Conformational Analysis of the DFG-Out Kinase Motif and Biochemical Profiling of Structurally Validated Type II Inhibitors

PubMed Central

2015-01-01

Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as “classical DFG-out” conformations in order to distinguish them from conformations that have also been referred to as DFG-out in the literature but that do not have a fully formed allosteric pocket. We have completed a structural analysis of almost 200 small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that nonclassical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two data sets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity. PMID:25478866

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors.

PubMed

Grimster, Neil P; Anderson, Erica; Alimzhanov, Marat; Bebernitz, Geraldine; Bell, Kirsten; Chuaqui, Claudio; Deegan, Tracy; Ferguson, Andrew D; Gero, Thomas; Harsch, Andreas; Huszar, Dennis; Kawatkar, Aarti; Kettle, Jason Grant; Lyne, Paul D; Read, Jon A; Rivard Costa, Caroline; Ruston, Linette; Schroeder, Patricia; Shi, Jie; Su, Qibin; Throner, Scott; Toader, Dorin; Vasbinder, Melissa Marie; Woessner, Richard; Wang, Haixia; Wu, Allan; Ye, Minwei; Zheng, Weijia; Zinda, Michael

2018-06-01

Herein, we report the discovery and characterization of a novel series of pyrimidine based JAK1 inhibitors. Optimization of these ATP competitive compounds was guided by X-ray crystallography and a structure-based drug design approach, focusing on selectivity, potency, and pharmaceutical properties. The best compound, 24, displayed remarkable JAK1 selectivity (~1000-fold vs JAK2,3 and TYK2), as well as a good kinase selectivity profile. Moreover, a dose-dependent reduction in pSTAT3, a downstream marker of JAK1 inhibition, was observed when 24 was examined in vivo.

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors

PubMed Central

2014-01-01

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure. PMID:25589934

Discovery of N-[4-(1H-Pyrazolo[3,4-b]pyrazin-6-yl)-phenyl]-sulfonamides as Highly Active and Selective SGK1 Inhibitors.

PubMed

Halland, Nis; Schmidt, Friedemann; Weiss, Tilo; Saas, Joachim; Li, Ziyu; Czech, Jörg; Dreyer, Matthias; Hofmeister, Armin; Mertsch, Katharina; Dietz, Uwe; Strübing, Carsten; Nazare, Marc

2015-01-08

From a virtual screening starting point, inhibitors of the serum and glucocorticoid regulated kinase 1 were developed through a combination of classical medicinal chemistry and library approaches. This resulted in highly active small molecules with nanomolar activity and a good overall in vitro and ADME profile. Furthermore, the compounds exhibited unusually high kinase and off-target selectivity due to their rigid structure.

Discovery of 8-Amino-imidazo[1,5- a ]pyrazines as Reversible BTK Inhibitors for the Treatment of Rheumatoid Arthritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Jian; Guiadeen, Deodial; Krikorian, Arto

Bruton’s tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition and for the treatment of B cell related diseases. We report a series of compounds based on 8-amino-imidazo[1,5-a]pyrazine that are potent reversible BTK inhibitors with excellent kinase selectivity. Selectivity is achieved through specific interactions of the ligand with the kinase hinge and driven by aminopyridine hydrogen bondings with Ser538 and Asp539, and by hydrophobic interaction of trifluoropyridine in the back pocket. These interactions are evident in the X-raymore » crystal structure of the lead compounds 1 and 3 in the complex with the BTK enzyme. Our lead compounds show desirable PK profiles and efficacy in the preclinical rat collagen induced arthritis model.« less

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

PubMed

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.

Selective JAK inhibitors in development for rheumatoid arthritis.

PubMed

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.

Targeting kinase signaling pathways with constrained peptide scaffolds

PubMed Central

Hanold, Laura E.; Fulton, Melody D.; Kennedy, Eileen J.

2017-01-01

Kinases are amongst the largest families in the human proteome and serve as critical mediators of a myriad of cell signaling pathways. Since altered kinase activity is implicated in a variety of pathological diseases, kinases have become a prominent class of proteins for targeted inhibition. Although numerous small molecule and antibody-based inhibitors have already received clinical approval, several challenges may still exist with these strategies including resistance, target selection, inhibitor potency and in vivo activity profiles. Constrained peptide inhibitors have emerged as an alternative strategy for kinase inhibition. Distinct from small molecule inhibitors, peptides can provide a large binding surface area that allows them to bind shallow protein surfaces rather than defined pockets within the target protein structure. By including chemical constraints within the peptide sequence, additional benefits can be bestowed onto the peptide scaffold such as improved target affinity and target selectivity, cell permeability and proteolytic resistance. In this review, we highlight examples of diverse chemistries that are being employed to constrain kinase-targeting peptide scaffolds and highlight their application to modulate kinase signaling as well as their potential clinical implications. PMID:28185915

Discovery of Imidazoquinolines as a Novel Class of Potent, Selective, and in Vivo Efficacious Cancer Osaka Thyroid (COT) Kinase Inhibitors.

PubMed

Glatthar, Ralf; Stojanovic, Aleksandar; Troxler, Thomas; Mattes, Henri; Möbitz, Henrik; Beerli, Rene; Blanz, Joachim; Gassmann, Ernst; Drückes, Peter; Fendrich, Gabriele; Gutmann, Sascha; Martiny-Baron, Georg; Spence, Fiona; Hornfeld, Jeff; Peel, John Edmonson; Sparrer, Helmut

2016-08-25

Cancer Osaka thyroid (COT) kinase is an important regulator of pro-inflammatory cytokines in macrophages. Thus, pharmacologic inhibition of COT should be a valid approach to therapeutically intervene in the pathogenesis of macrophage-driven inflammatory diseases such as rheumatoid arthritis. We report the discovery and chemical optimization of a novel series of COT kinase inhibitors, with unprecedented nanomolar potency for the inhibition of TNFα. Pharmacological profiling in vivo revealed a high metabolism of these compounds in rats which was demonstrated to be predominantly attributed to aldehyde oxidase. Due to the very low activity of hepatic AO in the dog, the selected candidate 32 displayed significant blood exposure in dogs which resulted in a clear prevention of inflammation-driven lameness. Taken together, the described compounds both potently and selectively inhibit COT kinase in primary human cells and ameliorate inflammatory pathologies in vivo, supporting the notion that COT is an appropriate therapeutic target for inflammatory diseases.

Development of a 3D Tissue Culture-Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases.

PubMed

Booij, Tijmen H; Klop, Maarten J D; Yan, Kuan; Szántai-Kis, Csaba; Szokol, Balint; Orfi, Laszlo; van de Water, Bob; Keri, Gyorgy; Price, Leo S

2016-10-01

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting. © 2016 Society for Laboratory Automation and Screening.

p21-Activated kinase inhibitors: a patent review.

PubMed

Crawford, James J; Hoeflich, Klaus P; Rudolph, Joachim

2012-03-01

The p21-activated kinase (PAK) family of serine/threonine protein kinases is activated by binding to the small (p21) GTP-binding proteins Cdc42 and Rac. The PAK family plays important roles in cytoskeletal organisation, cellular morphogenesis and survival, and members of this family have been implicated in a wide range of diseases including cancer, infectious diseases, neurological disorders and arthritis. The present review seeks to summarise recent (up to 2011) reports of small-molecule inhibitors of p21-activated kinases. Where patent applications describe activity against a broad range of kinases and no information was provided specifically on PAK inhibition, these are excluded from this review. In patents considered to be relevant, exemplary compounds were selected and highlighted based on their representation of the chemical matter claimed, potencies, structural features and subsequent disclosure of their properties. Selected information from non-patent literature was also included. A considerable amount of research has been devoted over the past 15 years to exploring the role of PAKs in a wide range of diseases, with a focus on oncology. Published PAK inhibitors are still comparatively rare and few exhibit satisfactory kinase selectivity and 'drug-like' properties. A key question is which profile, pan-PAK, group selective or isoform selective, holds the most promise from both therapeutic and safety standpoints. To investigate this question, isoform-selective, as well as kinome-selective, PAK inhibitor tool compounds will be needed. Pfizer was the first company to progress a PAK inhibitor (pan-PAK) to clinical development; it is expected that, despite the difficulties, other PAK inhibitors will soon follow.

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

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

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

PubMed Central

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

Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

DOE PAGES

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

Computational methods for analysis and inference of kinase/inhibitor relationships

PubMed Central

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

Bis-Aryl Urea Derivatives as Potent and Selective LIM Kinase (Limk) Inhibitors

PubMed Central

Yin, Yan; Zheng, Ke; Eid, Nibal; Howard, Shannon; Jeong, Ji-Hak; Yi, Fei; Guo, Jia; Park, Chul M; Bibian, Mathieu; Wu, Weilin; Hernandez, Pamela; Park, HaJeung; Wu, Yuntao; Luo, Jun-Li; LoGrasso, Philip V.; Feng, Yangbo

2015-01-01

The discovery/optimization of bis-aryl ureas as Limk inhibitors to obtain high potency and selectivity, and appropriate pharmacokinetic properties through systematic SAR studies is reported. Docking studies supported the observed SAR. Optimized Limk inhibitors had high biochemical potency (IC50 < 25 nM), excellent selectivity against ROCK and JNK kinases (> 400-fold), potent inhibition of cofilin phosphorylation in A7r5,PC-3, and CEM-SS T cells (IC50 < 1 μM), and good in vitro and in vivo pharmacokinetic properties. In the profiling against a panel of 61 kinases, compound 18b at 1 μM inhibited only Limk1 and STK16 with ≥ 80% inhibition. Compounds 18b and 18f were highly efficient in inhibiting cell-invasion/migration in PC-3 cells. In addition, compound 18w was demonstrated to be effective on reducing intraocular pressure (IOP) on rat eyes. Taken together, these data demonstrated that we had developed a novel class of bis-aryl urea derived potent and selective Limk inhibitors. PMID:25621531

Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer.

PubMed

Chen, Ina; Mathews-Greiner, Lesley; Li, Dandan; Abisoye-Ogunniyan, Abisola; Ray, Satyajit; Bian, Yansong; Shukla, Vivek; Zhang, Xiaohu; Guha, Raj; Thomas, Craig; Gryder, Berkley; Zacharia, Athina; Beane, Joal D; Ravichandran, Sarangan; Ferrer, Marc; Rudloff, Udo

2017-05-01

Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function. Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-3α.

PubMed

Lo Monte, Fabio; Kramer, Thomas; Gu, Jiamin; Anumala, Upendra Rao; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Franco, Bénédicte; Demedts, David; Van Leuven, Fred; Fuertes, Ana; Dominguez, Juan Manuel; Plotkin, Batya; Eldar-Finkelman, Hagit; Schmidt, Boris

2012-05-10

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimer's disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

Structure–Activity Relationships and Molecular Modeling of Sphingosine Kinase Inhibitors

PubMed Central

2013-01-01

The design, synthesis, and evaluation of the potency of new isoform-selective inhibitors of sphingosine kinases 1 and 2 (SK1 and SK2), the enzyme that catalyzes the phosphorylation of d-erythro-sphingosine to produce the key signaling lipid, sphingosine 1-phosphate, are described. Recently, we reported that 1-(4-octylphenethyl)piperidin-4-ol (RB-005) is a selective inhibitor of SK1. Here we report the synthesis of 43 new analogues of RB-005, in which the lipophilic tail, polar headgroup, and linker region were modified to extend the structure–activity relationship profile for this lead compound, which we explain using modeling studies with the recently published crystal structure of SK1. We provide a basis for the key residues targeted by our profiled series and provide further evidence for the ability to discriminate between the two isoforms using pharmacological intervention. PMID:24164513

Large-scale Proteomics Analysis of the Human Kinome

PubMed Central

Oppermann, Felix S.; Gnad, Florian; Olsen, Jesper V.; Hornberger, Renate; Greff, Zoltán; Kéri, György; Mann, Matthias; Daub, Henrik

2009-01-01

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido[2,3-d]pyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis. PMID:19369195

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors

PubMed Central

Ravikumar, Balaguru; Parri, Elina; Timonen, Sanna; Airola, Antti; Wennerberg, Krister

2017-01-01

Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001) between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel-based modeling approach offers practical benefits for probing novel insights into the mode of action of investigational compounds, and for the identification of new target selectivities for drug repurposing applications. PMID:28787438

Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.

Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data providesmore » information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.« less

The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer .

PubMed

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.

Genome-wide identification, phylogeny and expressional profiles of mitogen activated protein kinase kinase kinase (MAPKKK) gene family in bread wheat (Triticum aestivum L.).

PubMed

Wang, Meng; Yue, Hong; Feng, Kewei; Deng, Pingchuan; Song, Weining; Nie, Xiaojun

2016-08-22

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are the important components of MAPK cascades, which play the crucial role in plant growth and development as well as in response to diverse stresses. Although this family has been systematically studied in many plant species, little is known about MAPKKK genes in wheat (Triticum aestivum L.), especially those involved in the regulatory network of stress processes. In this study, we identified 155 wheat MAPKKK genes through a genome-wide search method based on the latest available wheat genome information, of which 29 belonged to MEKK, 11 to ZIK and 115 to Raf subfamily, respectively. Then, chromosome localization, gene structure and conserved protein motifs and phylogenetic relationship as well as regulatory network of these TaMAPKKKs were systematically investigated and results supported the prediction. Furthermore, a total of 11 homologous groups between A, B and D sub-genome and 24 duplication pairs among them were detected, which contributed to the expansion of wheat MAPKKK gene family. Finally, the expression profiles of these MAPKKKs during development and under different abiotic stresses were investigated using the RNA-seq data. Additionally, 10 tissue-specific and 4 salt-responsive TaMAPKKK genes were selected to validate their expression level through qRT-PCR analysis. This study for the first time reported the genome organization, evolutionary features and expression profiles of the wheat MAPKKK gene family, which laid the foundation for further functional analysis of wheat MAPKKK genes, and contributed to better understanding the roles and regulatory mechanism of MAPKKKs in wheat.

