparing normal cells. Additionally SPB07479 did not show significant cytotoxicity in normal cells. In conclusion we report that SPB07479 appear promising for further development of Chk1 inhibitors. This study also highlights the role of conserved water molecules in the active site of Chk1 for the successful identification of novel inhibitors.
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Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K
2006-01-01
Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598
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Erdmann, Tabea; Klener, Pavel; Lynch, James T; Grau, Michael; Vočková, Petra; Molinsky, Jan; Tuskova, Diana; Hudson, Kevin; Polanska, Urszula M; Grondine, Michael; Mayo, Michele; Dai, Beiying; Pfeifer, Matthias; Erdmann, Kristian; Schwammbach, Daniela; Zapukhlyak, Myroslav; Staiger, Annette M; Ott, German; Berdel, Wolfgang E; Davies, Barry R; Cruzalegui, Francisco; Trneny, Marek; Lenz, Peter; Barry, Simon T; Lenz, Georg
2017-07-20
Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors. © 2017 by The American Society of Hematology.
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Judge, Russell A; Vasudevan, Anil; Scott, Victoria E; Simler, Gricelda H; Pratt, Steve D; Namovic, Marian T; Putman, C Brent; Aguirre, Ana; Stoll, Vincent S; Mamo, Mulugeta; Swann, Steven I; Cassar, Steven C; Faltynek, Connie R; Kage, Karen L; Boyce-Rustay, Janel M; Hobson, Adrian D
2018-03-16
We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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2013-01-01
PIK3CA is the most frequently mutated oncogene in human cancers. PIK3CA is phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha. It controls cell growth, proliferation, motility, survival, differentiation and intracellular trafficking. In most of human cancer alteration occurred frequently in the alpha isoform of phosphatidylinositol 3 kinase. PIK3CA mutations were most frequent in endometrial, ovarian, colorectal, breast, cervical, squamous cell cancer of the head and neck, chondroma, thyroid carcinoma and in cancer family syndrome. Inhibition of PI3K signaling can diminish cell proliferation, and in some circumstances, promote cell death. Consequently, components of this pathway present attractive targets for cancer therapeutics. A number of PI3K pathway inhibitors have been developed and used. PI3K inhibitors (both pan-PI3K and isoform-specific PI3K inhibitors), dual PI3K-mTOR inhibitors that are catalytic site inhibitors of the p110 isoforms and mTOR (the kinase component of both mTORC1 and mTORC2), mTOR catalytic site inhibitors, and AKT inhibitors are the most advanced in the clinic. They are approved for the treatment of several carcinomas. PMID:23768168
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Woo, Tae-Gyun; Yoon, Min-Ho; Hong, Shin-Deok; Choi, Jiyun; Ha, Nam-Chul; Sun, Hokeun; Park, Bum-Joon
2017-04-04
Hyper-activation of PAK1 (p21-activated kinase 1) is frequently observed in human cancer and speculated as a target of novel anti-tumor drug. In previous, we also showed that PAK1 is highly activated in the Smad4-deficient condition and suppresses PUMA (p53 upregulated modulator of apoptosis) through direct binding and phosphorylation. On the basis of this result, we have tried to find novel PAK1-PUMA binding inhibitors. Through ELISA-based blind chemical library screening, we isolated single compound, IPP-14 (IPP; Inhibitor of PAK1-PUMA), which selectively blocks the PAK1-PUMA binding and also suppresses cell proliferation via PUMA-dependent manner. Indeed, in PUMA-deficient cells, this chemical did not show anti-proliferating effect. This chemical possessed very strong PAK1 inhibition activity that it suppressed BAD (Bcl-2-asoociated death promoter) phosphorylation and meta-phase arrest via Aurora kinase inactivation in lower concentration than that of previous PAK1 kinase, FRAX486 and AG879. Moreover, our chemical obviously induced p21/WAF1/CIP1 (Cyclin-dependent kinase inhibitor 1A) expression by releasing from Bcl-2 (B-cell lymphoma-2) and by inhibition of AKT-mediated p21 suppression. Considering our result, IPP-14 and its derivatives would be possible candidates for PAK1 and p21 induction targeted anti-cancer drug.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Nishiguchi, Gisele A.; Rico, Alice; Tanner, Huw
RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [Aversa, Biaryl amide compounds as kinase inhibitors and their preparation. WO 2014151616, 2014], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in themore » medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.« less
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Targeting invadopodia-mediated breast cancer metastasis by using ABL kinase inhibitors
Meirson, Tomer; Genna, Alessandro; Lukic, Nikola; Makhnii, Tetiana; Alter, Joel; Sharma, Ved P.; Wang, Yarong; Samson, Abraham O.; Condeelis, John S.; Gil-Henn, Hava
2018-01-01
Metastatic dissemination of cancer cells from the primary tumor and their spread to distant sites in the body is the leading cause of mortality in breast cancer patients. While researchers have identified treatments that shrink or slow metastatic tumors, no treatment that permanently eradicates metastasis exists at present. Here, we show that the ABL kinase inhibitors imatinib, nilotinib, and GNF-5 impede invadopodium precursor formation and cortactin-phosphorylation dependent invadopodium maturation, leading to decreased actin polymerization in invadopodia, reduced extracellular matrix degradation, and impaired matrix proteolysis-dependent invasion. Using a mouse xenograft model we demonstrate that, while primary tumor size is not affected by ABL kinase inhibitors, the in vivo matrix metalloproteinase (MMP) activity, tumor cell invasion, and consequent spontaneous metastasis to lungs are significantly impaired in inhibitor-treated mice. Further proteogenomic analysis of breast cancer patient databases revealed co-expression of the Abl-related gene (Arg) and cortactin across all hormone- and human epidermal growth factor receptor 2 (HER2)-receptor status tumors, which correlates synergistically with distant metastasis and poor patient prognosis. Our findings establish a prognostic value for Arg and cortactin as predictors of metastatic dissemination and suggest that therapeutic inhibition of ABL kinases may be used for blocking breast cancer metastasis. PMID:29774130
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Botting, Carolyn; Lu, Xu; Triezenberg, Steven J
2016-01-27
Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xie, Peng; Streu, Craig; Qin, Jie
Substitution mutations in the BRAF serine/threonine kinase are found in a variety of human cancers. Such mutations occur in 70% of human malignant melanomas, and a single hyperactivating V600E mutation is found in the activation segment of the kinase domain and accounts for more than 90% of these mutations. Given this correlation, the molecular mechanism for BRAF regulation as well as oncogenic activation has attracted considerable interest, and activated forms of BRAF, such as BRAF{sup V600E}, have become attractive targets for small molecule inhibition. Here we report on the identification and subsequent optimization of a potent BRAF inhibitor, CS292, basedmore » on an organometallic kinase inhibitor scaffold. A cocrystal structure of CS292 in complex with the BRAF kinase domain reveals that CS292 binds to the ATP binding pocket of the kinase and is an ATP competitive inhibitor. The structure of the kinase-inhibitor complex also demonstrates that CS292 binds to BRAF in an active conformation and suggests a mechanism for regulation of BRAF by phosphorylation and BRAF{sup V600E} oncogene-induced activation. The structure of CS292 bound to the active form of the BRAF kinase also provides a novel scaffold for the design of BRAF{sup V600E} oncogene selective BRAF inhibitors for therapeutic application.« less
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Uitdehaag, Joost C M; de Man, Jos; Willemsen-Seegers, Nicole; Prinsen, Martine B W; Libouban, Marion A A; Sterrenburg, Jan Gerard; de Wit, Joeri J P; de Vetter, Judith R F; de Roos, Jeroen A D M; Buijsman, Rogier C; Zaman, Guido J R
2017-07-07
The protein kinase threonine tyrosine kinase (TTK; also known as Mps1) is a critical component of the spindle assembly checkpoint and a promising drug target for the treatment of aggressive cancers, such as triple negative breast cancer. While the first TTK inhibitors have entered clinical trials, little is known about how the inhibition of TTK with small-molecule compounds affects cellular activity. We studied the selective TTK inhibitor NTRC 0066-0, which was developed in our own laboratory, together with 11 TTK inhibitors developed by other companies, including Mps-BAY2b, BAY 1161909, BAY 1217389 (Bayer), TC-Mps1-12 (Shionogi), and MPI-0479605 (Myrexis). Parallel testing shows that the cellular activity of these TTK inhibitors correlates with their binding affinity to TTK and, more strongly, with target residence time. TTK inhibitors are therefore an example where target residence time determines activity in in vitro cellular assays. X-ray structures and thermal stability experiments reveal that the most potent compounds induce a shift of the glycine-rich loop as a result of binding to the catalytic lysine at position 553. This "lysine trap" disrupts the catalytic machinery. Based on these insights, we developed TTK inhibitors, based on a (5,6-dihydro)pyrimido[4,5-e]indolizine scaffold, with longer target residence times, which further exploit an allosteric pocket surrounding Lys553. Their binding mode is new for kinase inhibitors and can be classified as hybrid Type I/Type III. These inhibitors have very potent anti-proliferative activity that rivals classic cytotoxic therapy. Our findings will open up new avenues for more applications for TTK inhibitors in cancer treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Desplats, Paula; Patel, Pruthul; Kosberg, Kori; Mante, Michael; Patrick, Christina; Rockenstein, Edward; Fujita, Masayo; Hashimoto, Makoto; Masliah, Eliezer
2012-09-28
Parkinson's disease (PD) is a multifactorial disease where environmental factors act on genetically predisposed individuals. Although only 5% of PD manifestations are associated with specific mutations, majority of PD cases are of idiopathic origin, where environment plays a prominent role. Concurrent exposure to Paraquat (PQ) and Maneb (MB) in rural workers increases the risk for PD and exposure of adult mice to MB/PQ results in dopamine fiber loss and decreased locomotor activity. While PD is characterized by neuronal loss in the substantia nigra, we previously showed that accumulation of α-synuclein in the limbic system contributes to neurodegeneration by interfering with adult neurogenesis. We investigated the effect of pesticides on adult hippocampal neurogenesis in two transgenic models: Line 61, expressing the human wild type SNCA gene and Line LRRK2(G2019S), expressing the human LRRK2 gene with the mutation G2019S. Combined exposure to MB/PQ resulted in significant reduction of neuronal precursors and proliferating cells in non-transgenic animals, and this effect was increased in transgenic mice, in particular for Line 61, suggesting that α-synuclein accumulation and environmental toxins have a synergistic effect. We further investigated the transcription of 84 genes with direct function on neurogenesis. Overexpresion of α-synuclein resulted in the downregulation of 12% of target genes, most of which were functionally related to cell differentiation, while LRRK2 mutation had a minor impact on gene expression. MB/PQ also affected transcription in non-transgenic backgrounds, but when transgenic mice were exposed to the pesticides, profound alterations in gene expression affecting 27% of the studied targets were observed in both transgenic lines. Gene enrichment analysis showed that 1:3 of those genes were under the regulation of FoxF2 and FoxO3A, suggesting a primary role of these proteins in the response to genetic and environmental cues. We report that adult neurogenesis is highly susceptible to multiple "risk factors" for PD, including α-synuclein accumulation, LRRK2 G2019 mutation and exposure to environmental toxins. We identified specific groups of genes that are responsive to each stressor, while uncovering a novel function for Fox transcription factors in PD.