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity

PubMed Central

Kaltenmeier, Christof T.; Vollmer, Laura L.; Vernetti, Lawrence A.; Caprio, Lindsay; Davis, Keanu; Korotchenko, Vasiliy N.; Day, Billy W.; Tsang, Michael; Hulkower, Keren I.; Lotze, Michael T.

2017-01-01

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration. PMID:28154014

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

PubMed Central

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

State-of-the-art of small molecule inhibitors of the TAM family: the point of view of the chemist.

PubMed

Baladi, Tom; Abet, Valentina; Piguel, Sandrine

2015-11-13

The TAM family of tyrosine kinases receptors (Tyro3, Axl and Mer) is implicated in cancer development, autoimmune reactions and viral infection and is therefore emerging as an effective and attractive therapeutic target. To date, only a few small molecules have been intentionally designed to block the TAM kinases, while most of the inhibitors were developed for blocking different protein kinases and then identified through selectivity profile studies. This minireview will examine in terms of chemical structure the different compounds able to act on either one, two or three TAM kinases with details about structure-activity relationships, drug-metabolism and pharmacokinetics properties where they exist. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

PubMed Central

Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

2012-01-01

A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

Novel CDK inhibition profiles of structurally varied 1-aza-9-oxafluorenes.

PubMed

Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Totzke, Frank; Hilgeroth, Andreas

2005-02-01

A series of 1-aza-9-oxafluorenes with functionally varied 3-substituents have been prepared from N-phenoxycarbonyl-4-phenyl-1,4-dihydropyridines and p-benzoquinone and biologically evaluated as inhibitors of various cyclin-dependant kinases. The absence of a 3-hydrogen bond acceptor function leads to a complete loss of inhibitory activity. Differing hydrogen bond acceptor functions surprisingly cause significant shifts in the selectivity of inhibition profiles.

Improved intra-array and interarray normalization of peptide microarray phosphorylation for phosphorylome and kinome profiling by rational selection of relevant spots

PubMed Central

Scholma, Jetse; Fuhler, Gwenny M.; Joore, Jos; Hulsman, Marc; Schivo, Stefano; List, Alan F.; Reinders, Marcel J. T.; Peppelenbosch, Maikel P.; Post, Janine N.

2016-01-01

Massive parallel analysis using array technology has become the mainstay for analysis of genomes and transcriptomes. Analogously, the predominance of phosphorylation as a regulator of cellular metabolism has fostered the development of peptide arrays of kinase consensus substrates that allow the charting of cellular phosphorylation events (often called kinome profiling). However, whereas the bioinformatical framework for expression array analysis is well-developed, no advanced analysis tools are yet available for kinome profiling. Especially intra-array and interarray normalization of peptide array phosphorylation remain problematic, due to the absence of “housekeeping” kinases and the obvious fallacy of the assumption that different experimental conditions should exhibit equal amounts of kinase activity. Here we describe the development of analysis tools that reliably quantify phosphorylation of peptide arrays and that allow normalization of the signals obtained. We provide a method for intraslide gradient correction and spot quality control. We describe a novel interarray normalization procedure, named repetitive signal enhancement, RSE, which provides a mathematical approach to limit the false negative results occuring with the use of other normalization procedures. Using in silico and biological experiments we show that employing such protocols yields superior insight into cellular physiology as compared to classical analysis tools for kinome profiling. PMID:27225531

A Potent and Selective Quinoxalinone-Based STK33 Inhibitor Does Not Show Synthetic Lethality in KRAS-Dependent Cells

PubMed Central

2012-01-01

The KRAS oncogene is found in up to 30% of all human tumors. In 2009, RNAi experiments revealed that lowering mRNA levels of a transcript encoding the serine/threonine kinase STK33 was selectively toxic to KRAS-dependent cancer cell lines, suggesting that small-molecule inhibitors of STK33 might selectively target KRAS-dependent cancers. To test this hypothesis, we initiated a high-throughput screen using compounds in the Molecular Libraries Small Molecule Repository (MLSMR). Several hits were identified, and one of these, a quinoxalinone derivative, was optimized. Extensive SAR studies were performed and led to the chemical probe ML281 that showed low nanomolar inhibition of purified recombinant STK33 and a distinct selectivity profile as compared to other STK33 inhibitors that were reported in the course of these studies. Even at the highest concentration tested (10 μM), ML281 had no effect on the viability of KRAS-dependent cancer cells. These results are consistent with other recent reports using small-molecule STK33 inhibitors. Small molecules having different chemical structures and kinase-selectivity profiles are needed to fully understand the role of STK33 in KRAS-dependent cancers. In this regard, ML281 is a valuable addition to small-molecule probes of STK33. PMID:23256033

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stachel, Shawn J.; Sanders, John M.; Henze, Darrell A.

We have identified several series of small molecule inhibitors of TrkA with unique binding modes. The starting leads were chosen to maximize the structural and binding mode diversity derived from a high throughput screen of our internal compound collection. These leads were optimized for potency and selectivity employing a structure based drug design approach adhering to the principles of ligand efficiency to maximize binding affinity without overly relying on lipophilic interactions. This endeavor resulted in the identification of several small molecule pan-Trk inhibitor series that exhibit high selectivity for TrkA/B/C versus a diverse panel of kinases. We have also demonstratedmore » efficacy in both inflammatory and neuropathic pain models upon oral dosing. Herein we describe the identification process, hit-to-lead progression, and binding profiles of these selective pan-Trk kinase inhibitors.« less

Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent.

PubMed

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.

First macrocyclic 3rd-generation ALK inhibitor for treatment of ALK/ROS1 cancer: Clinical and designing strategy update of lorlatinib.

PubMed

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.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

PubMed

Zhang, Sen; Anjum, Rana; Squillace, Rachel; Nadworny, Sara; Zhou, Tianjun; Keats, Jeff; Ning, Yaoyu; Wardwell, Scott D; Miller, David; Song, Youngchul; Eichinger, Lindsey; Moran, Lauren; Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Wang, Yihan; Mohemmad, Qurish; Jang, Hyun Gyung; Ye, Emily; Narasimhan, Narayana; Wang, Frank; Miret, Juan; Zhu, Xiaotian; Clackson, Tim; Dalgarno, David; Shakespeare, William C; Rivera, Victor M

2016-11-15

Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK + ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK + cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK + tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK + , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR. ©2016 American Association for Cancer Research.

Kinome and Transcriptome Profiling Reveal Broad and Distinct Activities of Erlotinib, Sunitinib, and Sorafenib in the Mouse Heart and Suggest Cardiotoxicity From Combined Signal Transducer and Activator of Transcription and Epidermal Growth Factor Receptor Inhibition.

PubMed

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.

Targeting the cyclin D-cyclin-dependent kinase (CDK) 4/6-retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions.

PubMed

Spring, Laura; Bardia, Aditya; Modi, Shanu

2016-01-01

Dysregulation of the cyclin D-cyclin-dependent kinase (CDK) 4/6-INK4-retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively non-selective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicated promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II-III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D-CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combination strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer.

Hit-to-lead optimization and kinase selectivity of imidazo[1,2-a]quinoxalin-4-amine derived JNK1 inhibitors.

PubMed

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.

Target identification, lead optimization and antitumor evaluation of some new 1,2,4-triazines as c-Met kinase inhibitors.

PubMed

El-Wakil, Marwa H; Ashour, Hayam M; Saudi, Manal N; Hassan, Ahmed M; Labouta, Ibrahim M

2017-08-01

In silico target fishing approach using PharmMapper server identified c-Met kinase as the selective target for our previously synthesized compound NCI 748494/1. This approach was validated by in vitro kinase assay which showed that NCI 748494/1 possessed promising inhibitory activity against c-Met kinase (IC 50 =31.70μM). Assessment of ADMET profiling, drug-likeness, drug score as well as docking simulation for the binding pose of that compound in the active site of c-Met kinase domain revealed that NCI 748494/1 could be considered as a promising drug lead. Based on target identification and validation, it was observed that there is structure similarity between NCI 748494/1 and the reported type II c-Met kinase inhibitor BMS-777607. Optimization of our lead NCI 748494/1 furnished newly synthesized 1,2,4-triazine derivatives based on well-established structure-activity relationships, whereas three compounds namely; 4d, 7a and 8c displayed excellent in vitro cytotoxicity against three c-Met addicted cancer cell lines; A549 (lung adenocarcinoma), HT-29 (colon cancer) and MKN-45 (gastric carcinoma); with IC 50 values in the range 0.01-1.86µM. In vitro c-Met kinase assay showed 8c to possess the highest c-Met kinase inhibition profile (IC 50 =4.31µM). Docking of the active compounds in c-Met kinase active site revealed strong binding interactions comparable to the lead NCI 748494/1 and BMS-777607, suggesting that c-Met inhibition is very likely to be the mechanism of the antitumor effect of these derivatives. Copyright © 2017 Elsevier Inc. All rights reserved.

Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition.

PubMed

Fearon, Daren; Westwood, Isaac M; van Montfort, Rob L M; Bayliss, Richard; Jones, Keith; Bavetsias, Vassilios

2018-07-15

Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode

PubMed Central

Chen, Cheng; Yu, Kailin; Zou, Fengming; Wang, Wenchao; Wang, Wei; Wu, Jiaxin; Liu, Juan; Wang, Beilei; Wang, Li; Ren, Tao; Zhang, Shanchun; Yun, Cai-Hong; Liu, Jing; Liu, Qingsong

2017-01-01

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct “DFG-in” and “cHelix-out” inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose. PMID:28407693

Discovery of 6-Fluoro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (BMS-986142): A Reversible Inhibitor of Bruton's Tyrosine Kinase (BTK) Conformationally Constrained by Two Locked Atropisomers.

PubMed

Watterson, Scott H; De Lucca, George V; Shi, Qing; Langevine, Charles M; Liu, Qingjie; Batt, Douglas G; Beaudoin Bertrand, Myra; Gong, Hua; Dai, Jun; Yip, Shiuhang; Li, Peng; Sun, Dawn; Wu, Dauh-Rurng; Wang, Chunlei; Zhang, Yingru; Traeger, Sarah C; Pattoli, Mark A; Skala, Stacey; Cheng, Lihong; Obermeier, Mary T; Vickery, Rodney; Discenza, Lorell N; D'Arienzo, Celia J; Zhang, Yifan; Heimrich, Elizabeth; Gillooly, Kathleen M; Taylor, Tracy L; Pulicicchio, Claudine; McIntyre, Kim W; Galella, Michael A; Tebben, Andy J; Muckelbauer, Jodi K; Chang, ChiehYing; Rampulla, Richard; Mathur, Arvind; Salter-Cid, Luisa; Barrish, Joel C; Carter, Percy H; Fura, Aberra; Burke, James R; Tino, Joseph A

2016-10-13

Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a member of the Tec family of kinases. BTK plays an essential role in B cell receptor (BCR)-mediated signaling as well as Fcγ receptor signaling in monocytes and Fcε receptor signaling in mast cells and basophils, all of which have been implicated in the pathophysiology of autoimmune disease. As a result, inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as lupus and rheumatoid arthritis. This article details the structure-activity relationships (SAR) leading to a novel series of highly potent and selective carbazole and tetrahydrocarbazole based, reversible inhibitors of BTK. Of particular interest is that two atropisomeric centers were rotationally locked to provide a single, stable atropisomer, resulting in enhanced potency and selectivity as well as a reduction in safety liabilities. With significantly enhanced potency and selectivity, excellent in vivo properties and efficacy, and a very desirable tolerability and safety profile, 14f (BMS-986142) was advanced into clinical studies.

Comparison of Quantitative Mass Spectrometry Platforms for Monitoring Kinase ATP Probe Uptake in Lung Cancer.

PubMed

Hoffman, Melissa A; Fang, Bin; Haura, Eric B; Rix, Uwe; Koomen, John M

2018-01-05

Recent developments in instrumentation and bioinformatics have led to new quantitative mass spectrometry platforms including LC-MS/MS with data-independent acquisition (DIA) and targeted analysis using parallel reaction monitoring mass spectrometry (LC-PRM), which provide alternatives to well-established methods, such as LC-MS/MS with data-dependent acquisition (DDA) and targeted analysis using multiple reaction monitoring mass spectrometry (LC-MRM). These tools have been used to identify signaling perturbations in lung cancers and other malignancies, supporting the development of effective kinase inhibitors and, more recently, providing insights into therapeutic resistance mechanisms and drug repurposing opportunities. However, detection of kinases in biological matrices can be challenging; therefore, activity-based protein profiling enrichment of ATP-utilizing proteins was selected as a test case for exploring the limits of detection of low-abundance analytes in complex biological samples. To examine the impact of different MS acquisition platforms, quantification of kinase ATP uptake following kinase inhibitor treatment was analyzed by four different methods: LC-MS/MS with DDA and DIA, LC-MRM, and LC-PRM. For discovery data sets, DIA increased the number of identified kinases by 21% and reduced missingness when compared with DDA. In this context, MRM and PRM were most effective at identifying global kinome responses to inhibitor treatment, highlighting the value of a priori target identification and manual evaluation of quantitative proteomics data sets. We compare results for a selected set of desthiobiotinylated peptides from PRM, MRM, and DIA and identify considerations for selecting a quantification method and postprocessing steps that should be used for each data acquisition strategy.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML.