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Haiying, Gao; Mingjie, Han; Lingyu, Zhang; Qingxiang, Wang; Haisong, Wang; Bingxi, Zhang
2017-02-01
Extracellular signal-regulated kinase 1/2 (ERK1/2) has been implicated in learning and memory; however, whether intravenous anesthetics modulate ERK1/2 remains unknown. The aim of this study was to examine the effect of several intravenous anesthetics on the phosphorylation of ERK1/2 in the hippocampus of adult mice. Western blotting was used to examine cellular levels of phosphorylated and unphosphorylated ERK1/2 in mouse hippocampus slices, which were incubated with or without anesthetics including propofol, etomidate, ketamine and midazolam, a protein kinase C (PKC) activator or inhibitor, or phospholipase C (PLC) activator or inhibitor. Propofol, etomidate, ketamine and midazolam reduced phosphorylation of ERK1/2 in a time-dependent manner. Washing out propofol after 5 min increased ERK1/2 phosphorylation. The anesthetic-induced depression of ERK1/2 phosphorylation was blocked by 0.1 μM phorbol-12-myristate 13-acetate (an activator of PKC), 50 μM U73122 (an inhibitor of PLC). The anesthetic-induced depression of ERK1 phosphorylation was blocked by 1 mMN-methyl-d-aspartate (NMDA). Whereas 100 μM chelerythrine (an inhibitor of PKC) and 100 μM carbachol (an activator of PLC) and 20 μM PD-98059 (an inhibitor of MEK) had additive effects on propofol-induced inhibition of ERK1/2 phosphorylation. In contrast, 10 μM MK801 (a NMDA receptor antagonist) did not block anesthetic-induced inhibition of ERK1/2 phosphorylation. Intravenous anesthetics markedly decreased phosphorylation of ERK1/2 in mouse hippocampal slices, most likely via the NMDA receptor, and PLC- and PKC-dependent pathways. Thus, ERK1/2 represents a target for anesthetics in the brain. Copyright © 2016. Published by Elsevier Ltd.
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Skin problems and EGFR-tyrosine kinase inhibitor.
Kozuki, Toshiyuki
2016-04-01
Epidermal growth factor receptor inhibition is a good target for the treatment of lung, colon, pancreatic and head and neck cancers. Epidermal growth factor receptor-tyrosine kinase inhibitor was first approved for the treatment of advanced lung cancer in 2002. Epidermal growth factor receptor-tyrosine kinase inhibitor plays an essential role in the treatment of cancer, especially for patients harbouring epidermal growth factor receptor activating mutation. Hence, skin toxicity is the most concerning issue for the epidermal growth factor receptor-tyrosine kinase inhibitor treatment. Skin toxicity is bothersome and sometimes affects the quality of life and treatment compliance. Thus, it is important for physicians to understand the background and how to manage epidermal growth factor receptor-tyrosine kinase inhibitor-associated skin toxicity. Here, the author reviewed the mechanism and upfront preventive and reactive treatments for epidermal growth factor receptor inhibitor-associated skin toxicities. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Kim, Jung Nam; Kim, Hyun Jung; Kim, Iksung; Kim, Yun Tai; Kim, Byung Joo
2018-01-01
Zingerone, a major component found in ginger root, is clinically effective for the treatment of various diseases. Interstitial cells of Cajal (ICCs) are the pacemaker cells responsible for slow waves in the gastrointestinal (GI) tract. We investigated the effects of zingerone on the pacemaker potentials of ICCs to assess its mechanisms of action and its potential as a treatment for GI tract motility disorder. We isolated ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record the pacemaker potentials in cultured ICCs. Under the current clamping mode, zingerone inhibited pacemaker potentials of ICCs concentration-dependently. These effects were blocked not by capsazepine, a transient receptor potential vanilloid 1 (TRPV1) channel blocker, but by glibenclamide, a specific ATP-sensitive K+ channel blocker. Pretreatment with SQ-22536 (an adenylate cyclase inhibitor), LY294002 (a phosphoinositide 3-kinase inhibitor), and calphostin C (a protein kinase C (PKC) inhibitor) did not block the effects of zingerone on the pacemaker potentials relative to treatment with zingerone alone. However, zingerone-induced pacemaker potential inhibition was blocked by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor), KT5823 (a protein kinase G (PKG) inhibitor), and L-NAME (a non-selective nitric oxide synthase (NOS) inhibitor). In addition, zingerone stimulated cyclic guanosine monophosphate (cGMP) production in ICCs. Finally, pretreatment with PD98059 (a p42/44 mitogen-activated protein kinase (MAPK) inhibitor), SB203580 (a p38 MAPK inhibitor), and SP600125 (c-Jun N-terminal kinases (JNK)-specific inhibitor) blocked the zingerone-induced pacemaker potential inhibition. These results suggest that zingerone concentration-dependently inhibits pacemaker potentials of ICCs via NO/cGMP-dependent ATP-sensitive K+ channels through MAPK-dependent pathways. Taken together, this study shows that zingerone may have the potential for development as a GI regulation agent. © 2018 The Author(s). Published by S. Karger AG, Basel.
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Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen
2017-01-12
The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.
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Crowther, Gregory J.; Hillesland, Heidi K.; Keyloun, Katelyn R.; Reid, Molly C.; Lafuente-Monasterio, Maria Jose; Ghidelli-Disse, Sonja; Leonard, Stephen E.; He, Panqing; Jones, Jackson C.; Krahn, Mallory M.; Mo, Jack S.; Dasari, Kartheek S.; Fox, Anna M. W.; Boesche, Markus; El Bakkouri, Majida; Rivas, Kasey L.; Leroy, Didier; Hui, Raymond; Drewes, Gerard; Maly, Dustin J.; Van Voorhis, Wesley C.; Ojo, Kayode K.
2016-01-01
In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible. PMID:26934697
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Purine inhibitors of protein kinases, G proteins and polymerases
Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent
2004-10-12
The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.
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Lee, Wendy; Ortwine, Daniel F; Bergeron, Philippe; Lau, Kevin; Lin, Lichuan; Malek, Shiva; Nonomiya, Jim; Pei, Zhonghua; Robarge, Kirk D; Schmidt, Stephen; Sideris, Steve; Lyssikatos, Joseph P
2013-09-15
A series of N-7-methyl-imidazolopyrimidine inhibitors of the mTOR kinase have been designed and prepared, based on the hypothesis that the N-7-methyl substituent on imidazolopyrimidine would impart selectivity for mTOR over the related PI3Kα and δ kinases. The corresponding N-Me substituted pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines also show potent mTOR inhibition with selectivity toward both PI3α and δ kinases. The most potent compound synthesized is pyrazolo[4,3-d]pyrimidine 21c. Compound 21c shows a Ki of 2 nM against mTOR inhibition, remarkable selectivity (>2900×) over PI3 kinases, and excellent potency in cell-based assays. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Kim, Okseon; Jeong, Yujeong; Lee, Hyunseung; Hong, Sun-Sun; Hong, Sungwoo
2011-04-14
Phosphatidylinositol 3-kinase α (PI3Kα) is an important regulator of intracellular signaling pathways, controlling remarkably diverse arrays of physiological processes. Because the PI3K pathway is frequently up-regulated in human cancers, the inhibition of PI3Kα can be a promising approach to cancer therapy. In this study, we have designed and synthesized a new series of imidazo[1,2-a]pyridine derivatives as PI3Kα inhibitors through the fragment-growing strategy. By varying groups at the 3- and 6-positions of imidazo[1,2-a]pyridines, we studied the structure-activity relationships (SAR) profiles and identified a series of potent PI3Kα inhibitors. Representative derivatives showed good activity in cellular proliferation and apoptosis assays. Moreover, these inhibitors exhibited noteworthy antiangiogenic activity.
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Zhang, Bing; Tan, Vincent B C; Lim, Kian Meng; Tay, Tong Earn
2006-06-01
Interests in CDK2 and CDK5 have stemmed mainly from their association with cancer and neuronal migration or differentiation related diseases and the need to design selective inhibitors for these kinases. Molecular dynamics (MD) simulations have not only become a viable approach to drug design because of advances in computer technology but are increasingly an integral part of drug discovery processes. It is common in MD simulations of inhibitor/CDK complexes to exclude the activator of the CDKs in the structural models to keep computational time tractable. In this paper, we present simulation results of CDK2 and CDK5 with roscovitine using models with and without their activators (cyclinA and p25). While p25 was found to induce slight changes in CDK5, the calculations support that cyclinA leads to significant conformational changes near the active site of CDK2. This suggests that detailed and structure-based inhibitor design targeted at these CDKs should employ activator-included models of the kinases. Comparisons between P/CDK2/cyclinA/roscovitine and CDK5/p25/roscovitine complexes reveal differences in the conformations of the glutamine around the active sites, which may be exploited to find highly selective inhibitors with respect to CDK2 and CDK5.
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Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C
Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar
2003-01-01
DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165
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Machrouhi, Fouzia; Ouhamou, Nouara; Laderoute, Keith; Calaoagan, Joy; Bukhtiyarova, Marina; Ehrlich, Paula J.; Klon, Anthony E.
2010-01-01
We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK. PMID:20932747
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Foote, Kevin M; Mortlock, Andrew A; Heron, Nicola M; Jung, Frédéric H; Hill, George B; Pasquet, Georges; Brady, Madeleine C; Green, Stephen; Heaton, Simon P; Kearney, Sarah; Keen, Nicholas J; Odedra, Rajesh; Wedge, Stephen R; Wilkinson, Robert W
2008-03-15
A new class of 1-acetanilide-4-aminopyrazole-substituted quinazoline Aurora kinase inhibitors has been discovered possessing highly potent cellular activity. Continuous infusion into athymic mice bearing SW620 tumors of the soluble phosphate derivative 2 led to dose-proportional exposure of the des-phosphate compound 8 with a high-unbound fraction. The combination of potent cell activity and high free-drug exposure led to pharmacodynamic changes in the tumor at low doses, indicative of Aurora B-kinase inhibition and a reduction in tumor volume.
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Discovery and Structure Enabled Synthesis of 2,6-Diaminopyrimidin-4-one IRAK4 Inhibitors
DOE Office of Scientific and Technical Information (OSTI.GOV)
Seganish, W. Michael; Fischmann, Thierry O.; Sherborne, Brad
2015-08-13
We report the identification and synthesis of a series of aminopyrimidin-4-one IRAK4 inhibitors. Through high throughput screening, an aminopyrimidine hit was identified and modified via structure enabled design to generate a new, potent, and kinase selective pyrimidin-4-one chemotype. This chemotype is exemplified by compound 16, which has potent IRAK4 inhibition activity (IC50 = 27 nM) and excellent kinase selectivity (>100-fold against 99% of 111 tested kinases), and compound 31, which displays potent IRAK4 activity (IC50 = 93 nM) and good rat bioavailability (F = 42%).