PubMed

Liyanage, Sanduni U; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D; Bader, Gary D; Laposa, Rebecca; Schimmer, Aaron D

2017-05-11

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. © 2017 by The American Society of Hematology.

Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML

PubMed Central

Liyanage, Sanduni U.; Hurren, Rose; Voisin, Veronique; Bridon, Gaëlle; Wang, Xiaoming; Xu, ChangJiang; MacLean, Neil; Siriwardena, Thirushi P.; Gronda, Marcela; Yehudai, Dana; Sriskanthadevan, Shrivani; Avizonis, Daina; Shamas-Din, Aisha; Minden, Mark D.; Bader, Gary D.; Laposa, Rebecca

2017-01-01

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2′3′-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2′3′-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. PMID:28283480

Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia.

PubMed

Neumann, Theresa; Benajiba, Lina; Göring, Stefan; Stegmaier, Kimberly; Schmidt, Boris

2015-11-25

The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The scorpion shaped GSK-3 inhibitors developed by our group achieved the highest GSK-3α selectivity reported so far but suffered from insufficient aqueous solubility. This work presents the solubility-driven optimization of our isoform-selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed high activity against GSK-3α/β with the highest GSK-3α selectivity reported to date. Compound 27 was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSK-3α targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation phenotype and colony formation impairment, confirming the potential of GSK-3α inhibition in AML therapy.

G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives.

PubMed

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.

Structure-Activity Relationship Studies and Molecular Modeling of Naphthalene-Based Sphingosine Kinase 2 Inhibitors.

PubMed

Congdon, Molly D; Kharel, Yugesh; Brown, Anne M; Lewis, Stephanie N; Bevan, David R; Lynch, Kevin R; Santos, Webster L

2016-03-10

The two isoforms of sphingosine kinase (SphK1 and SphK2) are the only enzymes that phosphorylate sphingosine to sphingosine-1-phosphate (S1P), which is a pleiotropic lipid mediator involved in a broad range of cellular processes including migration, proliferation, and inflammation. SphKs are targets for various diseases such as cancer, fibrosis, and Alzheimer's and sickle cell disease. Herein, we disclose the structure-activity profile of naphthalene-containing SphK inhibitors and molecular modeling studies that reveal a key molecular switch that controls SphK selectivity.

Design, synthesis and biological evaluation of 7H-pyrrolo[2,3-d]pyrimidin-4-amine derivatives as selective Btk inhibitors with improved pharmacokinetic properties for the treatment of rheumatoid arthritis.

PubMed

He, Linhong; Pei, Heying; Zhang, Chufeng; Shao, Mingfeng; Li, Dan; Tang, Mingli; Wang, Taijing; Chen, Xiaoxin; Xiang, Mingli; Chen, Lijuan

2018-02-10

Bruton's tyrosine kinase (Btk) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) and Fcγ receptor (FcR) signaling pathways, which makes it a uniquely attractive target for the treatment of autoimmune diseases, such as rheumatoid arthritis (RA). We reported a series of compounds bearing 7H-pyrrolo [2,3-d]pyrimidin-4-amine scaffold that potently inhibited Btk in vitro. Analysis of the structure-activity relationships (SAR) and drug-like profiles led to the discovery of the optimal compound B16. B16 preferentially inhibited Btk (IC 50  = 21.70 ± 0.82 nM) over closely related kinases with moderate selectivity. Cell-based tests also confirmed that B16 significantly inhibited Btk Y223 auto-phosphorylation and PLCγ2 Y1217 phosphorylation. MTT revealed that B16 displayed weak suppression against normal LO2, HEK293 and THP-1 cell lines with IC 50 values over 30 μM. Moreover, B16 showed very weak potential to block the hERG channel (IC 50  = 11.10 μM) in comparison to ibrutinib (IC 50  = 0.97 μM). Owing to its favorable physicochemical properties (ClogP = 2.53, aqueous solubility ≈ 0.1 mg/mL), pharmacokinetic profiles (F = 49.15%, t 1/2  = 7.02 h) and reasonable CYP450 profile, B16 exhibited potent anti-arthritis activity and similar efficacy to ibrutinib in reducing paw thickness in CIA mice. In conclusion, B16 is a potent, selective and durable inhibitor of Btk and has the potential to a safe and efficacious treatment for arthritis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

PubMed

Pomel, Vincent; Klicic, Jasna; Covini, David; Church, Dennis D; Shaw, Jeffrey P; Roulin, Karen; Burgat-Charvillon, Fabienne; Valognes, Delphine; Camps, Montserrat; Chabert, Christian; Gillieron, Corinne; Françon, Bernard; Perrin, Dominique; Leroy, Didier; Gretener, Denise; Nichols, Anthony; Vitte, Pierre Alain; Carboni, Susanna; Rommel, Christian; Schwarz, Matthias K; Rückle, Thomas

2006-06-29

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase Domain*

PubMed Central

Tokarski, John S.; Zupa-Fernandez, Adriana; Tredup, Jeffrey A.; Pike, Kristen; Chang, ChiehYing; Xie, Dianlin; Cheng, Lihong; Pedicord, Donna; Muckelbauer, Jodi; Johnson, Stephen R.; Wu, Sophie; Edavettal, Suzanne C.; Hong, Yang; Witmer, Mark R.; Elkin, Lisa L.; Blat, Yuval; Pitts, William J.; Weinstein, David S.; Burke, James R.

2015-01-01

Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity. PMID:25762719

The Guareschi Pyridine Scaffold as a Valuable Platform for the Identification of Selective PI3K Inhibitors.

PubMed

Galli, Ubaldina; Ciraolo, Elisa; Massarotti, Alberto; Margaria, Jean Piero; Sorba, Giovanni; Hirsch, Emilio; Tron, Gian Cesare

2015-09-18

A novel series of 4-aryl-3-cyano-2-(3-hydroxyphenyl)-6-morpholino-pyridines have been designed as potential phosphatidylinositol-3-kinase (PI3K) inhibitors. The compounds have been synthesized using the Guareschi reaction to prepare the key 4-aryl-3-cyano-2,6-dihydroxypyridine intermediate. A different selectivity according to the nature of the aryl group has been observed. Compound 9b is a selective inhibitor against the PI3Kα isoform, maintaining a good inhibitory activity. Docking studies were also performed in order to rationalize its profile of selectivity.

Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Ken; Levell, Julian; Yoon, Taeyong

The observed structure–activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirmingmore » that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.« less

The discovery of thienopyridine analogues as potent IkappaB kinase beta inhibitors. Part II.

PubMed

Wu, Jiang-Ping; Fleck, Roman; Brickwood, Janice; Capolino, Alison; Catron, Katrina; Chen, Zhidong; Cywin, Charles; Emeigh, Jonathan; Foerst, Melissa; Ginn, John; Hrapchak, Matt; Hickey, Eugene; Hao, Ming-Hong; Kashem, Mohammed; Li, Jun; Liu, Weimin; Morwick, Tina; Nelson, Richard; Marshall, Daniel; Martin, Leslie; Nemoto, Peter; Potocki, Ian; Liuzzi, Michel; Peet, Gregory W; Scouten, Erika; Stefany, David; Turner, Michael; Weldon, Steve; Zimmitti, Clare; Spero, Denise; Kelly, Terence A

2009-10-01

An SAR study that identified a series of thienopyridine-based potent IkappaB Kinase beta (IKKbeta) inhibitors is described. With focuses on the structural optimization at C4 and C6 of structure 1 (Fig. 1), the study reveals that small alkyl and certain aromatic groups are preferred at C4, whereas polar groups with proper orientation at C6 efficiently enhance compound potency. The most potent analogues inhibit IKKbeta with IC50s as low as 40 nM, suppress LPS-induced TNF-alpha production in vitro and in vivo, display good kinase selectivity profiles, and are active in a HeLa cell NF-kappaB reporter gene assay, demonstrating that they directly interfere with the NF-kappaB signaling pathway.

Chemoproteomics-Aided Medicinal Chemistry for the Discovery of EPHA2 Inhibitors.

PubMed

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.

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

Discovery of a Potent BTK Inhibitor with a Novel Binding Mode by Using Parallel Selections with a DNA-Encoded Chemical Library.

PubMed

Cuozzo, John W; Centrella, Paolo A; Gikunju, Diana; Habeshian, Sevan; Hupp, Christopher D; Keefe, Anthony D; Sigel, Eric A; Soutter, Holly H; Thomson, Heather A; Zhang, Ying; Clark, Matthew A

2017-05-04

We have identified and characterized novel potent inhibitors of Bruton's tyrosine kinase (BTK) from a single DNA-encoded library of over 110 million compounds by using multiple parallel selection conditions, including variation in target concentration and addition of known binders to provide competition information. Distinct binding profiles were observed by comparing enrichments of library building block combinations under these conditions; one enriched only at high concentrations of BTK and was competitive with ATP, and another enriched at both high and low concentrations of BTK and was not competitive with ATP. A compound representing the latter profile showed low nanomolar potency in biochemical and cellular BTK assays. Results from kinetic mechanism of action studies were consistent with the selection profiles. Analysis of the co-crystal structure of the most potent compound demonstrated a novel binding mode that revealed a new pocket in BTK. Our results demonstrate that profile-based selection strategies using DNA-encoded libraries form the basis of a new methodology to rapidly identify small molecule inhibitors with novel binding modes to clinically relevant targets. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms

PubMed Central

2015-01-01

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile. PMID:26288683

Interstitial lung disease induced by alectinib (CH5424802/RO5424802).

PubMed

Ikeda, Satoshi; Yoshioka, Hiroshige; Arita, Machiko; Sakai, Takahiro; Sone, Naoyuki; Nishiyama, Akihiro; Niwa, Takashi; Hotta, Machiko; Tanaka, Tomohiro; Ishida, Tadashi

2015-02-01

A 75-year-old woman with anaplastic lymphoma kinase (ALK)-rearranged Stage IV lung adenocarcinoma was administered the selective anaplastic lymphoma kinase inhibitor, alectinib, as a third-line treatment in a Phase 1-2 study. On the 102nd day, chest computed tomography showed diffuse ground glass opacities. Laboratory data revealed high serum levels of KL-6, SP-D and lactate dehydrogenase without any clinical symptoms. There was no evidence of infection. Marked lymphocytosis was seen in bronchoalveolar lavage fluid analysis, and transbronchial lung biopsy showed mild thickening of alveolar septa and lymphocyte infiltration. Interstitial lung disease was judged to be related to alectinib based on improvements in imaging findings and serum biomarkers after discontinuation of alectinib. To our knowledge, this is the first reported case of alectinib-induced interstitial lung disease. Alectinib is a promising drug for ALK-rearranged non-small cell lung cancer. Clinical trials of this selective anaplastic lymphoma kinase inhibitor will facilitate the meticulous elucidation of its long-term safety profile. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of Biomarkers of Impaired Sensory Profiles among Autistic Patients

PubMed Central

El-Ansary, Afaf; Hassan, Wail M.; Qasem, Hanan; Das, Undurti N.

2016-01-01

Background Autism is a neurodevelopmental disorder that displays significant heterogeneity. Comparison of subgroups within autism, and analyses of selected biomarkers as measure of the variation of the severity of autistic features such as cognitive dysfunction, social interaction impairment, and sensory abnormalities might help in understanding the pathophysiology of autism. Methods and Participants In this study, two sets of biomarkers were selected. The first included 7, while the second included 6 biomarkers. For set 1, data were collected from 35 autistic and 38 healthy control participants, while for set 2, data were collected from 29 out of the same 35 autistic and 16 additional healthy subjects. These markers were subjected to a principal components analysis using either covariance or correlation matrices. Moreover, libraries composed of participants categorized into units were constructed. The biomarkers used include, PE (phosphatidyl ethanolamine), PS (phosphatidyl serine), PC (phosphatidyl choline), MAP2K1 (Dual specificity mitogen-activated protein kinase kinase 1), IL-10 (interleukin-10), IL-12, NFκB (nuclear factor-κappa B); PGE2 (prostaglandin E2), PGE2-EP2, mPGES-1 (microsomal prostaglandin synthase E-1), cPLA2 (cytosolic phospholipase A2), 8-isoprostane, and COX-2 (cyclo-oxygenase-2). Results While none of the studied markers correlated with CARS and SRS as measure of cognitive and social impairments, six markers significantly correlated with sensory profiles of autistic patients. Multiple regression analysis identifies a combination of PGES, mPGES-1, and PE as best predictors of the degree of sensory profile impairment. Library identification resulted in 100% correct assignments of both autistic and control participants based on either set 1 or 2 biomarkers together with a satisfactory rate of assignments in case of sensory profile impairment using different sets of biomarkers. Conclusion The two selected sets of biomarkers were effective to separate autistic from healthy control subjects, demonstarting the possibility to accurately predict the severity of autism using the selected biomarkers. The effectiveness of the identified libraries lied in the fact that they were helpful in correctly assigning the study population as control or autistic patients and in classifying autistic patients with different degree of sensory profile impairment. PMID:27824861

Journey of the ALK-inhibitor CH5424802 to phase II clinical trial.