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Kinase Pathway Dependence in Primary Human Leukemias Determined by Rapid Inhibitor Screening
Tyner, Jeffrey W.; Yang, Wayne F.; Bankhead, Armand; Fan, Guang; Fletcher, Luke B.; Bryant, Jade; Glover, Jason M.; Chang, Bill H.; Spurgeon, Stephen E.; Fleming, William H.; Kovacsovics, Tibor; Gotlib, Jason R.; Oh, Stephen T.; Deininger, Michael W.; Zwaan, C. Michel; Den Boer, Monique L.; van den Heuvel-Eibrink, Marry M.; O’Hare, Thomas; Druker, Brian J.; Loriaux, Marc M.
2012-01-01
Kinases are dysregulated in most cancer but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of gene targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options. PMID:23087056
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Clemens, Michael J; Elia, Androulla; Morley, Simon J
2013-01-01
The protein kinase mammalian target of rapamycin (mTOR) regulates the phosphorylation and activity of several proteins that have the potential to control translation, including p70S6 kinase and the eIF4E binding proteins 4E-BP1 and 4E-BP2. In spite of this, in exponentially growing cells overall protein synthesis is often resistant to mTOR inhibitors. We report here that sensitivity of wild-type mouse embryonic fibroblasts (MEFs) to mTOR inhibitors can be greatly increased when the cells are subjected to the physiological stress imposed by hypertonic conditions. In contrast, protein synthesis in MEFs with a double knockout of 4E-BP1 and 4E-BP2 remains resistant to mTOR inhibitors under these conditions. Phosphorylation of p70S6 kinase and protein kinase B (Akt) is blocked by the mTOR inhibitor Ku0063794 equally well in both wild-type and 4E-BP knockout cells, under both normal and hypertonic conditions. The response of protein synthesis to hypertonic stress itself does not require the 4E-BPs. These data suggest that under certain stress conditions: (i) translation has a greater requirement for mTOR activity and (ii) there is an absolute requirement for the 4E-BPs for regulation by mTOR. Importantly, dephosphorylation of p70S6 kinase and Akt is not sufficient to affect protein synthesis acutely.
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Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho
2002-09-30
We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.
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Badrinarayan, Preethi; Sastry, G Narahari
2012-04-01
In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.
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Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.
Van Voorhis, Wesley C; Doggett, J Stone; Parsons, Marilyn; Hulverson, Matthew A; Choi, Ryan; Arnold, Samuel L M; Riggs, Michael W; Hemphill, Andrew; Howe, Daniel K; Mealey, Robert H; Lau, Audrey O T; Merritt, Ethan A; Maly, Dustin J; Fan, Erkang; Ojo, Kayode K
2017-09-01
Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
De Lucca, George V.; Shi, Qing; Liu, Qingjie
Bruton’s tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure–activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]-9H-carbazole-1-carboxamide 6 (BMS-935177) was selected to advance into clinical development.
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Rattan, Satish; Fan, Ya-Ping; Puri, Rajinder N
2002-03-22
Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.
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Pergola, Carlo; Gaboriaud-Kolar, Nicolas; Jestädt, Nadine; König, Stefanie; Kritsanida, Marina; Schaible, Anja M; Li, Haokun; Garscha, Ulrike; Weinigel, Christina; Barz, Dagmar; Albring, Kai F; Huber, Otmar; Skaltsounis, Alexios L; Werz, Oliver
2014-05-08
The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 μM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention.
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Malínková, Veronika; Řezníčková, Eva; Jorda, Radek; Gucký, Tomáš; Kryštof, Vladimír
2017-12-15
Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G 1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Malki-Feldman, Laura; Jaffe, Charles L
2009-09-01
Effect of modulators on protein kinase A (PKA) activity, promastigote growth and their ability to infect peritoneal macrophages was monitored. PKA inhibitors reduced [Protein Kinase Inhibitor (PKI) - 56%; H89 - 54.5%] kemptide phosphorylation by Leishmania major promastigote lysates, while activators increased phosphorylation (8-CPT-cAMP - 88%; Sp-cAMPS-AM - 152%). Activation was specifically inhibited by PKI. Phosphodiesterase inhibitors also increased kemptide phosphorylation (dipyridamole - 171%; rolipram - 106%; and 3-isobutyl-1-methyl-xanthine - 154%). Parasite proliferation was significantly retarded (200 nM H89; 100 microM myristoylated-PKI) or completely inhibited (500 nM H89) by culturing with PKA inhibitors. Incubation with dipyridamole or Sp-cAMPS-AM also inhibited proliferation. Brief treatment (2h) with either H89, myristoylated-PKI, dipyridamole or Sp-cAMPS-AM reduced initial macrophage infection at days 1 and 2 (>40%) and on day 3 (>78% only for 100 microM myr-PKI). Characterization of leishmanial cAMP mediated signal transduction pathways will serve as the basis for the new drug design.
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Bae, Hyemi; Lee, Donghee; Kim, Young-Won; Choi, Jeongyoon; Lee, Hong Jun; Kim, Sang-Wook; Kim, Taeho; Noh, Yun-Hee; Ko, Jae-Hong; Bang, Hyoweon
2016-01-01
Human cardiac fibroblasts (HCFs) have various voltage-dependent K+ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H2O2) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H2O2 could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H2O2 stimulated Ca2+-activated K+ (KCa) currents but not delayed rectifier K+ or transient outward K+ currents, all of which are VDKCs. H2O2-stimulated KCa currents were blocked by iberiotoxin (IbTX, a large conductance KCa blocker). The H2O2-stimulating effect on large-conductance KCa (BKCa) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BKCa currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H2O2-stimulating effect on BKCa currents. Using RT-PCR and western blot analysis, three subtypes of KCa channels were detected in HCFs: BKCa channels, small-conductance KCa (SKCa) channels, and intermediate-conductance KCa (IKCa) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H2O2, but IbTX decreased H2O2-induced apoptosis. These data suggest that among the VDKCs of HCFs, H2O2 only enhances BKCa currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BKCa channels. PMID:27162486
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In vitro evaluation of the Aurora kinase inhibitor VX-680 for Hepatoblastoma.
Dewerth, Alexander; Wonner, Timo; Lieber, Justus; Ellerkamp, Verena; Warmann, Steven W; Fuchs, Jörg; Armeanu-Ebinger, Sorin
2012-06-01
Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors. Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation. Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 μM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei. The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.
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Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors
Davare, Monika A.; Vellore, Nadeem A.; Wagner, Jacob P.; Eide, Christopher A.; Goodman, James R.; Drilon, Alexander; Deininger, Michael W.; O’Hare, Thomas; Druker, Brian J.
2015-01-01
Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib’s dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1G2032R mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure–function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies. PMID:26372962
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Salgado, Eva; Maneiro, Jose R; Carmona, Loreto; Gomez-Reino, Juan J
2014-05-01
To summarise the adverse events (AE) reported in patients with rheumatoid arthritis (RA) treated with protein kinase inhibitors (PKi), and identify family and molecule-related AEs. Systematic review of the PKi used in clinical trials (CTs) in RA. Medline, Embase, Cochrane Library, Web of Knowledge, and international abstracts of congress were reviewed, (up to 31 October 2012). Search was limited to interventional studies of PKi used in CTs in RA, written in English, and reporting frequencies of AE. Diseases with similar comorbidity burden also were included. Frequency of AE, serious AE (SAE), death and discontinuation due to AEs (DCAE) were recorded. Risk of bias was assessed. Meta-analysis was carried using pooled relative risk (RR) with 95% CI as effect measure. The search produced 4410 hits. Forty-one articles reporting data on 21 PKi of the Janus kinase (JAK), SYK, p38 and cKit families were selected for detailed analysis. In patients treated with p38 inhibitors, RR for dizziness was 2.36 (1.20 to 4.63), and in patients treated with c-Kit inhibitors, RR for oedema was 3.43 (1.58 to 7.42). In patients treated with the JAK inhibitor tofacitinib, RR for hypercholesterolaemia was 1.70 (1.10 to 2.63) that was dose related. In patients treated with the Syk inhibitor fostamatinib, pooled RR for hypertransaminasaemia, hypertension, diarrhoea and neutropenia were 2.93 (1.02 to 8.43), 2.80 (1.58 to 5.99), 5.20 (3.19 to 8.49) and 9.24 (2.22 to 38.42), respectively. Serious infections and malignancies were not significantly more frequent in PKi-treated patients than in comparator groups. Event rates of serious infections and malignancies with PKi are not different from biologics. In addition, PKi have a unique safety profile related to target and off-target inhibition of kinases, at times dose related.
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Caumanns, Joseph J; Berns, Katrien; Wisman, G Bea A; Fehrmann, Rudolf S N; Tomar, Tushar; Klip, Harry; Meersma, Gert Jan; Hijmans, E Marielle; Gennissen, Annemiek; Duiker, Evelien W; Weening, Desiree; Itamochi, Hiroaki; Kluin, Roelof Jc; Reyners, An K L; Birrer, Michael J; Salvesen, Helga B; Vergote, Ignace; Van Nieuwenhuysen, Els; Brenton, James D; Braicu, Elena I; Kupryjanczyk, Jolanta; Spiewankiewicz, Beata; Mittempergher, Lorenza; Bernards, Rene; van der Zee, Ate G J; de Jong, Steven
2018-04-23
Advanced stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in OCCC patients and test druggability of downstream affected pathways in OCCC models. In a large set of OCCC patients (n=124), the human kinome (518 kinases) and additional cancer related genes were sequenced and copy number alterations were determined. Genetically characterized OCCC cell lines (n=17) and OCCC patient-derived xenografts (n=3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib and the mTORC1/2 inhibitor AZD8055. We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway or the ERBB family of receptor tyrosine kinases and 82% in the DNA repair pathway. Strong p-S6 staining in OCCC patients suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not towards drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models. These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Copyright ©2018, American Association for Cancer Research.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Weng, M.-S.; Ho, C.-T.; Ho, Y.-S.
Fatty acid synthase (FAS) is a major lipogenic enzyme catalyzing the synthesis of long-chain saturated fatty acids. Most breast cancers require lipogenesis for growth. Here, we demonstrated the effects of theanaphthoquinone (TNQ), a member of the thearubigins generated by the oxidation of theaflavin (TF-1), on the expression of FAS in human breast cancer cells. TNQ was found to suppress the EGF-induced expression of FAS mRNA and FAS protein in MDA-MB-231 cells. Expression of FAS has previously been shown to be regulated by the SREBP family of transcription factors. In this study, we demonstrated that the EGF-induced nuclear translocation of SREBP-1more » was blocked by TNQ. Moreover, TNQ also modulated EGF-induced ERK1/2 and Akt phosphorylation. Treatment of MDA-MB-231 cells with PI 3-kinase inhibitors, LY294002 and Wortmannin, inhibited the EGF-induced expression of FAS and nuclear translocation of SREBP-1. Treatment with TNQ inhibited EGF-induced EGFR/ErbB-2 phosphorylation and dimerization. Furthermore, treatment with kinase inhibitors of EGFR and ErbB-2 suggested that EGFR/ErbB-2 activation was involved in EGF-induced FAS expression. In constitutive FAS expression, TNQ inhibited FAS expression and Akt autophosphorylation in BT-474 cells. The PI 3-kinase inhibitors and tyrosine kinase inhibitors of EGFR and ErbB-2 also reduced constitutive FAS expression. In addition, pharmacological blockade of FAS by TNQ decreased cell viability and induced cell death in BT-474 cells. In summary, our findings suggest that TNQ modulates FAS expression by the regulation of EGFR/ErbB-2 pathways and induces cell death in breast cancer cells.« less
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Regulation of potassium transport in human lens epithelial cells.