PubMed

Latif, Muhammad; Saeed, Aamer; Kim, Seong Hwan

2013-09-01

The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase represents a potential therapeutic target. Specially, a variety of alterations in the ALK gene including mutations, overexpression, amplification, translocations and structural rearrangements, are involved in human cancer tumorigenesis. The second-generation ALK inhibitor CH5424802 (development code: AF802; Chugai Pharmaceutical, a subsidiary of Roche) achieves tumor regression with excellent tolerance and shows promising efficacy in patients with ALK-positive non-small cell lung cancer. CH5424802 shows good kinase selectivity, has a promising pharmacokinetics profile, and has strong antiproliferative activity in several ALK-driven tumor models. CH5424802 has also shown anti-tumor activity in mouse xenograft studies. Here, we summarize recent advances and the evidence that CH5424802 acts as an ALK inhibitor. We also discuss its potential for further development as an anticancer drug in clinical trials.

Discovery of benzofuran-3(2H)-one derivatives as novel DRAK2 inhibitors that protect islet β-cells from apoptosis.

PubMed

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.

Establishment of a structure-activity relationship of 1H-imidazo[4,5-c]quinoline-based kinase inhibitor NVP-BEZ235 as a lead for African sleeping sickness.

PubMed

Seixas, João D; Luengo-Arratta, Sandra A; Diaz, Rosario; Saldivia, Manuel; Rojas-Barros, Domingo I; Manzano, Pilar; Gonzalez, Silvia; Berlanga, Manuela; Smith, Terry K; Navarro, Miguel; Pollastri, Michael P

2014-06-12

Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure-activity relationships for analogues of 1 and have prepared analogues of 1 with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases.

IL-17A Mediates a Selective Gene Expression Profile in Asthmatic Human Airway Smooth Muscle Cells

PubMed Central

Dragon, Stéphane; Hirst, Stuart J.; Lee, Tak H.

2014-01-01

Airway smooth muscle (ASM) cells are thought to contribute to the pathogenesis of allergic asthma by orchestrating and perpetuating airway inflammation and remodeling responses. In this study, we evaluated the IL-17RA signal transduction and gene expression profile in ASM cells from subjects with mild asthma and healthy individuals. Human primary ASM cells were treated with IL-17A and probed by the Affymetrix GeneChip array, and gene targets were validated by real-time quantitative RT-PCR. Genomic analysis underlined the proinflammatory nature of IL-17A, as multiple NF-κB regulatory factors and chemokines were induced in ASM cells. Transcriptional regulators consisting of primary response genes were overrepresented and displayed dynamic expression profiles. IL-17A poorly enhanced IL-1β or IL-22 gene responses in ASM cells from both subjects with mild asthma and healthy donors. Interestingly, protein modifications to the NF-κB regulatory network were not observed after IL-17A stimulation, although oscillations in IκBε expression were detected. ASM cells from subjects with mild asthma up-regulated more genes with greater overall variability in response to IL-17A than from healthy donors. Finally, in response to IL-17A, ASM cells displayed rapid activation of the extracellular signal–regulated kinase/ribosomal S6 kinase signaling pathway and increased nuclear levels of phosphorylated extracellular signal–regulated kinase. Taken together, our results suggest that IL-17A mediated modest gene expression response, which, in cooperation with the NF-κB signaling network, may regulate the gene expression profile in ASM cells. PMID:24393021

Inhibitors of cyclin-dependent kinases as cancer therapeutics.

PubMed

Whittaker, Steven R; Mallinger, Aurélie; Workman, Paul; Clarke, Paul A

2017-05-01

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

Targeting the cyclin D–cyclin-dependent kinase (CDK)4/6–retinoblastoma pathway with selective CDK 4/6 inhibitors in hormone receptor-positive breast cancer: rationale, current status, and future directions

PubMed Central

Spring, Laura; Bardia, Aditya; Modi, Shanu

2017-01-01

Dysregulation of the cyclin D–cyclin-dependent kinase (CDK)4/6–INK4–retinoblastoma (Rb) pathway is an important contributor to endocrine therapy resistance. Recent clinical development of selective inhibitors of CDK4 and CDK6 kinases has led to renewed interest in cell cycle regulators, following experience with relatively nonselective pan-CDK inhibitors that often resulted in limited activity and poor safety profiles in the clinic. The highly selective oral CDK 4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219) are able to inhibit the proliferation of Rb-positive tumor cells and have demonstrated dose-dependent growth inhibition in ER+ breast cancer models. In metastatic breast cancer, all three agents are being explored in combination with endocrine therapy in Phase III studies. Results so far indicate promising efficacy and manageable safety profiles, and led to the FDA approval of palbociclib. Phase II–III studies of these agents, in combination with endocrine therapy, are also underway in early breast cancer in the neoadjuvant and adjuvant settings. Selective CDK 4/6 inhibitors are also being investigated with other targeted agents or chemotherapy in the advanced setting. This article reviews the rationale for targeting cyclin D–CDK 4/6 in hormone receptor-positive (HR+) breast cancer, provides an overview of the available preclinical and clinical data with CDK 4/6 inhibitors in breast cancer to date, and summarizes the main features of ongoing clinical trials of these new agents in breast cancer. Future trials evaluating further combinations strategies with CDK 4/6 backbone and translational studies refining predictive biomarkers are needed to help personalize the optimal treatment regimen for individual patients with ER+ breast cancer. PMID:26896604

Changes in glycolytic enzyme activities in aging erythrocytes fractionated by counter-current distribution in aqueous polymer two-phase systems.

PubMed Central

Jimeno, P; Garcia-Perez, A I; Luque, J; Pinilla, M

1991-01-01

Human and rat erythrocytes were fractionated by counter-current distribution in charge-sensitive dextran/poly(ethylene glycol) two-phase systems. The specific activities of the key glycolytic enzymes (hexokinase, phosphofructokinase and pyruvate kinase) declined along the distribution profiles, although the relative positions of the activity profiles were reversed in the two species. These enzymes maintained their normal response to specific regulatory effectors in all cell fractions. No variations were observed for phosphoglycerate kinase and bisphosphoglycerate mutase activities. Some correlations between enzyme activities (pyruvate kinase/hexokinase, pyruvate kinase/phosphofructokinase, pyruvate kinase/pyruvate kinase plus phosphoglycerate kinase, pyruvate kinase/bisphosphoglycerate mutase and phosphoglycerate kinase/bisphosphoglycerate mutase ratios) were studied in whole erythrocyte populations as well as in cell fractions. These results strongly support the fractionation of human erythrocytes according to cell age, as occurs with rat erythrocytes. PMID:1656939

Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity

PubMed Central

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

Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

PubMed Central

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

Discovery of a potent, covalent BTK inhibitor for B-cell lymphoma.

PubMed

Wu, Hong; Wang, Wenchao; Liu, Feiyang; Weisberg, Ellen L; Tian, Bei; Chen, Yongfei; Li, Binhua; Wang, Aoli; Wang, Beilei; Zhao, Zheng; McMillin, Douglas W; Hu, Chen; Li, Hong; Wang, Jinhua; Liang, Yanke; Buhrlage, Sara J; Liang, Junting; Liu, Jing; Yang, Guang; Brown, Jennifer R; Treon, Steven P; Mitsiades, Constantine S; Griffin, James D; Liu, Qingsong; Gray, Nathanael S

2014-05-16

BTK is a member of the TEC family of non-receptor tyrosine kinases whose deregulation has been implicated in a variety of B-cell-related diseases. We have used structure-based drug design in conjunction with kinome profiling and cellular assays to develop a potent, selective, and irreversible BTK kinase inhibitor, QL47, which covalently modifies Cys481. QL47 inhibits BTK kinase activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of a downstream effector PLCγ2 (Tyr759) with an EC50 of 318 nM. In Ramos cells QL47 induces a G1 cell cycle arrest that is associated with pronounced degradation of BTK protein. QL47 inhibits the proliferation of B-cell lymphoma cancer cell lines at submicromolar concentrations.

Itraconazole decreases left ventricular contractility in isolated rabbit heart: Mechanism of action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu, Yusheng, E-mail: yqu@amgen.com; Fang, Mei; Gao, BaoXi

Itraconazole (ITZ) is an approved antifungal agent that carries a “black box warning” in its label regarding a risk of negative cardiac inotropy based on clinical findings. Since the mechanism of the negative inotropic effect is unknown, we performed a variety of preclinical and mechanistic studies to explore the pharmacological profile of ITZ and understand the negative inotropic mechanism. ITZ was evaluated in: (1) an isolated rabbit heart (IRH) preparation using Langendorff retrograde perfusion; (2) ion channel studies; (3) a rat heart mitochondrial function profiling screen; (4) a mitochondrial membrane potential (MMP) assay; (5) in vitro pharmacology profiling assays (148more » receptors, ion channels, transporters, and enzymes); and (6) a kinase selectivity panel (451 kinases). In the IRH, ITZ decreased cardiac contractility (> 30%) at 0.3 μM, with increasing effect at higher concentrations, which indicated a direct negative inotropic effect upon the heart. It also decreased heart rate and coronary flow (≥ 1 μM) and prolonged PR/QRS intervals (3 μM). In mechanistic studies, ITZ inhibited the cardiac NaV channel (IC{sub 50}: 4.2 μM) and was devoid of any functional inhibitory effect at the remaining pharmacological targets. Lastly, ITZ did not affect MMP, nor interfere with mitochondrial enzymes or processes involved with fuel substrate utilization or energy formation. Overall, the cardiovascular and mechanistic data suggest that ITZ-induced negative inotropy is a direct effect on the heart, in addition, the potential involvement of mitochondria function and L-type Ca{sup 2+} channels are eliminated. The exact mechanism underlying the negative inotropy is uncertain, and requires further study. - Highlights: ► Effect of itraconazole (ITZ) was assessed in the isolated rabbit heart (IRH) assay. ► ITZ decreased ventricular contractility in IRH, indicating a direct effect. ► IC{sub 50} of ITZ on L-type I{sub Ca} was greater than 30 μM, on I{sub Na} was 4.2 μM. ► ITZ had minimal effects on mitochondrial functions. ► ITZ had minimal hits in pharmacology profiling and kinase selectivity panel.« less

Discovery of highly selective inhibitors of p38alpha.

PubMed

Popa-Burke, Ioana; Birkos, Steve; Blackwell, Leonard; Cheatham, Lynn; Clark, Jennifer; Dickson, John K; Galasinski, Scott; Janzen, William P; Mendoza, Jose; Miller, Jennifer L; Mohney, Robert P; Steed, Paul M; Hodge, C Nicholas

2005-01-01

The p38 MAP kinases are a family of serine/threonine protein kinases that play a key role in cellular pathways leading to pro-inflammatory responses. We have developed and implemented a method for rapidly identifying and optimizing potent and selective p38alpha inhibitors, which is amenable to other targets and target classes. A diverse library of druggable, purified and quantitated molecules was assembled and standardized enzymatic assays were performed in a microfluidic format that provided very accurate and precise inhibition data allowing for development of SAR directly from the primary HTS. All compounds were screened against a collection of more than 60 enzymes (kinases, proteases and phosphatases), allowing for removal of promiscuous and non-selective inhibitors very early in the discovery process. Follow-up enzymological studies included measurement of concentration of compound in buffer, yielding accurate determination of K(i) and IC50 values, as well as mechanism of action. In addition, active compounds were screened against less desirable properties such as inhibition of the enzyme activity by aggregation, irreversible binding, and time-dependence. Screening of an 88,634-compound library through the above-described process led to the rapid identification of multiple scaffolds (>5 active compounds per scaffold) of potential drug leads for p38alpha that are highly selective against all other enzymes tested, including the three other p38 isoforms. Potency and selectivity data allowed prioritization of the identified scaffolds for optimization. Herein we present results around our 3-thio-1,2,4-triazole lead series of p38- selective inhibitors, including identification, SAR, synthesis, selectivity profile, enzymatic and cellular data in their progression towards drug candidates.

Identification of Atuveciclib (BAY 1143572), the First Highly Selective, Clinical PTEFb/CDK9 Inhibitor for the Treatment of Cancer

PubMed Central

Scholz, Arne; Lienau, Philip; Siemeister, Gerhard; Kosemund, Dirk; Bohlmann, Rolf; Briem, Hans; Terebesi, Ildiko; Meyer, Kirstin; Prelle, Katja; Denner, Karsten; Bömer, Ulf; Schäfer, Martina; Eis, Knut; Valencia, Ray; Ince, Stuart; von Nussbaum, Franz; Mumberg, Dominik; Ziegelbauer, Karl; Klebl, Bert; Choidas, Axel; Nussbaumer, Peter; Baumann, Matthias; Schultz‐Fademrecht, Carsten; Rühter, Gerd; Eickhoff, Jan; Brands, Michael

2017-01-01

Abstract Selective inhibition of exclusively transcription‐regulating PTEFb/CDK9 is a promising new approach in cancer therapy. Starting from lead compound BAY‐958, lead optimization efforts strictly focusing on kinase selectivity, physicochemical and DMPK properties finally led to the identification of the orally available clinical candidate atuveciclib (BAY 1143572). Structurally characterized by an unusual benzyl sulfoximine group, BAY 1143572 exhibited the best overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats. BAY 1143572 is the first potent and highly selective PTEFb/CDK9 inhibitor to enter clinical trials for the treatment of cancer. PMID:28961375

Identification of methyl violet 2B as a novel blocker of focal adhesion kinase signaling pathway in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hwan; Translational Research Center for Protein Function Control; Kim, Nam Doo

2013-07-26

Highlights: •FAK signaling cascade in cancer cells is profoundly inhibited by methyl violet 2B. •Methyl violet 2B identified by virtual screening is a novel allosteric FAK inhibitor. •Methyl violet 2B possesses extremely high kinase selectivity. •Methyl violet 2B suppresses strongly the proliferation of cancer cells. •Methyl violet 2B inhibits focal adhesion, invasion and migration of cancer cells. -- Abstract: The focal adhesion kinase (FAK) signaling cascade in cancer cells was profoundly inhibited by methyl violet 2B identified with the structure-based virtual screening. Methyl violet 2B was shown to be a non-competitive inhibitor of full-length FAK enzyme vs. ATP. It turnedmore » out that methyl violet 2B possesses extremely high kinase selectivity in biochemical kinase profiling using a large panel of kinases. Anti-proliferative activity measurement against several different cancer cells and Western blot analysis showed that this substance is capable of suppressing significantly the proliferation of cancer cells and is able to strongly block FAK/AKT/MAPK signaling pathways in a dose dependent manner at low nanomolar concentration. Especially, phosphorylation of Tyr925-FAK that is required for full activation of FAK was nearly completely suppressed even with 1 nM of methyl violet 2B in A375P cancer cells. To the best of our knowledge, it has never been reported that methyl violet possesses anti-cancer effects. Moreover, methyl violet 2B significantly inhibited FER kinase phosphorylation that activates FAK in cell. In addition, methyl violet 2B was found to induce cell apoptosis and to exhibit strong inhibitory effects on the focal adhesion, invasion, and migration of A375P cancer cells at low nanomolar concentrations. Taken together, these results show that methyl violet 2B is a novel, potent and selective blocker of FAK signaling cascade, which displays strong anti-proliferative activities against a variety of human cancer cells and suppresses adhesion/migration/invasion of tumor cells.« less

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

PubMed

Proszkowiec-Weglarz, M; Richards, M P

2009-01-01

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Comparison of acalabrutinib, a selective Bruton tyrosine kinase inhibitor, with ibrutinib in chronic lymphocytic leukemia cells

PubMed Central

Patel, Viralkumar; Balakrishnan, Kumudha; Bibikova, Elena; Ayres, Mary; Keating, Michael J.; Wierda, William G.; Gandhi, Varsha

2017-01-01

Purpose Ibrutinib inhibits Bruton tyrosine kinase (BTK) by irreversibly binding to the Cys-481 residue in the enzyme. However, ibrutinib also inhibits several other enzymes that contain cysteine residues homologous to Cys-481 in BTK. Patients with relapsed/refractory or previously untreated chronic lymphocytic leukemia (CLL) demonstrate a high overall response rate to ibrutinib with prolonged survival. Acalabrutinib, a selective BTK inhibitor developed to minimize off-target activity, has shown promising overall response rates in patients with relapsed/refractory CLL. A head-to-head comparison of ibrutinib and acalabrutinib in CLL cell cultures and healthy T cells is needed to understand preclinical biologic and molecular effects. Experimental Design Using samples from patients with CLL, we compared the effects of both BTK inhibitors on biologic activity, chemokine production, cell migration, BTK phosphorylation, and downstream signaling in primary CLL lymphocytes and on normal T-cell signaling to determine effects on other kinases. Results Both BTK inhibitors induced modest cell death accompanied by cleavage of PARP and caspase 3. Production of CCL3 and CCL4 chemokines and pseudoemperipolesis were inhibited by both drugs to a similar degree. These drugs also showed similar inhibitory effects on phosphorylation of BTK and downstream S6 and ERK kinases. By contrast, off-target effects on SRC-family kinases were more pronounced with ibrutinib than acalabrutinib in healthy T lymphocytes. Conclusion Both BTK inhibitors show similar biological and molecular profile in primary CLL cells but appear different on their effect on normal T-cells. PMID:28034907

Structure-Based Design and Synthesis of Harmine Derivatives with Different Selectivity Profiles in Kinase versus Monoamine Oxidase Inhibition.

PubMed

Bálint, Balázs; Wéber, Csaba; Cruzalegui, Francisco; Burbridge, Mike; Kotschy, Andras

2017-06-21

Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is an emerging biological target with implications in diverse therapeutic areas such as neurological disorders (Down syndrome, in particular), metabolism, and oncology. Harmine, a natural product that selectively inhibits DYRK1A amongst kinases, could serve as a tool compound to better understand the biological processes that arise from DYRK1A inhibition. On the other hand, harmine is also a potent inhibitor of monoamine oxidase A (MAO-A). Using structure-based design, we synthesized a collection of harmine analogues with tunable selectivity toward these two enzymes. Modifications at the 7-position typically decreased affinity for DYRK1A, whereas substitution at the 9-position had a similar effect on MAO-A inhibition but DYRK1A inhibition was maintained. The resulting collection of compounds can help to understand the biological role of DYRK1A and also to assess the interference in the biological effect originating in MAO-A inhibition. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The selectivity of protein kinase inhibitors: a further update

PubMed Central

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

Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

PubMed

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.

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

PubMed Central

Tahiri, Andliena; Røe, Kathrine; Ree, Anne H.; de Wijn, Rik; Risberg, Karianne; Busch, Christian; Lønning, Per E.; Kristensen, Vessela; Geisler, Jürgen

2013-01-01

Background Treatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed. Methodology In this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status. Results Overall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro. Conclusions Our findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers. PMID:24023633

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

PubMed

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.

Discovery of a highly selective glycogen synthase kinase-3 inhibitor (PF-04802367) that modulates tau phosphorylation in brain: Translation for PET neuroimaging

PubMed Central

Liang, Steven H.; Chen, Jinshan Michael; Normandin, Marc D.; Chang, Jeanne S.; Chang, George C.; Taylor, Christine K.; Trapa, Patrick; Plummer, Mark S.; Para, Kimberly S.; Conn, Edward L.; Lopresti-Morrow, Lori; Lanyon, Lorraine F.; Cook, James M.; Richter, Karl E. G.; Nolan, Charlie E.; Schachter, Joel B.; Janat, Fouad; Che, Ye; Shanmugasundaram, Veerabahu; Lefker, Bruce A.; Enerson, Bradley E.; Livni, Elijahu; Wang, Lu; Guehl, Nicolas; Patnaik, Debasis; Wagner, Florence F.; Perlis, Roy; Holson, Edward B.; Haggarty, Stephen J.; Fakhri, Georges El

2016-01-01

Glycogen synthase kinase-3 (GSK-3) regulates multiple cellular processes in diabetes, oncology and neurology. We have identified N-(3-(1H-1,2,4-triazol-1-yl)propyl)-5-(3-chloro-4-methoxyphenyl)oxazole-4-carboxamide (PF-04802367 or PF-367) as a highly potent inhibitor, which is among the most selective antagonists of GSK-3 to date. We demonstrated its efficacy in modulation of tau phosphorylation in vitro and in vivo. Whereas the kinetics of PF-367 binding in brain tissues are too fast for an effective therapeutic agent, the pharmacokinetic profile of PF-367 is ideal for discovery of radiopharmaceuticals for GSK-3 in the central nervous system. A 11C-isotopologue of PF-367 was synthesized and preliminary PET imaging studies in non-human primates confirmed that we have overcome the two major obstacles for imaging GSK-3, namely, reasonable brain permeability and displaceable binding. PMID:27355874

A systematic profile of clinical inhibitors responsive to EGFR somatic amino acid mutations in lung cancer: implication for the molecular mechanism of drug resistance and sensitivity.

PubMed

Ai, Xinghao; Sun, Yingjia; Wang, Haidong; Lu, Shun

2014-07-01

Human epidermal growth factor receptor (EGFR) has become a well-established target for the treatment of patients with non-small cell lung cancer (NSCLC). However, a large number of somatic mutations in such protein have been observed to cause drug resistance or sensitivity during pathological progression, limiting the application of reversible EGFR tyrosine kinase inhibitor therapy in NSCLC. In the current work, we describe an integration of in silico analysis and in vitro assay to profile six representative EGFR inhibitors against a panel of 71 observed somatic mutations in EGFR tyrosine kinase domain. In the procedure, the changes in interaction free energy of inhibitors with EGFR upon various mutations were calculated one by one using a rigorous computational scheme, which was preoptimized based on a set of structure-solved, affinity-known samples to improve its performance in characterizing the EGFR-inhibitor system. This method was later demonstrated to be effective in inferring drug response to the classical L858R and G719S mutations that confer constitutive activation for the EGFR kinase. It is found that the Staurosporine, a natural product isolated from the bacterium Streptomyces staurosporeus, exhibits selective inhibitory activity on the T790M and T790M/L858R mutants. This finding was subsequently solidified by in vitro kinase assay experiment; the inhibitory IC50 values of Staurosporine against wild-type, T790M and T790M/L858R mutant EGFR were measured to be 937, 12 and 3 nM, respectively.

The First Pentacyclic Triterpenoid Gypsogenin Derivative Exhibiting Anti-ABL1 Kinase and Anti-chronic Myelogenous Leukemia Activities.

PubMed

Ciftci, Halil Ibrahim; Ozturk, Safiye Emirdag; Ali, Taha F S; Radwan, Mohamed O; Tateishi, Hiroshi; Koga, Ryoko; Ocak, Zeynep; Can, Mustafa; Otsuka, Masami; Fujita, Mikako

2018-04-01

The discovery of the chimeric tyrosine kinase breakpoint cluster region kinase-Abelson kinase (BCR-ABL)-targeted drug imatinib conceptually changed the treatment of chronic myelogenous leukemia (CML). However, some CML patients show drug resistance to imatinib. To address this issue, some artificial heterocyclic compounds have been identified as BCR-ABL inhibitors. Here we examined whether plant-derived pentacyclic triterpenoid gypsogenin and/or their derivatives show inhibitory activity against BCR-ABL. Among the three derivatives, benzyl 3-hydroxy-23-oxoolean-12-en-28-oate (1c) was found to be the most effective anticancer agent on the CML cell line K562, with an IC 50 value of 9.3 µM. In contrast, the IC 50 against normal peripheral blood mononuclear cells was 276.0 µM, showing better selectivity than imatinib. Compound 1c had in vitro inhibitory activity against Abelson kinase 1 (ABL1) (IC 50 =8.7 µM), the kinase component of BCR-ABL. In addition, compound 1c showed a different inhibitory profile against eight kinases compared with imatinib. The interaction between ATP binding site of ABL and 1c was examined by molecular docking study, and the binding mode was different from imatinib and newer generation inhibitors. Furthermore, 1c suppressed signaling downstream of BCR-ABL. This study suggests the possibility that plant extracts may be a source for CML treatment and offer a strategy to overcome drug resistance to known BCR-ABL inhibitors.

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

PubMed Central

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

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2).

PubMed

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.

Spectrum of activity and mechanisms of resistance of various nucleoside derivatives against gammaherpesviruses.

PubMed

Coen, Natacha; Duraffour, Sophie; Topalis, Dimitri; Snoeck, Robert; Andrei, Graciela

2014-12-01

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Spectrum of Activity and Mechanisms of Resistance of Various Nucleoside Derivatives against Gammaherpesviruses

PubMed Central

Coen, Natacha; Duraffour, Sophie; Topalis, Dimitri; Snoeck, Robert

2014-01-01

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-d-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2′-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase. PMID:25267682

Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK) inhibitor.

PubMed

Liang, Qianmao; Chen, Yongfei; Yu, Kailin; Chen, Cheng; Zhang, Shouxiang; Wang, Aoli; Wang, Wei; Wu, Hong; Liu, Xiaochuan; Wang, Beilei; Wang, Li; Hu, Zhenquan; Wang, Wenchao; Ren, Tao; Zhang, Shanchun; Liu, Qingsong; Yun, Cai-Hong; Liu, Jing

2017-05-05

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC 50 : 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 μM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC 50 : <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Bioinformatics identification and transcript profile analysis of the mitogen-activated protein kinase gene family in the diploid woodland strawberry Fragaria vesca

PubMed Central

Wei, Wei; Chai, Zhuangzhuang; Xie, Yinge; Gao, Kuan; Cui, Mengyuan; Jiang, Ying

2017-01-01

Mitogen-activated protein kinases (MAPKs) play essential roles in mediating biotic and abiotic stress responses in plants. However, the MAPK gene family in strawberry has not been systematically characterized. Here, we performed a genome-wide survey and identified 12 MAPK genes in the Fragaria vesca genome. Protein domain analysis indicated that all FvMAPKs have typical protein kinase domains. Sequence alignments and phylogenetic analysis classified the FvMAPK genes into four different groups. Conserved motif and exon-intron organization supported the evolutionary relationships inferred from the phylogenetic analysis. Analysis of the stress-related cis-regulatory element in the promoters and subcellular localization predictions of FvMAPKs were also performed. Gene transcript profile analysis showed that the majority of the FvMAPK genes were ubiquitously transcribed in strawberry leaves after Podosphaera aphanis inoculation and after treatment with cold, heat, drought, salt and the exogenous hormones abscisic acid, ethephon, methyl jasmonate, and salicylic acid. RT-qPCR showed that six selected FvMAPK genes comprehensively responded to various stimuli. Additionally, interaction networks revealed that the crucial signaling transduction controlled by FvMAPKs may be involved in the biotic and abiotic stress responses. Our results may provide useful information for future research on the function of the MAPK gene family and the genetic improvement of strawberry resistance to environmental stresses. PMID:28562633

Therapeutic Blockade of Immune Complex-Mediated Glomerulonephritis by Highly Selective Inhibition of Bruton’s Tyrosine Kinase

PubMed Central

Chalmers, Samantha A.; Doerner, Jessica; Bosanac, Todd; Khalil, Sara; Smith, Dustin; Harcken, Christian; Dimock, Janice; Der, Evan; Herlitz, Leal; Webb, Deborah; Seccareccia, Elise; Feng, Di; Fine, Jay S.; Ramanujam, Meera; Klein, Elliott; Putterman, Chaim

2016-01-01

Lupus nephritis (LN) is a potentially dangerous end organ pathology that affects upwards of 60% of lupus patients. Bruton’s tyrosine kinase (BTK) is important for B cell development, Fc receptor signaling, and macrophage polarization. In this study, we investigated the effects of a novel, highly selective and potent BTK inhibitor, BI-BTK-1, in an inducible model of LN in which mice receive nephrotoxic serum (NTS) containing anti-glomerular antibodies. Mice were treated once daily with vehicle alone or BI-BTK-1, either prophylactically or therapeutically. When compared with control treated mice, NTS-challenged mice treated prophylactically with BI-BTK-1 exhibited significantly attenuated kidney disease, which was dose dependent. BI-BTK-1 treatment resulted in decreased infiltrating IBA-1+ cells, as well as C3 deposition within the kidney. RT-PCR on whole kidney RNA and serum profiling indicated that BTK inhibition significantly decreased levels of LN-relevant inflammatory cytokines and chemokines. Renal RNA expression profiling by RNA-seq revealed that BI-BTK-1 dramatically modulated pathways related to inflammation and glomerular injury. Importantly, when administered therapeutically, BI-BTK-1 reversed established proteinuria and improved renal histopathology. Our results highlight the important role for BTK in the pathogenesis of immune complex-mediated nephritis, and BTK inhibition as a promising therapeutic target for LN. PMID:27192942

Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance.