Lauf, Peter K; Warwar, Ronald; Brown, Thomas L; Adragna, Norma C
2006-01-01
The major K influx pathways and their response to thiol modification by N-ethylmaleimide (NEM) and protein kinase and phosphatase inhibitors were characterized in human lens epithelial B3 (HLE-B3) cells with Rb as K congener. Ouabain (0.1 mM) and bumetanide (5 microM) discriminated between the Na/K pump ( approximately 35% of total Rb influx) and Na-K-2Cl cotransport (NKCC) ( approximately 50%). Cl-replacement with nitrate or sulfamate revealed <10% residual [ouabain+bumetanide]-insensitive K-Cl cotransport (KCC). At 0.3-0.5 mM, NEM stimulated the Na/K pump by 2-fold independent of external Na, KCC between 2 and 4-fold, and abolished approximately 90% of NKCC. Calyculin-A, a serine/threonine protein phosphatase-1 inhibitor, did not affect NKCC but inhibited KCC, whereas 10 microM staurosporine, a serine/threonine kinase inhibitor, abolished NKCC, and stimulated KCC only when followed by NEM treatment. The tyrosine-kinase inhibitor genistein, at concentrations >100 microM, activated the Na/K pump and abolished NKCC but did not affect KCC. The data suggest at least partial inverse regulation of KCC and NKCC in HLE-B3 cells by signaling cascades involving serine, threonine and tyrosine phosphorylation/dephosphorylation equilibria.
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Macrophage differentiation increases expression of the ascorbate transporter (SVCT2)
Qiao, Huan; May, James M.
2013-01-01
To determine whether macrophage differentiation involves increased uptake of vitamin C, or ascorbic acid, we assessed the expression and function of its transporter SVCT2 during phorbol ester-induced differentiation of human-derived THP-1 monocytes. Induction of THP-1 monocyte differentiation by phorbol 12-myristate 13-acetate (PMA) markedly increased SVCT2 mRNA, protein, and function. When ascorbate was present during PMA-induced differentiation, the increase in SVCT2 protein expression was inhibited, but differentiation was enhanced. PMA-induced SVCT2 protein expression was blocked by inhibitors of protein kinase C (PKC), with most of the affect due to the PKCβI and βII isoforms. Activation of MEK/ERK was sustained up to 48 h after PMA treatment, and the inhibitors completely blocked PMA-stimulated SVCT2 protein expression, indicating an exclusive role for the classical MAP kinase pathway. However, inhibitors of NF-κB activation, NADPH oxidase inhibitors, and several antioxidants also partially prevented SVCT2 induction, suggesting diverse distal routes for control of SVCT2 transcription. Both known promoters for the SVCT2 were involved in these effects. In conclusion, PMA-induced monocyte-macrophage differentiation is enhanced by ascorbate and associated with increased expression and function of the SVCT2 protein through a pathway involving sustained activation of PKCβI/II, MAP kinase, NADPH oxidase, and NF-κB. PMID:19232538
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Kimura, Yukihiro; Chihara, Kazuyasu; Takeuchi, Kenji; Sada, Kiyonao
2013-07-01
Non-receptor type of protein-tyrosine kinase Syk (spleen tyrosine kinase) was isolated in the University of Fukui in 1991. Syk is known to be essential for the various physiological functions, especially in hematopoietic lineage cells. Moreover, ectopic expression of Syk by epigenetic changes is reported to cause retinoblastoma. Recently, novel Syk inhibitors were developed and its usefulness has been evaluated in the treatment of allergic rhinitis, rheumatoid arthritis, and idiopathic thrombocytopenic purpura. In this review, we will summarize the history, structure, and function of Syk, and then describe the novel Syk inhibitors and their current status. Furthermore, we will introduce our findings of the adaptor protein 3BP2 (c-Abl SH3 domain-binding protein-2), as a novel target of Syk.
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Thress, Kenneth; Macintyre, Terry; Wang, Haiyun; Whitston, Dave; Liu, Zhong-Ying; Hoffmann, Ethan; Wang, Tao; Brown, Jeffrey L; Webster, Kevin; Omer, Charles; Zage, Peter E; Zeng, Lizhi; Zweidler-McKay, Patrick A
2009-07-01
Tropomyosin-related kinases (TrkA, TrkB, and TrkC) are receptor tyrosine kinases that, along with their ligands, the neurotrophins, are involved in neuronal cell growth, development, and survival. The Trk-neurotrophin pathway may also play a role in tumorigenesis through oncogenic fusions, mutations, and autocrine signaling, prompting the development of novel Trk inhibitors as agents for cancer therapy. This report describes the identification of AZ-23, a novel, potent, and selective Trk kinase inhibitor. In vitro studies with AZ-23 showed improved selectivity over previous compounds and inhibition of Trk kinase activity in cells at low nanomolar concentrations. AZ-23 showed in vivo TrkA kinase inhibition and efficacy in mice following oral administration in a TrkA-driven allograft model and significant tumor growth inhibition in a Trk-expressing xenograft model of neuroblastoma. AZ-23 represents a potent and selective Trk kinase inhibitor from a novel series with the potential for use as a treatment for cancer.
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Ribosomal S6 kinase (RSK) modulators: a patent review.
Ludwik, Katarzyna A; Lannigan, Deborah A
2016-09-01
The p90 ribosomal S6 kinases (RSK) are a family of Ser/Thr protein kinases that are downstream effectors of MEK1/2-ERK1/2. Increased RSK activation is implicated in the etiology of multiple pathologies, including numerous types of cancers, cardiovascular disease, liver and lung fibrosis, and infections. The review summarizes the patent and scientific literature on small molecule modulators of RSK and their potential use as therapeutics. The patents were identified using World Intellectual Property Organization and United States Patent and Trademark Office databases. The compounds described are predominantly RSK inhibitors, but a RSK activator is also described. The majority of the inhibitors are not RSK-specific. Based on the overwhelming evidence that RSK is involved in a number of diseases that have high mortalities it seems surprising that there are no RSK modulators that have pharmacokinetic properties suitable for in vivo use. MEK1/2 inhibitors are in the clinic, but the efficacy of these compounds appears to be limited by their side effects. We hypothesize that targeting the downstream effectors of MEK1/2, like RSK, are an untapped source of drug targets and that they will generate less side effects than MEK1/2 inhibitors because they regulate fewer effectors.
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Agarwal, Anupriya; MacKenzie, Ryan J.; Pippa, Raffaella; Eide, Christopher A.; Oddo, Jessica; Tyner, Jeffrey W.; Sears, Rosalie; Vitek, Michael P.; Odero, María D.; Christensen, Dale; Druker, Brian J.
2014-01-01
Purpose The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A. Experimental Design In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis and colonogenic assays. Efficacy of target inhibition by OP449 is evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model. Results We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1-mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells. Conclusions We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. PMID:24436473
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Lawson, Marie; Rodrigo, Jordi; Baratte, Blandine; Robert, Thomas; Delehouzé, Claire; Lozach, Olivier; Ruchaud, Sandrine; Bach, Stéphane; Brion, Jean-Daniel; Alami, Mouad; Hamze, Abdallah
2016-11-10
We report here the synthesis, the biological evaluation and the molecular modeling studies of new imidazo[1,2-a]pyridines derivatives designed as potent kinase inhibitors. This collection was obtained from 2-aminopyridines and 2-bromoacetophenone which afforded final compound in only one step. The bioactivity of this family of new compounds was tested using protein kinase and ATP competition assays. The structure-activity relationship (SAR) revealed that six compounds inhibit DYRK1A and CLK1 at a micromolar range. Docking studies provided possible explanations that correlate with the SAR data. The most active compound 4c inhibits CLK1 (IC50 of 0.7 μM) and DYRK1A (IC50 of 2.6 μM). Copyright © 2016 Elsevier Masson SAS. All rights reserved.
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PI3K regulates MEK/ERK signaling in breast cancer via the Rac-GEF, P-Rex1
Ebi, Hiromichi; Costa, Carlotta; Faber, Anthony C.; Nishtala, Madhuri; Kotani, Hiroshi; Juric, Dejan; Della Pelle, Patricia; Song, Youngchul; Yano, Seiji; Mino-Kenudson, Mari; Benes, Cyril H.; Engelman, Jeffrey A.
2013-01-01
The PI3K pathway is genetically altered in excess of 70% of breast cancers, largely through PIK3CA mutation and HER2 amplification. Preclinical studies have suggested that these subsets of breast cancers are particularly sensitive to PI3K inhibitors; however, the reasons for this heightened sensitivity are mainly unknown. We investigated the signaling effects of PI3K inhibition in PIK3CA mutant and HER2 amplified breast cancers using PI3K inhibitors currently in clinical trials. Unexpectedly, we found that in PIK3CA mutant and HER2 amplified breast cancers sensitive to PI3K inhibitors, PI3K inhibition led to a rapid suppression of Rac1/p21-activated kinase (PAK)/protein kinase C-RAF (C-RAF)/ protein kinase MEK (MEK)/ERK signaling that did not involve RAS. Furthermore, PI3K inhibition led to an ERK-dependent up-regulation of the proapoptotic protein, BIM, followed by induction of apoptosis. Expression of a constitutively active form of Rac1 in these breast cancer models blocked PI3Ki-induced down-regulation of ERK phosphorylation, apoptosis, and mitigated PI3K inhibitor sensitivity in vivo. In contrast, protein kinase AKT inhibitors failed to block MEK/ERK signaling, did not up-regulate BIM, and failed to induce apoptosis. Finally, we identified phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) as the PI(3,4,5)P3-dependent guanine exchange factor for Rac1 responsible for regulation of the Rac1/C-RAF/MEK/ERK pathway in these cells. The expression level of P-Rex1 correlates with sensitivity to PI3K inhibitors in these breast cancer cell lines. Thus, PI3K inhibitors have enhanced activity in PIK3CA mutant and HER2 amplified breast cancers in which PI3K inhibition down-regulates both the AKT and Rac1/ERK pathways. In addition, P-Rex1 may serve as a biomarker to predict response to single-agent PI3K inhibitors within this subset of breast cancers. PMID:24327733
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Horiuchi, Dai; Camarda, Roman; Zhou, Alicia Y.; Yau, Christina; Momcilovic, Olga; Balakrishnan, Sanjeev; Corella, Alexandra N.; Eyob, Henok; Kessenbrock, Kai; Lawson, Devon A.; Marsh, Lindsey A.; Anderton, Brittany N.; Rohrberg, Julia; Kunder, Ratika; Bazarov, Alexey V.; Yaswen, Paul; McManus, Michael T.; Rugo, Hope S.; Werb, Zena; Goga, Andrei
2017-01-01
Triple-negative breast cancer (TNBC), which lacks the expression of the estrogen, progesterone, and HER2 receptors, represents the breast cancer subtype with the poorest outcome1. No targeted therapy is available against this subtype due to lack of validated molecular targets. We previously reported that MYC signaling is disproportionally elevated in triple-negative (TN) tumors compared to receptor-positive (RP) tumors2. MYC is an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes3. Direct inhibition of oncogenic MYC transcriptional activity has remained challenging4,5. The present study conducted an shRNA screen against all kinases to uncover novel MYC-dependent synthetic lethal combinations, and identified PIM1, a non-essential kinase. Here we demonstrate that PIM1 expression was elevated in TN tumors and was associated with poor prognosis in patients with hormone and HER2 receptor-negative tumors. Small molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic breast cancer models by inhibiting oncogenic transcriptional activity of MYC while simultaneously restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that exhibit elevated MYC expression. PMID:27775705
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Substituted 2H-isoquinolin-1-one as potent Rho-Kinase inhibitors. Part 1: Hit-to-lead account.
Wu, Frank; Büttner, Frank H; Chen, Rhonda; Hickey, Eugene; Jakes, Scott; Kaplita, Paul; Kashem, Mohammed A; Kerr, Steven; Kugler, Stanley; Paw, Zofia; Prokopowicz, Anthony; Shih, Cheng-Kon; Snow, Roger; Young, Erick; Cywin, Charles L
2010-06-01
Two closely related scaffolds were identified through an uHTS campaign as desirable starting points for the development of Rho-Kinase (ROCK) inhibitors. Here, we describe our hit-to-lead evaluation process which culminated in the rapid discovery of potent leads such as 22 which successfully demonstrated an early in vivo proof of concept for anti-hypertensive activity. Copyright 2010 Elsevier Ltd. All rights reserved.
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Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola
2009-06-15
Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu, Zhiwei; Cui, Binbin; Jin, Yinghu
2011-08-12
Highlights: {yields} This article described the effects of the EGFR tyrosine kinase inhibitor on the cell proliferation and the apoptosis induction of the colon carcinoma cell lines. {yields} Demonstrated that 326474 is a more potent EGFR inhibitor on colon cancer cells than other three TKIs. {yields} It can be important when considering chemotherapy for colonic cancer patients. -- Abstract: Background: Epidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. Methods: In this study,more » using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. Results: Among the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. Conclusion: Our studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.« less
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Shin, Hwa Kyoung; Salomone, Salvatore; Potts, E Michelle; Lee, Sae-Won; Millican, Eric; Noma, Kensuke; Huang, Paul L; Boas, David A; Liao, James K; Moskowitz, Michael A; Ayata, Cenk
2007-05-01
Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS-/-) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS-/- mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS-/- mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.
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In Silico Identification of a Novel Hinge-Binding Scaffold for Kinase Inhibitor Discovery.
Wang, Yanli; Sun, Yuze; Cao, Ran; Liu, Dan; Xie, Yuting; Li, Li; Qi, Xiangbing; Huang, Niu
2017-10-26
To explore novel kinase hinge-binding scaffolds, we carried out structure-based virtual screening against p38α MAPK as a model system. With the assistance of developed kinase-specific structural filters, we identify a novel lead compound that selectively inhibits a panel of kinases with threonine as the gatekeeper residue, including BTK and LCK. These kinases play important roles in lymphocyte activation, which encouraged us to design novel kinase inhibitors as drug candidates for ameliorating inflammatory diseases and cancers. Therefore, we chemically modified our substituted triazole-class lead compound to improve the binding affinity and selectivity via a "minimal decoration" strategy, which resulted in potent and selective kinase inhibitors against LCK (18 nM) and BTK (8 nM). Subsequent crystallographic experiments validated our design. These rationally designed compounds exhibit potent on-target inhibition against BTK in B cells or LCK in T cells, respectively. Our work demonstrates that structure-based virtual screening can be applied to facilitate the development of novel chemical entities in crowded chemical space in the field of kinase inhibitor discovery.
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Vijayakumar, Balakrishnan; Velmurugan, Devadasan
2012-01-01
Protein Kinase C β-II (PKC β-II) is an important enzyme in the development of diabetic complications like cardiomyopathy, retinopathy, neuropathy, nephropathy and angiopathy. PKC β-II is activated in vascular tissues during diabetic vascular abnormalities. Thus, PKC β-II is considered as a potent drug target and the crystal structure of the kinase domain of PKC β-II (PDB id: 2I0E) was used to design inhibitors using Structure-Based Drug Design (SBDD) approach. Sixty inhibitors structurally similar to Staurosporine were retrieved from PubChem Compound database and High Throughput Virtual screening (HTVs) was carried out with PKC β-II. Based on the HTVs results and the nature of active site residues of PKC β-II, Staurosporine inhibitors were designed using SBDD. Induced Fit Docking (IFD) studies were carried out between kinase domain of PKC β-II and the designed inhibitors. These IFD complexes showed favorable docking score, glide energy, glide emodel and hydrogen bond and hydrophobic interactions with the active site of PKC β-II. Binding free energy was calculated for IFD complexes using Prime MM-GBSA method. The conformational changes induced by the inhibitor at the active site of PKC β-II were observed for the back bone Cα atoms and side-chain chi angles. PASS prediction tool was used to analyze the biological activities for the designed inhibitors. The various physicochemical properties were calculated for the compounds. One of the designed inhibitors successively satisfied all the in silico parameters among the others and seems to be a potent inhibitor against PKC β-II. PMID:22829732
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Ritchie, E; Nichols, G
2006-12-01
One of the challenges of cancer therapeutics is to discover targets unique to the tumor cell population. Constitutively activated tyrosine kinases play a role in the malignant phenotype in a number of different cancers. While the kinases may be present in the normal cell, the cancer cell is often dependent upon the activation of the kinase for the maintenance of malignant growth. Inhibition of kinase activation may therefore selectively inhibit malignant proliferation. In the case of chronic myelogenous leukemia (CML), the activated tyrosine kinase (BCR-ABL) is due to a chromosomal translocation that defines this disease, and is necessary for malignant transformation. Imatinib mesylate (Gleevec, Novartis) is a small molecule tyrosine kinase inhibitor, developed through the chemical modification to be selected for a small number of tyrosine kinases present in human cells. This agent is also orally bioavailable and has been found to be effective in clinical trials. We have learned much through the clinical use of this agent. 1) Specific targeting of activated signal transduction pathways may be effective in inhibiting cancer cells. 2) Cancer cells may not only be inherently resistant to small molecule inhibitors, but may also develop resistance after exposure to the inhibitor. 3) Increased knowledge regarding critical signal transduction pathways, the structure of the molecules that are being targeted and the inhibitors themselves, will allow us to understand resistance as it develops and create new molecules to bypass resistance. We will discuss imatinib as an important example of the success and pitfalls of targeted therapeutics for cancer.
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Yan, Fengxia; Wang, Haitao; Gao, Yang; Xu, Jiangping; Zheng, Wenhua
2017-08-16
Oxidative stress plays a key role in the pathogenesis of age-related macular degeneration (AMD), a leading cause of severe visual loss and blindness in the aging population which lacks any effective treatments currently. In this study, artemisinin, a well-known antimalarial drug was found to suppress hydrogen peroxide (H 2 O 2 )-induced cell death in retinal neuronal RGC-5 cells. Artemisinin, in the therapeutically relevant dosage, concentration-dependently attenuated the accumulation of intracellular reactive oxygen species (ROS), increased mitochondrial membrane potential and decreased cell apoptosis in RGC-5 cells induced by H 2 O 2 . Western blot analysis showed that artemisinin upregulated the phosphorylation of p38 and extracellular signal-regulated kinases1/2 (ERK1/2) and reversed the inhibitory effect of H 2 O 2 on the phosphorylation of these two kinases. Moreover, protective effect of artemisinin was blocked by the p38 kinase inhibitor PD169316 or ERK1/2 kinase pathway inhibitor PD98059, respectively. In contrast, c-Jun N-terminal kinase inhibitor and rapamycin had no effect in the protective effect of artemisinin. Taken together, these results demonstrated that artemisinin promoted the survival of RGC-5 cells from H 2 O 2 toxicity via the activation of the p38 and ERK1/2 pathways. Interestingly, intravitreous injection of artimisinin, concentration-dependently reversed light exposed-damage (a dry AMD animal model) of rat retinal physiological function detected by flash electroretinogram. These results indicate that artemisinin can protect retinal neuronal functions from H 2 O 2 -induced damage in vitro and in vivo and suggest the potential application of artemisinin as a new drug in the treatment of retinal disorders like AMD.
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Sajan, M P; Farese, R V
2012-05-01
We examined the role of protein kinase C-ι (PKC-ι) in mediating alterations in the abundance of enzymes in hepatocytes of type 2 diabetic humans that contribute importantly to the development of lipid and carbohydrate abnormalities in type 2 diabetes. We examined (1) insulin signalling in isolated hepatocytes of non-diabetic and type 2 diabetic humans and (2) the effects of two newly developed small molecule PKC-ι inhibitors on aberrant signalling and downstream processes. In contrast with PKC-ι deficiency in diabetic muscle, which diminishes glucose transport, PKC-ι in diabetic hepatocytes was overproduced and overactive, basally and after insulin treatment, and, moreover, was accompanied by increased abundance of PKC-ι-dependent lipogenic, proinflammatory and gluconeogenic enzymes. Heightened PKC-ι activity most likely reflected heightened activity of IRS-2-dependent phosphatidylinositol 3-kinase (PI3K), as IRS-1 levels and IRS-1/PI3K activity were markedly diminished. Importantly, insulin-stimulated PKC-ι abundance and its overabundance in diabetic hepatocytes was reversed in vitro by both insulin deprivation and PKC-ι inhibitors; this suggested operation of an insulin-driven, feed-forward/positive-feedback mechanism. In contrast with PKC-ι, protein kinase B (Akt2) activity and activation by insulin was diminished, apparently reflecting IRS-1 deficiency. Treatment of diabetic hepatocytes with PKC-ι/λ inhibitors diminished abundance of lipogenic, proinflammatory and gluconeogenic enzymes. Our findings suggest that a vicious cycle of PKC-ι overactivity and overproduction exists in hepatocytes of humans with type 2 diabetes and contributes importantly to maintaining overactivity of lipogenic, proinflammatory and gluconeogenic pathways, which underlies the lipid and carbohydrate abnormalities in type 2 diabetes.
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Effects of selective inhibitors of Aurora kinases on anaplastic thyroid carcinoma cell lines.