PubMed

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.

The Selective PI3K Inhibitor XL147 (SAR245408) Inhibits Tumor Growth and Survival and Potentiates the Activity of Chemotherapeutic Agents in Preclinical Tumor Models.

PubMed

Foster, Paul; Yamaguchi, Kyoko; Hsu, Pin P; Qian, Fawn; Du, Xiangnan; Wu, Jianming; Won, Kwang-Ai; Yu, Peiwen; Jaeger, Christopher T; Zhang, Wentao; Marlowe, Charles K; Keast, Paul; Abulafia, Wendy; Chen, Jason; Young, Jenny; Plonowski, Artur; Yakes, F Michael; Chu, Felix; Engell, Kelly; Bentzien, Frauke; Lam, Sanh T; Dale, Stephanie; Yturralde, Olivia; Matthews, David J; Lamb, Peter; Laird, A Douglas

2015-04-01

Dysregulation of PI3K/PTEN pathway components, resulting in hyperactivated PI3K signaling, is frequently observed in various cancers and correlates with tumor growth and survival. Resistance to a variety of anticancer therapies, including receptor tyrosine kinase (RTK) inhibitors and chemotherapeutic agents, has been attributed to the absence or attenuation of downregulating signals along the PI3K/PTEN pathway. Thus, PI3K inhibitors have therapeutic potential as single agents and in combination with other therapies for a variety of cancer indications. XL147 (SAR245408) is a potent and highly selective inhibitor of class I PI3Ks (α, β, γ, and δ). Moreover, broad kinase selectivity profiling of >130 protein kinases revealed that XL147 is highly selective for class I PI3Ks over other kinases. In cellular assays, XL147 inhibits the formation of PIP3 in the membrane, and inhibits phosphorylation of AKT, p70S6K, and S6 in multiple tumor cell lines with diverse genetic alterations affecting the PI3K pathway. In a panel of tumor cell lines, XL147 inhibits proliferation with a wide range of potencies, with evidence of an impact of genotype on sensitivity. In mouse xenograft models, oral administration of XL147 results in dose-dependent inhibition of phosphorylation of AKT, p70S6K, and S6 with a duration of action of at least 24 hours. Repeat-dose administration of XL147 results in significant tumor growth inhibition in multiple human xenograft models in nude mice. Administration of XL147 in combination with chemotherapeutic agents results in antitumor activity in xenograft models that is enhanced over that observed with the corresponding single agents. ©2015 American Association for Cancer Research.

Synthesis and structure-activity relationship study of pyrazolo[3,4-d]pyrimidines as tyrosine kinase RET inhibitors.

PubMed

Wang, Chengyan; Liu, Hongchun; Song, Zilan; Ji, Yinchun; Xing, Li; Peng, Xia; Wang, Xisheng; Ai, Jing; Geng, Meiyu; Zhang, Ao

2017-06-01

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhibitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile. Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity, 23c did not show significant KDR-inhibition even at the concentration of 1μM. These results demonstrated that 23c is a potent and selective RET inhibitor. Copyright © 2017 Elsevier Ltd. All rights reserved.

Predictive biomarkers of sensitivity to the phosphatidylinositol 3' kinase inhibitor GDC-0941 in breast cancer preclinical models.

PubMed

O'Brien, Carol; Wallin, Jeffrey J; Sampath, Deepak; GuhaThakurta, Debraj; Savage, Heidi; Punnoose, Elizabeth A; Guan, Jane; Berry, Leanne; Prior, Wei Wei; Amler, Lukas C; Belvin, Marcia; Friedman, Lori S; Lackner, Mark R

2010-07-15

The class I phosphatidylinositol 3' kinase (PI3K) plays a major role in proliferation and survival in a wide variety of human cancers. A key factor in successful development of drugs targeting this pathway is likely to be the identification of responsive patient populations with predictive diagnostic biomarkers. This study sought to identify candidate biomarkers of response to the selective PI3K inhibitor GDC-0941. We used a large panel of breast cancer cell lines and in vivo xenograft models to identify candidate predictive biomarkers for a selective inhibitor of class I PI3K that is currently in clinical development. The approach involved pharmacogenomic profiling as well as analysis of gene expression data sets from cells profiled at baseline or after GDC-0941 treatment. We found that models harboring mutations in PIK3CA, amplification of human epidermal growth factor receptor 2, or dual alterations in two pathway components were exquisitely sensitive to the antitumor effects of GDC-0941. We found that several models that do not harbor these alterations also showed sensitivity, suggesting a need for additional diagnostic markers. Gene expression studies identified a collection of genes whose expression was associated with in vitro sensitivity to GDC-0941, and expression of a subset of these genes was found to be intimately linked to signaling through the pathway. Pathway focused biomarkers and the gene expression signature described in this study may have utility in the identification of patients likely to benefit from therapy with a selective PI3K inhibitor. Copyright 2010 AACR.

Synthesis, biological evaluation, and molecular dynamics (MD) simulation studies of three novel F-18 labeled and focal adhesion kinase (FAK) targeted 5-bromo pyrimidines as radiotracers for tumor.

PubMed

Fang, Yu; Wang, Dawei; Xu, Xingyu; Liu, Jianping; Wu, Aiqin; Li, Xiang; Xue, Qianqian; Wang, Huan; Wang, Hang; Zhang, Huabei

2017-02-15

Focal adhesion kinase (FAK) is considered as an attractive target for oncology. A series of F-18 labeled 5-bromo-N 2 -(4-(2-fluoro-pegylated (FPEG))-3,5-dimethoxyphenyl)-N 4 -(4-methoxyphenyl)pyrimidine-2,4-diamine derivatives were prepared and evaluated as the FAK targeted radiotracers for the early diagnoses of tumor. For the study of the FAK targeted drug molecules, this was the first attempt to develop the tumor diagnostic imaging agents on the radiopharmaceutical level. They inhibited the activity of FAK with IC 50 in the range of 91.4-425.7 nM, and among which the result of the [ 19 F]2 was relatively good and had a modest IC 50 of 91.4 nM. The [ 19 F]2 was also profiled in vitro against some other kinds of cancer-related kinases (including two kinds of non-receptor tyrosine kinase: PYK2 and JAK2, and three kinds of receptor tyrosine kinase: IGF-1R, EGFR and PDGFRβ). It displayed 25.2 folds selectivity against PYK2, 35.1 folds selectivity against EGFR, and more than 100 folds selectivity against IGF-1R, JAK2 and PDGFRβ. For the biodistribution in S180 bearing mice, the corresponding [ 18 F]2 were also relatively good, with modest tumor uptake of 5.47 ± 0.19 and 5.80 ± 0.06 %ID/g at 15 and 30 min post-injection, respectively. Furthermore, its tumor/muscle, tumor/bone and tumor/blood ratio at 15 min post-injection were 3.16, 2.53 and 4.52, respectively. And its tumor/muscle, tumor/bone and tumor/blood ratio at 30 min post-injection were 3.14, 2.76 and 4.43, respectively. In addition, coronal micro-PET/CT images of a mouse bearing S180 tumor clearly confirmed that [ 18 F]2 could be accumulated in tumor, especially at 30 min post-injection. Besides, for the [ 18 F]2, both the biodistribution data and the micro-PET/CT imaging study showed significantly reduced uptake of the radiotracer in the tumor tissue at 30 min post-injection in mice that received PF-562,271 (one of the reported best selective FAK inhibitor which was developed by Pfitzer Inc. and inhibited the activity of FAK with IC 50 value of 1.5 nM) at 1 h before the injection of radiotracer. In combination with the above kinase profiling assay, it could be indicated that the uptake of [ 18 F]2 in tumor of the mouse model was due to FAK expression, and that [ 18 F]2 might be a kind of selectively FAK targeted tumor imaging agents. What's more, the results of the MD (molecular dynamics) simulations were in agreement with the changing trends of the interaction between the different F-19 standards and the FAK (expressed as the in vitro inhibitory abilities of enzymatic activities of FAK in this article), which was also in agreement with and had great effect on the changing trends of the uptake of the corresponding F-18 labeled tracers in tumor and some of theirs target/non-target ratios. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Promiscuity and selectivity of small-molecule inhibitors across TAM receptor tyrosine kinases in pediatric leukemia.

PubMed

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.

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

PubMed

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.

Simultaneous inhibition assay for human and microbial kinases via MALDI-MS/MS.

PubMed

Smith, Anne Marie E; Brennan, John D

2014-03-03

Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein Tyrosine Kinase Signaling During Oocyte Maturation and Fertilization

PubMed Central

McGinnis, Lynda K.; Carroll, David J.; Kinsey, William H.

2011-01-01

The oocyte is a highly specialized cell capable of accumulating and storing energy supplies as well as maternal transcripts and pre-positioned signal transduction components needed for zygotic development, undergoing meiosis under control of paracrine signals from the follicle, fusing with a single sperm during fertilization, and zygotic development. The oocyte accomplishes this diverse series of events by establishing an array of signal transduction pathway components that include a select collection of protein tyrosine kinases (PTKs) that are expressed at levels significantly higher than most other cell types. This array of PTKs includes cytosolic kinases such as SRC-family PTKs (FYN and YES), and FAK kinases, as well as FER. These kinases typically exhibit distinct patterns of localization and in some cases are translocated from one subcellular compartment to another during meiosis. Significant differences exist in the extent to which PTK-mediated pathways are used by oocytes from species that fertilize externally versus internally. The PTK activation profiles as well as calcium signaling pattern seems to correlate with the extent to which a rapid block to polyspermy is required by the biology of each species. Suppression of each of the SRC-family PTKs as well as FER kinase results in failure of meiotic maturation or zygote development, indicating that these PTKs are important for oocyte quality and developmental potential. Future studies will hopefully reveal the extent to which these factors impact clinical assisted reproductive techniques in domestic animals and humans. PMID:21681843

BI 885578, a Novel IGF1R/INSR Tyrosine Kinase Inhibitor with Pharmacokinetic Properties That Dissociate Antitumor Efficacy and Perturbation of Glucose Homeostasis.

PubMed

Sanderson, Michael P; Apgar, Joshua; Garin-Chesa, Pilar; Hofmann, Marco H; Kessler, Dirk; Quant, Jens; Savchenko, Alexander; Schaaf, Otmar; Treu, Matthias; Tye, Heather; Zahn, Stephan K; Zoephel, Andreas; Haaksma, Eric; Adolf, Günther R; Kraut, Norbert

2015-12-01

Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosine kinases represents an attractive approach of pharmacologic intervention in cancer, owing to the roles of the IGF1R and INSRA in promoting cell proliferation and survival. However, the central role of the INSRB isoform in glucose homeostasis suggests that prolonged inhibition of this kinase could result in metabolic toxicity. We describe here the profile of the novel compound BI 885578, a potent and selective ATP-competitive IGF1R/INSR tyrosine kinase inhibitor distinguished by rapid intestinal absorption and a short in vivo half-life as a result of rapid metabolic clearance. BI 885578, administered daily per os, displayed an acceptable tolerability profile in mice at doses that significantly reduced the growth of xenografted human GEO and CL-14 colon carcinoma tumors. We found that treatment with BI 885578 is accompanied by increases in circulating glucose and insulin levels, which in turn leads to compensatory hyperphosphorylation of muscle INSRs and subsequent normalization of blood glucose within a few hours. In contrast, the normalization of IGF1R and INSR phosphorylation in GEO tumors occurs at a much slower rate. In accordance with this, BI 885578 led to a prolonged inhibition of cell proliferation and induction of apoptosis in GEO tumors. We propose that the remarkable therapeutic window observed for BI 885578 is achieved by virtue of the distinctive pharmacokinetic properties of the compound, capitalizing on the physiologic mechanisms of glucose homeostasis and differential levels of IGF1R and INSR expression in tumors and normal tissues. ©2015 American Association for Cancer Research.

Genetic predictors of MEK dependence in non-small cell lung cancer.

PubMed

Pratilas, Christine A; Hanrahan, Aphrothiti J; Halilovic, Ensar; Persaud, Yogindra; Soh, Junichi; Chitale, Dhananjay; Shigematsu, Hisayuki; Yamamoto, Hiromasa; Sawai, Ayana; Janakiraman, Manickam; Taylor, Barry S; Pao, William; Toyooka, Shinichi; Ladanyi, Marc; Gazdar, Adi; Rosen, Neal; Solit, David B

2008-11-15

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.

Dynamics of human protein kinase Aurora A linked to drug selectivity

DOE PAGES

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee; ...

2018-06-14

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Dynamics of human protein kinase Aurora A linked to drug selectivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pitsawong, Warintra; Buosi, Vanessa; Otten, Renee

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Auroramore » A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.« less

Structure-Based Design of Orally Bioavailable 1H-Pyrrolo[3,2-c]pyridine Inhibitors of Mitotic Kinase Monopolar Spindle 1 (MPS1)

PubMed Central

2013-01-01

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition. PMID:24256217

Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of mitotic kinase monopolar spindle 1 (MPS1).

PubMed

Naud, Sébastien; Westwood, Isaac M; Faisal, Amir; Sheldrake, Peter; Bavetsias, Vassilios; Atrash, Butrus; Cheung, Kwai-Ming J; Liu, Manjuan; Hayes, Angela; Schmitt, Jessica; Wood, Amy; Choi, Vanessa; Boxall, Kathy; Mak, Grace; Gurden, Mark; Valenti, Melanie; de Haven Brandon, Alexis; Henley, Alan; Baker, Ross; McAndrew, Craig; Matijssen, Berry; Burke, Rosemary; Hoelder, Swen; Eccles, Suzanne A; Raynaud, Florence I; Linardopoulos, Spiros; van Montfort, Rob L M; Blagg, Julian

2013-12-27

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Evaluating the Role of p38 MAPK in the Accelerated Cell Senescence of Werner Syndrome Fibroblasts

PubMed Central

Davis, Terence; Brook, Amy J. C.; Rokicki, Michal J.; Bagley, Mark C.; Kipling, David

2016-01-01

Progeroid syndromes show features of accelerated ageing and are used as models for human ageing, of which Werner syndrome (WS) is one of the most widely studied. WS fibroblasts show accelerated senescence that may result from p38 MAP kinase activation since it is prevented by the p38 inhibitor SB203580. Thus, small molecule inhibition of p38-signalling may be a therapeutic strategy for WS. To develop this approach issues such as the in vivo toxicity and kinase selectivity of existing p38 inhibitors need to be addressed, so as to strengthen the evidence that p38 itself plays a critical role in mediating the effect of SB203580, and to find an inhibitor suitable for in vivo use. In this work we used a panel of different p38 inhibitors selected for: (1) having been used successfully in vivo in either animal models or human clinical trials; (2) different modes of binding to p38; and (3) different off-target kinase specificity profiles, in order to critically address the role of p38 in the premature senescence seen in WS cells. Our findings confirmed the involvement of p38 in accelerated cell senescence and identified p38 inhibitors suitable for in vivo use in WS, with BIRB 796 the most effective. PMID:27136566

Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

PubMed

Stavenger, Robert A; Cui, Haifeng; Dowdell, Sarah E; Franz, Robert G; Gaitanopoulos, Dimitri E; Goodman, Krista B; Hilfiker, Mark A; Ivy, Robert L; Leber, Jack D; Marino, Joseph P; Oh, Hye-Ja; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Zhang, Daohua; Zhao, Yongdong; Jolivette, Larry J; Head, Martha S; Semus, Simon F; Elkins, Patricia A; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Doe, Christopher P; Bentley, Ross; Chen, Zunxuan X; Hu, Erding; Lee, Dennis

2007-01-11

The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

Identification and preclinical characterization of AZ-23, a novel, selective, and orally bioavailable inhibitor of the Trk kinase pathway.

PubMed

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.

Discovery of DNA repair inhibitors by combinatorial library profiling

PubMed Central

Moeller, Benjamin J.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2011-01-01

Small molecule inhibitors of DNA repair are emerging as potent and selective anti-cancer therapies, but the sheer magnitude of the protein networks involved in DNA repair processes poses obstacles to discovery of effective candidate drugs. To address this challenge, we used a subtractive combinatorial selection approach to identify a panel of peptide ligands that bind DNA repair complexes. Supporting the concept that these ligands have therapeutic potential, we show that one selected peptide specifically binds and non-competitively inactivates DNA-PKcs, a protein kinase critical in double-strand DNA break repair. In doing so, this ligand sensitizes BRCA-deficient tumor cells to genotoxic therapy. Our findings establish a platform for large-scale parallel screening for ligand-directed DNA repair inhibitors, with immediate applicability to cancer therapy. PMID:21343400

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition

PubMed Central

Juss, Jatinder K.; House, David; Amour, Augustin; Begg, Malcolm; Herre, Jurgen; Storisteanu, Daniel M. L.; Hoenderdos, Kim; Bradley, Glyn; Lennon, Mark; Summers, Charlotte; Hessel, Edith M.; Condliffe, Alison

2016-01-01

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease’s complex pathophysiology, yet these cells have been little studied. Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. Measurements and Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase–dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells. PMID:27064380

Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

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

BCR-ABL1 tyrosine kinase inhibitors for the treatment of chronic myeloid leukemia.

PubMed

Cuellar, Sandra; Vozniak, Michael; Rhodes, Jill; Forcello, Nicholas; Olszta, Daniel

2017-01-01

The management of chronic myeloid leukemia with BCR-ABL1 tyrosine kinase inhibitors has evolved chronic myeloid leukemia into a chronic, manageable disease. A patient-centered approach is important for the appropriate management of chronic myeloid leukemia and optimization of long-term treatment outcomes. The pharmacist plays a key role in treatment selection, monitoring drug-drug interactions, identification and management of adverse events, and educating patients on adherence. The combination of tyrosine kinase inhibitors with unique safety profiles and individual patients with unique medical histories can make managing treatment difficult. This review will provide up-to-date information regarding tyrosine kinase inhibitor-based treatment of patients with chronic myeloid leukemia. Management strategies for adverse events and considerations for drug-drug interactions will not only vary among patients but also across tyrosine kinase inhibitors. Drug-drug interactions can be mild to severe. In instances where co-administration of concomitant medications cannot be avoided, it is critical to understand how drug levels are impacted and how subsequent dose modifications ensure therapeutic drug levels are maintained. An important component of patient-centered management of chronic myeloid leukemia also includes educating patients on the significance of early and regular monitoring of therapeutic milestones, emphasizing the importance of adhering to treatment in achieving these targets, and appropriately modifying treatment if these clinical goals are not being met. Overall, staying apprised of current research, utilizing the close pharmacist-patient relationship, and having regular interactions with patients, will help achieve successful long-term treatment of chronic myeloid leukemia in the age of BCR-ABL1 tyrosine kinase inhibitors.

Global Impact of Oncogenic Src on a Phosphotyrosine Proteome

PubMed Central

Luo, Weifeng; Slebos, Robbert J.; Hill, Salisha; Li, Ming; Brábek, Jan; Amanchy, Ramars; Chaerkady, Raghothama; Pandey, Akhilesh; Ham, Amy-Joan L.; Hanks, Steven K.

2008-01-01

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype. PMID:18563927

Prediction of Response to Preoperative Chemoradiotherapy in Rectal Cancer by Multiplex Kinase Activity Profiling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folkvord, Sigurd; Flatmark, Kjersti; Department of Cancer and Surgery, Norwegian Radium Hospital, Oslo University Hospital

2010-10-01

Purpose: Tumor response of rectal cancer to preoperative chemoradiotherapy (CRT) varies considerably. In experimental tumor models and clinical radiotherapy, activity of particular subsets of kinase signaling pathways seems to predict radiation response. This study aimed to determine whether tumor kinase activity profiles might predict tumor response to preoperative CRT in locally advanced rectal cancer (LARC). Methods and Materials: Sixty-seven LARC patients were treated with a CRT regimen consisting of radiotherapy, fluorouracil, and, where possible, oxaliplatin. Pretreatment tumor biopsy specimens were analyzed using microarrays with kinase substrates, and the resulting substrate phosphorylation patterns were correlated with tumor response to preoperative treatmentmore » as assessed by histomorphologic tumor regression grade (TRG). A predictive model for TRG scores from phosphosubstrate signatures was obtained by partial-least-squares discriminant analysis. Prediction performance was evaluated by leave-one-out cross-validation and use of an independent test set. Results: In the patient population, 73% and 15% were scored as good responders (TRG 1-2) or intermediate responders (TRG 3), whereas 12% were assessed as poor responders (TRG 4-5). In a subset of 7 poor responders and 12 good responders, treatment outcome was correctly predicted for 95%. Application of the prediction model on the remaining patient samples resulted in correct prediction for 85%. Phosphosubstrate signatures generated by poor-responding tumors indicated high kinase activity, which was inhibited by the kinase inhibitor sunitinib, and several discriminating phosphosubstrates represented proteins derived from signaling pathways implicated in radioresistance. Conclusions: Multiplex kinase activity profiling may identify functional biomarkers predictive of tumor response to preoperative CRT in LARC.« less

Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

PubMed

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.

Identification and Analysis of Mitogen-Activated Protein Kinase (MAPK) Cascades in Fragaria vesca.

PubMed

Zhou, Heying; Ren, Suyue; Han, Yuanfang; Zhang, Qing; Qin, Ling; Xing, Yu

2017-08-13

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, including yeasts, plants and animals. MAPK cascades are responsible for protein phosphorylation during signal transduction events, and typically consist of three protein kinases: MAPK, MAPK kinase, and MAPK kinase kinase. In this current study, we identified a total of 12 FvMAPK , 7 FvMAPKK , 73 FvMAPKKK , and one FvMAPKKKK genes in the recently published Fragaria vesca genome sequence. This work reported the classification, annotation and phylogenetic evaluation of these genes and an assessment of conserved motifs and the expression profiling of members of the gene family were also analyzed here. The expression profiles of the MAPK and MAPKK genes in different organs and fruit developmental stages were further investigated using quantitative real-time reverse transcription PCR (qRT-PCR). Finally, the MAPK and MAPKK expression patterns in response to hormone and abiotic stresses (salt, drought, and high and low temperature) were investigated in fruit and leaves of F. vesca . The results provide a platform for further characterization of the physiological and biochemical functions of MAPK cascades in strawberry.

Deja Vu: EGF receptors drive resistance to BRAF inhibitors.

PubMed

Girotti, Maria Romina; Marais, Richard

2013-05-01

The promise of personalized medicine is upon us, and in some cancers, targeted therapies are rapidly becoming the mainstay of treatment for selected patients based on their molecular profile. The protein kinase BRAF is a driver oncogene in both thyroid cancer and melanoma, but while drugs that target BRAF and its downstream signaling pathway are effective in melanoma, they are ineffective in thyroid cancer. In this issue of Cancer Discovery, Montero-Conde and colleagues investigate why thyroid cancer is resistant to BRAF inhibitors despite the presence of BRAF mutation.

BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia

PubMed Central

Zabriskie, Matthew S.; Eide, Christopher A.; Tantravahi, Srinivas K.; Vellore, Nadeem A.; Estrada, Johanna; Nicolini, Franck E.; Khoury, Hanna J.; Larson, Richard A.; Konopleva, Marina; Cortes, Jorge E.; Kantarjian, Hagop; Jabbour, Elias J.; Kornblau, Steven M.; Lipton, Jeffrey H.; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J.; Press, Richard D.; Chuah, Charles; Goldberg, Stuart L.; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R.; Heaton, William L.; Eiring, Anna M.; Pomicter, Anthony D.; Khorashad, Jamshid S.; Kelley, Todd W.; Baron, Riccardo; Druker, Brian J.; Deininger, Michael W.; O'Hare, Thomas

2014-01-01

Summary Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph+) leukemia, including the recalcitrant BCR-ABL1T315I mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph+ leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome. PMID:25132497

Multiplexed Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry Quantification of Cancer Signaling Proteins

PubMed Central

Chen, Yi; Fisher, Kate J.; Lloyd, Mark; Wood, Elizabeth R.; Coppola, Domenico; Siegel, Erin; Shibata, David; Chen, Yian A.; Koomen, John M.

2017-01-01

Quantitative evaluation of protein expression across multiple cancer-related signaling pathways (e.g. Wnt/β-catenin, TGF-β, receptor tyrosine kinases (RTK), MAP kinases, NF-κB, and apoptosis) in tumor tissues may enable the development of a molecular profile for each individual tumor that can aid in the selection of appropriate targeted cancer therapies. Here, we describe the development of a broadly applicable protocol to develop and implement quantitative mass spectrometry assays using cell line models and frozen tissue specimens from colon cancer patients. Cell lines are used to develop peptide-based assays for protein quantification, which are incorporated into a method based on SDS-PAGE protein fractionation, in-gel digestion, and liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM/MS). This analytical platform is then applied to frozen tumor tissues. This protocol can be broadly applied to the study of human disease using multiplexed LC-MRM assays. PMID:28808993

Structure-based design of novel quinoxaline-2-carboxylic acids and analogues as Pim-1 inhibitors.