Baldini, Enke; Tuccilli, Chiara; Prinzi, Natalie; Sorrenti, Salvatore; Antonelli, Alessandro; Gnessi, Lucio; Morrone, Stefania; Moretti, Costanzo; Bononi, Marco; Arlot-Bonnemains, Yannick; D'Armiento, Massimino; Ulisse, Salvatore
2014-10-01
Aurora kinases are serine/threonine kinases that play an essential role in cell division. Their aberrant expression and/or function induce severe mitotic abnormalities, resulting in either cell death or aneuploidy. Overexpression of Aurora kinases is often found in several malignancies, among which is anaplastic thyroid carcinoma (ATC). We have previously demonstrated the in vitro efficacy of Aurora kinase inhibitors in restraining cell growth and survival of different ATC cell lines. In this study, we sought to establish which Aurora might represent the preferential drug target for ATC. To this end, the effects of two selective inhibitors of Aurora-A (MLN8237) and Aurora-B (AZD1152) on four human ATC cell lines (CAL-62, BHT-101, 8305C, and 8505C) were analysed. Both inhibitors reduced cell proliferation in a time- and dose-dependent manner, with IC50 ranges of 44.3-134.2 nM for MLN8237 and of 9.2-461.3 nM for AZD1152. Immunofluorescence experiments and time-lapse videomicroscopy yielded evidence that each inhibitor induced distinct mitotic phenotypes, but both of them prevented the completion of cytokinesis. As a result, poliploidy increased in all AZD1152-treated cells, and in two out of four cell lines treated with MLN8237. Apoptosis was induced in all the cells by MLN8237, and in BHT-101, 8305C, and 8505C by AZD1152, while CAL-62 exposed to AZD1152 died through necrosis after multiple rounds of endoreplication. Both inhibitors were capable of blocking anchorage-independent cell growth. In conclusion, we demonstrated that either Aurora-A or Aurora-B might represent therapeutic targets for the ATC treatment, but inhibition of Aurora-A appears more effective for suppressing ATC cell proliferation and for inducing the apoptotic pathway. © 2014 Society for Endocrinology.
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Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.
Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio
2014-04-01
In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.
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Edgar, Alexander J; Trost, Matthias; Watts, Colin; Zaru, Rossana
2014-02-01
Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells. © 2014 The author(s).
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Dessalew, Nigus; Bharatam, Prasad V
2007-07-01
Selective glycogen synthase kinase 3 (GSK3) inhibition over cyclin dependent kinases such as cyclin dependent kinase 2 (CDK2) and cyclin dependent kinase 4 (CDK4) is an important requirement for improved therapeutic profile of GSK3 inhibitors. The concepts of selectivity and additivity fields have been employed in developing selective CoMFA models for these related kinases. Initially, sets of three individual CoMFA models were developed, using 36 compounds of bisarylmaleimide series to correlate with the GSK3, CDK2 and CDK4 inhibitory potencies. These models showed a satisfactory statistical significance: CoMFA-GSK3 (r(2)(con), r(2)(cv): 0.931, 0.519), CoMFA-CDK2 (0.937, 0.563), and CoMFA-CDK4 (0.892, 0.725). Three different selective CoMFA models were then developed using differences in pIC(50) values. These three models showed a superior statistical significance: (i) CoMFA-Selective1 (r(2)(con), r(2)(cv): 0.969, 0.768), (ii) CoMFA-Selective 2 (0.974, 0.835) and (iii) CoMFA-Selective3 (0.963, 0.776). The selective models were found to outperform the individual models in terms of the quality of correlation and were found to be more informative in pinpointing the structural basis for the observed quantitative differences of kinase inhibition. An in-depth comparative investigation was carried out between the individual and selective models to gain an insight into the selectivity criterion. To further validate this approach, a set of new compounds were designed which show selectivity and were docked into the active site of GSK3, using FlexX based incremental construction algorithm.
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Rosado, J A; Graves, D; Sage, S O
2000-01-01
We have recently reported that store-mediated Ca(2+) entry in platelets is likely to be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, a model termed 'secretion-like coupling'. In this model the actin cytoskeleton plays a key regulatory role. Since tyrosine kinases have been shown to be important for Ca(2+) entry in platelets and other cells, we have now investigated the possible involvement of tyrosine kinases in the secretion-like-coupling model. Treatment of platelets with thrombin or thapsigargin induced actin polymerization by a calcium-independent pathway. Methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor, prevented thrombin- or thapsigargin-induced actin polymerization. The effects of tyrosine kinases in store-mediated Ca(2+) entry were found to be entirely dependent on the actin cytoskeleton. PP1, an inhibitor of the Src family of proteins, partially inhibited store-mediated Ca(2+) entry. In addition, depletion of intracellular Ca(2+) stores stimulated cytoskeletal association of the cytoplasmic tyrosine kinase pp60(src), a process that was sensitive to treatment with cytochalasin D and PP1, but not to inhibition of Ras proteins using prenylcysteine analogues. Finally, combined inhibition of both Ras proteins and tyrosine kinases resulted in complete inhibition of Ca(2+) entry, suggesting that these two families of proteins have independent effects in the activation of store-mediated Ca(2+) entry in human platelets. PMID:11023829
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Ligand efficiency based approach for efficient virtual screening of compound libraries.
Ke, Yi-Yu; Coumar, Mohane Selvaraj; Shiao, Hui-Yi; Wang, Wen-Chieh; Chen, Chieh-Wen; Song, Jen-Shin; Chen, Chun-Hwa; Lin, Wen-Hsing; Wu, Szu-Huei; Hsu, John T A; Chang, Chung-Ming; Hsieh, Hsing-Pang
2014-08-18
Here we report for the first time the use of fit quality (FQ), a ligand efficiency (LE) based measure for virtual screening (VS) of compound libraries. The LE based VS protocol was used to screen an in-house database of 125,000 compounds to identify aurora kinase A inhibitors. First, 20 known aurora kinase inhibitors were docked to aurora kinase A crystal structure (PDB ID: 2W1C); and the conformations of docked ligand were used to create a pharmacophore (PH) model. The PH model was used to screen the database compounds, and rank (PH rank) them based on the predicted IC50 values. Next, LE_Scale, a weight-dependant LE function, was derived from 294 known aurora kinase inhibitors. Using the fit quality (FQ = LE/LE_Scale) score derived from the LE_Scale function, the database compounds were reranked (PH_FQ rank) and the top 151 (0.12% of database) compounds were assessed for aurora kinase A inhibition biochemically. This VS protocol led to the identification of 7 novel hits, with compound 5 showing aurora kinase A IC50 = 1.29 μM. Furthermore, testing of 5 against a panel of 31 kinase reveals that it is selective toward aurora kinase A & B, with <50% inhibition for other kinases at 10 μM concentrations and is a suitable candidate for further development. Incorporation of FQ score in the VS protocol not only helped identify a novel aurora kinase inhibitor, 5, but also increased the hit rate of the VS protocol by improving the enrichment factor (EF) for FQ based screening (EF = 828), compared to PH based screening (EF = 237) alone. The LE based VS protocol disclosed here could be applied to other targets for hit identification in an efficient manner. Copyright © 2014 Elsevier Masson SAS. All rights reserved.
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Computational methods for analysis and inference of kinase/inhibitor relationships
Ferrè, Fabrizio; Palmeri, Antonio; Helmer-Citterich, Manuela
2014-01-01
The central role of kinases in virtually all signal transduction networks is the driving motivation for the development of compounds modulating their activity. ATP-mimetic inhibitors are essential tools for elucidating signaling pathways and are emerging as promising therapeutic agents. However, off-target ligand binding and complex and sometimes unexpected kinase/inhibitor relationships can occur for seemingly unrelated kinases, stressing that computational approaches are needed for learning the interaction determinants and for the inference of the effect of small compounds on a given kinase. Recently published high-throughput profiling studies assessed the effects of thousands of small compound inhibitors, covering a substantial portion of the kinome. This wealth of data paved the road for computational resources and methods that can offer a major contribution in understanding the reasons of the inhibition, helping in the rational design of more specific molecules, in the in silico prediction of inhibition for those neglected kinases for which no systematic analysis has been carried yet, in the selection of novel inhibitors with desired selectivity, and offering novel avenues of personalized therapies. PMID:25071826
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Hennequin, Laurent F; Allen, Jack; Breed, Jason; Curwen, Jon; Fennell, Michael; Green, Tim P; Lambert-van der Brempt, Christine; Morgentin, Rémy; Norman, Richard A; Olivier, Annie; Otterbein, Ludovic; Plé, Patrick A; Warin, Nicolas; Costello, Gerard
2006-11-02
Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent pharmacokinetic parameters in animal preclinically and in man (t(1/2) = 40 h). AZD0530 is a potent inhibitor of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once daily. AZD0530 is currently undergoing clinical evaluation in man.
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Amen, Yhiya; Zhu, Qinchang; Tran, Hai-Bang; Afifi, Mohamed S; Halim, Ahmed F; Ashour, Ahmed; Shimizu, Kuniyoshi
2017-04-01
Recent studies identified Rho-kinase enzymes (ROCK-I and ROCK-II) as important targets that are involved in a variety of diseases. Synthetic Rho-kinase inhibitors have emerged as potential therapeutic agents to treat disorders such as hypertension, stroke, cancer, diabetes, glaucoma, etc. Our study is the first to screen the total ethanol extract of the medicinal mushroom Ganoderma lingzhi with thirty-five compounds for Rho-kinase inhibitory activity. Moreover, a molecular binding experiment was designed to investigate the binding affinity of the compounds at the active sites of Rho-kinase enzymes. The structure-activity relationship analysis was investigated. Our results suggest that the traditional uses of G. lingzhi might be in part due to the ROCK-I and ROCK-II inhibitory potential of this mushroom. Structure-activity relationship studies revealed some interesting features of the lanostane triterpenes that potentiate their Rho-kinase inhibition. These findings would be helpful for further studies on the design of Rho-kinase inhibitors from natural sources and open the door for contributions from other researchers for optimizing the development of natural Rho-kinase inhibitors.
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Sunitinib: from charge-density studies to interaction with proteins.
Malińska, Maura; Jarzembska, Katarzyna N; Goral, Anna M; Kutner, Andrzej; Woźniak, Krzysztof; Dominiak, Paulina M
2014-05-01
Protein kinases are targets for the treatment of a number of diseases. Sunitinib malate is a type I inhibitor of tyrosine kinases and was approved as a drug in 2006. This contribution constitutes the first comprehensive analysis of the crystal structures of sunitinib malate and of complexes of sunitinib with a series of protein kinases. The high-resolution single-crystal X-ray measurement and aspherical atom databank approach served as a basis for reconstruction of the charge-density distribution of sunitinib and its protein complexes. Hirshfeld surface and topological analyses revealed a similar interaction pattern in the sunitinib malate crystal structure to that in the protein binding pockets. Sunitinib forms nine preserved bond paths corresponding to hydrogen bonds and also to the C-H···O and C-H···π contacts common to the VEGRF2, CDK2, G2, KIT and IT kinases. In general, sunitinib interacts with the studied proteins with a similar electrostatic interaction energy and can adjust its conformation to fit the binding pocket in such a way as to enhance the electrostatic interactions, e.g. hydrogen bonds in ligand-kinase complexes. Such behaviour may be responsible for the broad spectrum of action of sunitinib as a kinase inhibitor.
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Regulation of 2-5A Dependent RNase at the Level of its Phosphorylation
1991-06-26
extract as follows: 25 ul wheat germ extract 10 ul H2O 1 ul RNasin ribonuclease inhibitor (40 u/ml) 7 ul ImM amino acid mixture 1 ul IM...diacylglycerol (DAG) 2. TPA 3. Indolactam Figure 6. Chemical structure of: 1. H-7 (A kinase inhibitor) 2. okadaic acid (A phosphatase inhibitor) Figure 7...elevating agents: Forskolin and Cholera toxin Figure 17. Down-regulation of 2-5A-depRNase by Okadaic 77 acid : A phosphatase inhibitor Figure 18
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Indole RSK inhibitors. Part 1: discovery and initial SAR.
Boyer, Stephen J; Burke, Jennifer; Guo, Xin; Kirrane, Thomas M; Snow, Roger J; Zhang, Yunlong; Sarko, Chris; Soleymanzadeh, Lida; Swinamer, Alan; Westbrook, John; Dicapua, Frank; Padyana, Anil; Cogan, Derek; Gao, Amy; Xiong, Zhaoming; Madwed, Jeffrey B; Kashem, Mohammed; Kugler, Stanley; O'Neill, Margaret M
2012-01-01
A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subsequent kinase selectivity optimization. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Zhu, Wei; Chen, Hui; Wang, Yulan; Wang, Jiang; Peng, Xia; Chen, Xianjie; Gao, Yinglei; Li, Chunpu; He, Yulong; Ai, Jing; Geng, Meiyu; Zheng, Mingyue; Liu, Hong
2017-07-27
A novel series of pyridin-3-amine derivatives were designed, synthesized, and evaluated as multitargeted protein kinase inhibitors for the treatment of non-small cell lung cancer (NSCLC). Hit 1 was first disclosed by in silico screening against fibroblast growth factor receptors (FGFR), which was subsequently validated by in vitro experiments. The structure-activity relationship (SAR) of its analogues was then explored to afford novel FGFR inhibitors 2a-2p and 3a-3q. Among them, 3m showed potent inhibition against FGFR1, 2, and 3. Interestingly, compound 3m not only inhibited various phosphorylation and downstream signaling across different oncogenic forms in FGFR-overactivated cancer cells but also showed nanomolar level inhibition against several other NSCLC-related oncogene kinases, including RET, EGFR, EGFR/T790M/L858R, DDR2, and ALK. Finally, in vivo pharmacology evaluations of 3m showed significant antitumor activity (TGI = 66.1%) in NCI-H1581 NSCLC xenografts with a good pharmacokinetic profile.
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Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.
Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M
2011-11-15
Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.
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Wender, Paul A; Axtman, Alison D; Golden, Jennifer E; Kee, Jung-Min; Sirois, Lauren E; Quiroz, Ryan V; Stevens, Matthew C
2014-12-29
The human kinome comprises over 500 protein kinases. When mutated or over-expressed, many play critical roles in abnormal cellular functions associated with cancer, cardiovascular disease and neurological disorders. Here we report a step-economical approach to designed kinase inhibitors inspired by the potent, but non-selective, natural product staurosporine, and synthetically enabled by a novel, complexity-increasing, serialized [5 + 2]/[4 + 2] cycloaddition strategy. This function-oriented synthesis approach rapidly affords tunable scaffolds, and produced a low nanomolar inhibitor of protein kinase C.
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Resistance to MEK inhibitors: should we co-target upstream?
Poulikakos, Poulikos I; Solit, David B
2011-03-29
Aberrant activation of the ERK pathway is common in human tumors. This pathway consists of a three-tiered kinase module [comprising the kinases RAF, mitogen-activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK)] that functions as a negative feedback amplifier to confer robustness and stabilization of pathway output. Because this pathway is frequently dysregulated in human cancers, intense efforts are under way to develop selective inhibitors of the ERK pathway as anticancer drugs. Although promising results have been reported in early trials for inhibitors of RAF or MEK, resistance invariably occurs. Amplification of the upstream oncogenic driver of ERK signaling has been identified as a mechanism for MEK inhibitor resistance in cells with mutant BRAF or KRAS. Increased abundance of the oncogenic driver (either KRAS or BRAF in the appropriate cellular context) in response to prolonged drug treatment results in increased flux through the ERK pathway and restoration of ERK activity above the threshold required for cell growth. For patients with BRAF mutant tumors, the results suggest that the addition of a RAF inhibitor to a MEK inhibitor may delay or overcome drug resistance. The data thus provide a mechanistic basis for ongoing trials testing concurrent treatment with RAF and MEK inhibitors.
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Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian
2010-01-01
Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396
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Brain penetrant kinase inhibitors: Learning from kinase neuroscience discovery.
Shi, Yuan; Mader, Mary
2018-06-15
A recent review of kinase inhibitors in clinical trials for brain cancer noted differences in the properties of these compounds relative to the mean property parameters associated with drugs marketed for CNS-associated conditions. However, many of these kinase drugs arose from opportunistic observations of brain activity, rather than design or flow schemes focused on optimizing CNS penetration. Thus, this digest examines kinase inhibitors that have been developed specifically for neurodegenerative indications such as Alzheimer's or Parkinson's disease, and considers design, flow scheme, and the physicochemical properties associated with compounds that have demonstrated brain penetrance. Copyright © 2018 Elsevier Ltd. All rights reserved.
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Reddy, M. V. Ramana; Akula, Balireddy; Jatiani, Shashidhar; Vasquez-Del Carpio, Rodrigo; Billa, Vinay K.; Mallireddigari, Muralidhar R.; Cosenza, Stephen C.; Subbaiah, D. R. C. Venkata; Bharathi, E. Vijaya; Pallela, Venkat R.; Ramkumar, Poornima; Jain, Rinku; Aggarwal, Aneel K.; Reddy, E. Premkumar
2018-01-01
Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao. PMID:26762835
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Reddy, M V Ramana; Akula, Balireddy; Jatiani, Shashidhar; Vasquez-Del Carpio, Rodrigo; Billa, Vinay K; Mallireddigari, Muralidhar R; Cosenza, Stephen C; Venkata Subbaiah, D R C; Bharathi, E Vijaya; Pallela, Venkat R; Ramkumar, Poornima; Jain, Rinku; Aggarwal, Aneel K; Reddy, E Premkumar
2016-02-15
Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Anticancer therapy: boosting the bang of Bim
Hendrickson, Andrea Wahner; Meng, Xue Wei; Kaufmann, Scott H.
2008-01-01
Even though activating mutations of B-Raf, a kinase atop the MAPK signaling cascade, reportedly sensitize tumor cells to MEK inhibitors, Raf and MEK inhibitors have exhibited limited clinical activity. In this issue of the JCI, Cragg et al. report that MEK inhibition upregulates the proapoptotic Bcl-2 family member Bim but induces little regression of human melanoma xenografts in mice unless the Bcl-2 antagonist ABT-737 is added (see the related article beginning on page 3651). These findings illustrate the potential benefit of simultaneously inhibiting oncogenic kinases and inhibiting Bcl-2 action in solid tumors. PMID:18949061
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Witters, Lois; Scherle, Peggy; Friedman, Steven; Fridman, Jordan; Caulder, Eian; Newton, Robert; Lipton, Allan
2008-09-01
The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.
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Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R; Combs, Andrew P; Vaddi, Kris; Haley, Patrick J; Burn, Timothy C; Rupar, Mark; Bromberg, Jacqueline F; Heaney, Mark L; de Stanchina, Elisa; Fridman, Jordan S; Levine, Ross L
2010-04-08
The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.
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Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R.; Combs, Andrew P.; Vaddi, Kris; Haley, Patrick J.; Burn, Timothy C.; Rupar, Mark; Bromberg, Jacqueline F.; Heaney, Mark L.; de Stanchina, Elisa; Fridman, Jordan S.
2010-01-01
The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model. PMID:20154217
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Development of Specific Inhibitors for Breast Cancer-Associated Variants of ErbB2
2015-10-01
Produce ErbB2 structures for drug-lead identification Months 1-12 Milestone #2: Production of computationally-derived pdb files of the structures of...crystallographic structures of the kinase domain of ErbB2 and its close relative EGFR (ErbB1). The kinase domains of ErbB2 and EGFR are highly...homologous as indicated by a sequence identity of ~ 78%. There are two currently available crystallographic structures of the ErbB2 kinase domain. One is
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Lueptow, Lindsay M; Zhan, Chang-Guo; O'Donnell, James M
2016-02-01
Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 μg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 μg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.
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ABT-199 (venetoclax) and BCL-2 inhibitors in clinical development.
Cang, Shundong; Iragavarapu, Chaitanya; Savooji, John; Song, Yongping; Liu, Delong
2015-11-20
With the advent of new agents targeting CD20, Bruton's tyrosine kinase, and phosphoinositol-3 kinase for chronic lymphoid leukemia (CLL), more treatment options exist than ever before. B-cell lymphoma-2 (BCL-2) plays a major role in cellular apoptosis and is a druggable target. Small molecule inhibitors of BCL-2 are in active clinical studies. ABT-199 (venetoclax, RG7601, GDC-0199) has been granted breakthrough designation by FDA for relapsed or refractory CLL with 17p deletion. In this review, we summarized the latest clinical development of ABT-199/venetoclax and other novel agents targeting the BCL-2 proteins.
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Kaplan, Jennifer M; Hake, Paul W; Denenberg, Alvin; Nowell, Marchele; Piraino, Giovanna; Zingarelli, Basilia
2010-01-01
Peroxisome proliferator–activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury. PMID:20809049
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Moon, Robert W.; Whalley, David; Bowyer, Paul W.; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K.; Howell, Steven A.; Grainger, Munira; Jones, Hayley M.; Ansell, Keith H.; Chapman, Timothy M.; Taylor, Debra L.; Osborne, Simon A.; Baker, David A.; Tatu, Utpal
2015-01-01
Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771
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Green, Judith L; Moon, Robert W; Whalley, David; Bowyer, Paul W; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K; Howell, Steven A; Grainger, Munira; Jones, Hayley M; Ansell, Keith H; Chapman, Timothy M; Taylor, Debra L; Osborne, Simon A; Baker, David A; Tatu, Utpal; Holder, Anthony A
2015-12-28
Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. Copyright © 2016 Green et al.
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NASA Astrophysics Data System (ADS)
Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham
1998-01-01
The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.
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Molecular Mechanisms of Hormone-Refractory Prostate Cancer
2006-02-01
FIN-00014 Helsinki, Finland Bmx/Etk, a member of the Tec/ Btk family of nonrecep- tor kinases, has recently been shown to mediate cell motility in...Bmx, which is a member of the Tec/ Btk family of tyrosine kinases (37). In addition to the SH2, SH3, and kinase domains, the Tec family of tyrosine...divided and resuspended in 18 l of kinase buffer with or without 1 M Src inhibitor SU6656 in the presence of either 2 g of GST or 2 g of GST-CasSD
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Tyrosine Kinase Display of Prostate Cancer Cells
2001-10-01
markers and important targets for intervention (2,4). Kinase inhibitors have recently shown tremendous efficacies and promises in the treatment of human...fully characterize this kinase. Etk is a new member of the Btk family of kinases (27), which distinguish themselves from others by having a pleckstrin- 5...Kung, Hsing-Jien DAMD 17-99-1-9021 homology (PH) domain at the N-terminus (27,28,29,30). Btk was uncovered as a kinase whose germ- line mutation
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The identification of new protein kinase inhibitors as targets in modern drug discovery.
Akritopoulou-Zanze, Irini
2006-07-01
In recent years there has been great interest in developing protein kinase inhibitors as therapeutic agents for a variety of diseases. This article provides an overview on the history, development and validity of kinases as drug targets, as well as a description of kinase research, including its limitations, challenges and successes.
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Marley, K; Maier, C S; Helfand, S C
2012-09-01
Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival. © 2012 Blackwell Publishing Ltd.
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Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Wales, Thomas E.; Engen, John R.; Smithgall, Thomas E.
2013-01-01
Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control. PMID:23303187
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Wang, Yen-Ling
2014-01-01
Checkpoint kinase 2 (Chk2) has a great effect on DNA-damage and plays an important role in response to DNA double-strand breaks and related lesions. In this study, we will concentrate on Chk2 and the purpose is to find the potential inhibitors by the pharmacophore hypotheses (PhModels), combinatorial fusion, and virtual screening techniques. Applying combinatorial fusion into PhModels and virtual screening techniques is a novel design strategy for drug design. We used combinatorial fusion to analyze the prediction results and then obtained the best correlation coefficient of the testing set (r test) with the value 0.816 by combining the BesttrainBesttest and FasttrainFasttest prediction results. The potential inhibitors were selected from NCI database by screening according to BesttrainBesttest + FasttrainFasttest prediction results and molecular docking with CDOCKER docking program. Finally, the selected compounds have high interaction energy between a ligand and a receptor. Through these approaches, 23 potential inhibitors for Chk2 are retrieved for further study. PMID:24864236
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Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol
Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; ...
2014-12-31
The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less
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Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol
DOE Office of Scientific and Technical Information (OSTI.GOV)
Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.
The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. In this paper, we have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). Wemore » found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC 50 < 100 nM) inhibit Jak3 activity in cell-based assays. Finally, these results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases.« less
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Effects of inorganic polyphosphate on bone sialoprotein gene expression.
Wang, Zhitao; Li, Xinyue; Li, Zhengyang; Yang, Li; Sasaki, Yoko; Wang, Shuang; Zhou, Liming; Araki, Shouta; Mezawa, Masaru; Takai, Hideki; Ogata, Yorimasa
2010-03-01
Inorganic polyphosphate (poly(P)) is a biopolymer existing in almost all cells and tissues. The biological functions of poly(P) in micro-organisms have been extensively investigated in studies of poly(P) in eukaryotic cells, especially osteoblasts, and are increasing. Bone sialoprotein (BSP) is thought to function in bone mineralization, and is selectively expressed by differentiated osteoblasts. In this study, application of sodium phosphate glass type 25 (SPG25, 12.5 and 125 microM) increased BSP mRNA levels at 12 h in osteoblast-like ROS 17/2.8 cells. In transient transfection assay, 12.5 and 125 microM SPG25 increased luciferase activities of the constructs pLUC3 (-116 to +60), pLUC4 (-425 to +60), pLUC5 (-801 to +60) and pLUC6 (-938 to +60). Introduction of 2 bp mutations to the luciferase constructs showed that the effects of SPG25 were mediated by a FGF2 response element (FRE) and a homeodomain protein binding site (HOX). Luciferase activities induced by SPG25 were blocked by tyrosine kinase inhibitor herbimycine A, MAP kinase kinase inhibitor U0126, PI3-kinase/Akt inhibitor LY249002 and inorganic phosphate transport inhibitor foscarnet. Gel shift analyses showed that both 12.5 and 125 microM SPG25 increased nuclear protein binding to FRE and HOX elements. These studies demonstrate that SPG25 stimulates BSP transcription by targeting FRE and HOX elements in the proximal promoter of the rat BSP gene. Copyright 2010 Elsevier B.V. All rights reserved.
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Woo, Tae-Gyun; Yoon, Min-Ho; Hong, Shin-Deok; Choi, Jiyun; Ha, Nam-Chul; Sun, Hokeun; Park, Bum-Joon
2017-01-01
Hyper-activation of PAK1 (p21-activated kinase 1) is frequently observed in human cancer and speculated as a target of novel anti-tumor drug. In previous, we also showed that PAK1 is highly activated in the Smad4-deficient condition and suppresses PUMA (p53 upregulated modulator of apoptosis) through direct binding and phosphorylation. On the basis of this result, we have tried to find novel PAK1-PUMA binding inhibitors. Through ELISA-based blind chemical library screening, we isolated single compound, IPP-14 (IPP; Inhibitor of PAK1-PUMA), which selectively blocks the PAK1-PUMA binding and also suppresses cell proliferation via PUMA-dependent manner. Indeed, in PUMA-deficient cells, this chemical did not show anti-proliferating effect. This chemical possessed very strong PAK1 inhibition activity that it suppressed BAD (Bcl-2-asoociated death promoter) phosphorylation and meta-phase arrest via Aurora kinase inactivation in lower concentration than that of previous PAK1 kinase, FRAX486 and AG879. Moreover, our chemical obviously induced p21/WAF1/CIP1 (Cyclin-dependent kinase inhibitor 1A) expression by releasing from Bcl-2 (B-cell lymphoma-2) and by inhibition of AKT-mediated p21 suppression. Considering our result, IPP-14 and its derivatives would be possible candidates for PAK1 and p21 induction targeted anti-cancer drug. PMID:28423593
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Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D.; Desai, Avanti; Metcalfe, Dean D.; Olivera, Ana
2018-01-01
Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation. PMID:29643855
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Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D; Desai, Avanti; Metcalfe, Dean D; Olivera, Ana
2018-01-01
Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.
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Kharel, Yugesh; Raje, Mithun R.; Gao, Ming; Tomsig, Jose L.; Lynch, Kevin R.; Santos, Webster L.
2015-01-01
Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P1–5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure–activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states. PMID:25643074
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Tandon, Manuj; Salamoun, Joseph M.; Carder, Evan J.; Farber, Elisa; Xu, Shuping; Deng, Fan; Tang, Hua; Wipf, Peter; Wang, Q. Jane
2015-01-01
Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment. PMID:25747583
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A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.
Espina, Virginia; Edmiston, Kirsten H; Heiby, Michael; Pierobon, Mariaelena; Sciro, Manuela; Merritt, Barbara; Banks, Stacey; Deng, Jianghong; VanMeter, Amy J; Geho, David H; Pastore, Lucia; Sennesh, Joel; Petricoin, Emanuel F; Liotta, Lance A
2008-10-01
Little is known about the preanalytical fluctuations of phosphoproteins during tissue procurement for molecular profiling. This information is crucial to establish guidelines for the reliable measurement of these analytes. To develop phosphoprotein profiles of tissue subjected to the trauma of excision, we measured the fidelity of 53 signal pathway phosphoproteins over time in tissue specimens procured in a community clinical practice. This information provides strategies for potential surrogate markers of stability and the design of phosphoprotein preservative/fixation solutions. Eleven different specimen collection time course experiments revealed augmentation (+/-20% from the time 0 sample) of signal pathway phosphoprotein levels as well as decreases over time independent of tissue type, post-translational modification, and protein subcellular location (tissues included breast, colon, lung, ovary, and uterus (endometrium/myometrium) and metastatic melanoma). Comparison across tissue specimens showed an >20% decrease of protein kinase B (AKT) Ser-473 (p < 0.002) and myristoylated alanine-rich C-kinase substrate protein Ser-152/156 (p < 0.0001) within the first 90-min postexcision. Proteins in apoptotic (cleaved caspase-3 Asp-175 (p < 0.001)), proliferation/survival/hypoxia (IRS-1 Ser-612 (p < 0.0003), AMP-activated protein kinase beta Ser-108 (p < 0.005), ERK Thr-202/Tyr-204 (p < 0.003), and GSK3alphabeta Ser-21/9 (p < 0.01)), and transcription factor pathways (STAT1 Tyr-701 (p < 0.005) and cAMP response element-binding protein Ser-133 (p < 0.01)) showed >20% increases within 90-min postprocurement. Endothelial nitric-oxide synthase Ser-1177 did not change over the time period evaluated with breast or leiomyoma tissue. Treatment with phosphatase or kinase inhibitors alone revealed that tissue kinase pathways are active ex vivo. Combinations of kinase and phosphatase inhibitors appeared to stabilize proteins that exhibited increases in the presence of phosphatase inhibitors alone (ATF-2 Thr-71, SAPK/JNK Thr-183/Tyr-185, STAT1 Tyr-701, JAK1 Tyr-1022/1023, and PAK1/PAK2 Ser-199/204/192/197). This time course study 1) establishes the dynamic nature of specific phosphoproteins in excised tissue, 2) demonstrates augmented phosphorylation in the presence of phosphatase inhibitors, 3) shows that kinase inhibitors block the upsurge in phosphorylation of phosphoproteins, 4) provides a rational strategy for room temperature preservation of proteins, and 5) constitutes a foundation for developing evidence-based tissue procurement guidelines.
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Pardanani, A
2008-01-01
The recent identification of somatic mutations such as JAK2V617F that deregulate Janus kinase (JAK)-signal transducer and activator of transcription signaling has spurred development of orally bioavailable small-molecule inhibitors that selectively target JAK2 kinase as an approach to pathogenesis-directed therapy of myeloproliferative disorders (MPD). In pre-clinical studies, these compounds inhibit JAK2V617F-mediated cell growth at nanomolar concentrations, and in vivo therapeutic efficacy has been demonstrated in mouse models of JAK2V617F-induced disease. In addition, ex vivo growth of progenitor cells from MPD patients harboring JAK2V617F or MPLW515L/K mutations is also potently inhibited. JAK2 inhibitors currently in clinical trials can be grouped into those designed to primarily target JAK2 kinase (JAK2-selective) and those originally developed for non-MPD indications, but that nevertheless have significant JAK2-inhibitory activity (non-JAK2 selective). This article discusses the rationale for using JAK2 inhibitors for the treatment of MPD, as well as relevant aspects of clinical trial development for these patients. For instance, which group of MPD patients is appropriate for initial Phase I studies? Should JAK2V617F-negative MPD patients be included in the initial studies? What are the likely consequences of 'off-target' JAK3 and wild-type JAK2 inhibition? How should treatment responses be monitored?