PubMed

Oyallon, Bruno; Brachet-Botineau, Marie; Logé, Cédric; Bonnet, Pascal; Souab, Mohamed; Robert, Thomas; Ruchaud, Sandrine; Bach, Stéphane; Berthelot, Pascal; Gouilleux, Fabrice; Viaud-Massuard, Marie-Claude; Denevault-Sabourin, Caroline

2018-05-11

We identified a new series of quinoxaline-2-carboxylic acid derivatives, targeting the human proviral integration site for Moloney murine leukemia virus-1 (HsPim-1) kinase. Seventeen analogues were synthesized providing useful insight into structure-activity relationships studied. Docking studies realized in the ATP pocket of HsPim-1 are consistent with an unclassical binding mode of these inhibitors. The lead compound 1 was able to block HsPim-1 enzymatic activity at nanomolar concentrations (IC 50 of 74 nM), with a good selectivity profile against a panel of mammalian protein kinases. In vitro studies on the human chronic myeloid leukemia cell line KU812 showed an antitumor activity at micromolar concentrations. As a result, compound 1 represents a promising lead for the design of novel anticancer targeted therapies. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Kinome-wide transcriptional profiling of uveal melanoma reveals new vulnerabilities to targeted therapeutics.

PubMed

Bailey, Fiona P; Clarke, Kim; Kalirai, Helen; Kenyani, Jenna; Shahidipour, Haleh; Falciani, Francesco; Coulson, Judy M; Sacco, Joseph J; Coupland, Sarah E; Eyers, Patrick A

2018-03-01

Metastatic uveal melanoma (UM) is invariably fatal, usually within a year of diagnosis. There are currently no effective therapies, and clinical studies employing kinase inhibitors have so far demonstrated limited success. This is despite common activating mutations in GNAQ/11 genes, which trigger signalling pathways that might predispose tumours to a variety of targeted drugs. In this study, we have profiled kinome expression network dynamics in various human ocular melanomas. We uncovered a shared transcriptional profile in human primary UM samples and across a variety of experimental cell-based models. The poor overall response of UM cells to FDA-approved kinase inhibitors contrasted with much higher sensitivity to the bromodomain inhibitor JQ1, a broad transcriptional repressor. Mechanistically, we identified a repressed FOXM1-dependent kinase subnetwork in JQ1-exposed cells that contained multiple cell cycle-regulated protein kinases. Consistently, we demonstrated vulnerability of UM cells to inhibitors of mitotic protein kinases within this network, including the investigational PLK1 inhibitor BI6727. We conclude that analysis of kinome-wide signalling network dynamics has the potential to reveal actionable drug targets and inhibitors of potential therapeutic benefit for UM patients. © 2017 The Authors. Pigment Cell & Melanoma Research Published by John Wiley & Sons.

Overcoming acquired BRAF inhibitor resistance in melanoma via targeted inhibition of Hsp90 with ganetespib.

PubMed

Acquaviva, Jaime; Smith, Donald L; Jimenez, John-Paul; Zhang, Chaohua; Sequeira, Manuel; He, Suqin; Sang, Jim; Bates, Richard C; Proia, David A

2014-02-01

Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors.

Structural Biology Insight for the Design of Sub-type Selective Aurora Kinase Inhibitors.

PubMed

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.

Profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells using MRM-based targeted proteomics.

PubMed

Guo, Lei; Xiao, Yongsheng; Fan, Ming; Li, Jian Jian; Wang, Yinsheng

2015-01-02

Ionizing radiation is widely used in cancer therapy; however, cancer cells often develop radioresistance, which compromises the efficacy of cancer radiation therapy. Quantitative assessment of the alteration of the entire kinome in radioresistant cancer cells relative to their radiosensitive counterparts may provide important knowledge to define the mechanism(s) underlying tumor adaptive radioresistance and uncover novel target(s) for effective prevention and treatment of tumor radioresistance. By employing a scheduled multiple-reaction monitoring analysis in conjunction with isotope-coded ATP affinity probes, we assessed the global kinome of radioresistant MCF-7/C6 cells and their parental MCF-7 human breast cancer cells. We rigorously quantified 120 kinases, of which (1)/3 exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response were found to be overexpressed or hyperactivated, including checkpoint kinase 1 (CHK1), cyclin-dependent kinases 1 and 2 (CDK1 and CDK2), and the catalytic subunit of DNA-dependent protein kinase. The elevated expression of CHK1, CDK1, and CDK2 in MCF-7/C6 cells was further validated by Western blot analysis. Thus, the altered kinome profile of radioresistant MCF-7/C6 cells suggests the involvement of kinases on cell cycle progression and DNA repair in tumor adaptive radioresistance. The unique kinome profiling results also afforded potential effective targets for resensitizing radioresistant cancer cells and counteracting deleterious effects of ionizing radiation exposure.

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C.

PubMed

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.

Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

PubMed Central

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

c-Jun N-terminal kinase inhibitors: a patent review (2010 - 2014).

PubMed

Gehringer, Matthias; Muth, Felix; Koch, Pierre; Laufer, Stefan A

2015-01-01

c-Jun N-terminal kinases (JNKs) are involved in the emergence and progression of diverse pathologies such as neurodegenerative, cardiovascular and metabolic disorders as well as inflammation and cancer. In recent years, several highly selective pan-JNK inhibitors have been characterized and three chemical entities targeting JNKs have been investigated in clinical trials. This review summarizes patents claiming inhibitors of all JNK isoforms published between 2010 and 2014. Although primarily focusing on the patent literature, relevant peer-reviewed publications related to the covered patents have also been included. Moreover, key patents claiming novel applications of previously published chemical entities are reviewed. The article highlights a total of 28 patents from nine pharmaceutical companies and academic research groups. Although some selective pan-JNK inhibitors with reasonable in vivo profiles are now available, little is known about the isoform selectivity required for each particular indication and the development of isoform-selective JNK inhibitors still represents a challenge in JNK drug discovery. Moreover, isoform-selective tool compounds are a prerequisite to a comprehensive understanding of the biology of each JNK isoform. Potential approaches towards such compounds include the design of type-II and type-I(1)/2 binders, which are absent in the current JNK inhibitor portfolios, as well as the design of novel allosteric inhibitors. Furthermore, covalent inhibition, which already led to the first high-quality probe for JNKs, might be further exploited for gaining selectivity and in vivo efficacy. With regard to a potential therapeutic application, the recently proposed concept of covalent reversible inhibitors is expected to be attractive.

Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

PubMed

Gaillard, Pascale; Jeanclaude-Etter, Isabelle; Ardissone, Vittoria; Arkinstall, Steve; Cambet, Yves; Camps, Montserrat; Chabert, Christian; Church, Dennis; Cirillo, Rocco; Gretener, Denise; Halazy, Serge; Nichols, Anthony; Szyndralewiez, Cedric; Vitte, Pierre-Alain; Gotteland, Jean-Pierre

2005-07-14

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

Design and synthesis of carbazole carboxamides as promising inhibitors of Bruton’s tyrosine kinase (BTK) and Janus kinase 2 (JAK2)

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).

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA

PubMed Central

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

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

PubMed

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.

p53 is a major component of the transcriptional and apoptotic program regulated by PI 3-kinase/Akt/GSK3 signaling.

PubMed

Nayak, G; Cooper, G M

2012-10-11

The phosphatidylinositol (PI) 3-kinase/Akt signaling pathway has a prominent role in cell survival and proliferation, in part, by regulating gene expression at the transcriptional level. Previous work using global expression profiling identified FOXOs and the E-box-binding transcription factors MITF and USF1 as key targets of PI 3-kinase signaling that lead to the induction of proapoptotic and cell cycle arrest genes in response to inhibition of PI 3-kinase. In this study, we investigated the role of p53 downstream of PI 3-kinase signaling by analyzing the effects of inhibition of PI 3-kinase in Rat-1 cells, which have wild-type p53, compared with Rat-1 cells expressing a dominant-negative p53 mutant. Expression of dominant-negative p53 conferred partial resistance to apoptosis induced by inhibition of PI 3-kinase. Global gene expression profiling combined with computational and experimental analysis of transcription factor binding sites demonstrated that p53, along with FOXO, MITF and USF1, contributed to gene induction in response to PI 3-kinase inhibition. Activation of p53 was mediated by phosphorylation of the histone acetyltransferase Tip60 by glycogen synthase kinase (GSK) 3, leading to activation of p53 by acetylation. Many of the genes targeted by p53 were also targeted by FOXO and E-box-binding transcription factors, indicating that p53 functions coordinately with these factors to regulate gene expression downstream of PI 3-kinase/Akt/GSK3 signaling.

Profile of neratinib and its potential in the treatment of breast cancer

PubMed Central

Feldinger, Katharina; Kong, Anthony

2015-01-01

The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s). PMID:26089701

Profile of neratinib and its potential in the treatment of breast cancer.

PubMed

Feldinger, Katharina; Kong, Anthony

2015-01-01

The HER (ErbB) receptor tyrosine kinase receptors are implicated in many cancers and several anti-HER treatments are now approved. In recent years, a new group of compounds that bind irreversibly to the adenosine triphosphate binding pocket of HER receptors have been developed. One of these compounds, neratinib, has passed preclinical phases and is currently undergoing various clinical trials. This manuscript reviews the preclinical as well as clinical data on neratinib. As a pan-HER inhibitor, this irreversible tyrosine kinase inhibitor binds and inhibits the tyrosine kinase activity of epidermal growth factor receptors, EGFR (or HER1), HER2 and HER4, which leads to reduced phosphorylation and activation of downstream signaling pathways. Neratinib has been shown to be effective against HER2-overexpressing or mutant tumors in vitro and in vivo. Neratinib is currently being investigated in various clinical trials in breast cancers and other solid tumors, including those with HER2 mutation. Earlier studies have already shown promising clinical activity for neratinib. However, more translational research is required to investigate biomarkers that could help to predict response and resistance for selection of appropriate patients for treatment with neratinib, either as monotherapy or in combination with other drug(s).

The MAPKKK gene family in cassava: Genome-wide identification and expression analysis against drought stress.

PubMed

Ye, Jianqiu; Yang, Hai; Shi, Haitao; Wei, Yunxie; Tie, Weiwei; Ding, Zehong; Yan, Yan; Luo, Ying; Xia, Zhiqiang; Wang, Wenquan; Peng, Ming; Li, Kaimian; Zhang, He; Hu, Wei

2017-11-02

Mitogen-activated protein kinase kinase kinases (MAPKKKs), an important unit of MAPK cascade, play crucial roles in plant development and response to various stresses. However, little is known concerning the MAPKKK family in the important subtropical and tropical crop cassava. In this study, 62 MAPKKK genes were identified in the cassava genome, and were classified into 3 subfamilies based on phylogenetic analysis. Most of MAPKKKs in the same subfamily shared similar gene structures and conserved motifs. The comprehensive transcriptome analysis showed that MAPKKK genes participated in tissue development and response to drought stress. Comparative expression profiles revealed that many MAPKKK genes were activated in cultivated varieties SC124 and Arg7 and the function of MeMAPKKKs in drought resistance may be different between SC124/Arg7 and W14. Expression analyses of the 7 selected MeMAPKKK genes showed that most of them were significantly upregulated by osmotic, salt and ABA treatments, whereas slightly induced by H 2 O 2 and cold stresses. Taken together, this study identified candidate MeMAPKKK genes for genetic improvement of abiotic stress resistance and provided new insights into MAPKKK -mediated cassava resistance to drought stress.

Icotinib (BPI-2009H), a novel EGFR tyrosine kinase inhibitor, displays potent efficacy in preclinical studies.

PubMed

Tan, Fenlai; Shen, Xiaoyan; Wang, Dechang; Xie, Guojian; Zhang, Xiaodong; Ding, Lieming; Hu, Yunyan; He, Wei; Wang, Yanping; Wang, Yinxiang

2012-05-01

Icotinib, one of the leading compounds selected from our compound library, was found to be a potent and specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) with an IC(50) of 5 nM. When profiled with 88 kinases, Icotinib only showed meaningful inhibitory activity to EGFR and its mutants. Icotinib blocked EGFR-mediated intracellular tyrosine phosphorylation (IC(50)=45 nM) in the human epidermoid carcinoma A431 cell line and inhibits tumor cell proliferation. In vivo studies demonstrated that Icotinib exhibited potent dose-dependent antitumor effects in nude mice carrying a variety of human tumor-derived xenografts. The drug was well tolerated at doses up to 120 mg/kg/day in mice without mortality or significant body weight loss during the treatment. A head to head randomized, double blind phase III trial using Gefitinib as an active control for patients with advanced non-small cell lung cancer (NSCLC) was finished recently (Trial registration ID: NCT01040780). The data shows that Icotinib was non-inferior to Gefitinib in terms of median progression free survival (PFS) and safety superior favor to Icotinib compared to Gefitinib. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

In Silico Identification of a Novel Hinge-Binding Scaffold for Kinase Inhibitor Discovery.

PubMed

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.

Safety profile of protein kinase inhibitors in rheumatoid arthritis: systematic review and meta-analysis.

PubMed

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.

Phosphoproteomics links glycogen synthase kinase-3 to RNA splicing.

PubMed

Khoa, Le Tran Phuc; Dou, Yali

2017-11-03

Protein kinases play essential biological roles by phosphorylating a diverse range of signaling molecules, but deciphering their direct physiological targets remains a challenge. A new study by Shinde et al. uses phosphoproteomics to identify glycogen synthase kinase-3 (GSK-3) substrates in mouse embryonic stem cells (mESCs), providing a broad profile of GSK-3 activity and defining a new role for this central kinase in regulating RNA splicing. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Elucidation of the molecular mechanisms underlying adverse reactions associated with a kinase inhibitor using systems toxicology

PubMed Central

Amemiya, Takahiro; Honma, Masashi; Kariya, Yoshiaki; Ghosh, Samik; Kitano, Hiroaki; Kurachi, Yoshihisa; Fujita, Ken-ichi; Sasaki, Yasutsuna; Homma, Yukio; Abernethy, Darrel R; Kume, Haruki; Suzuki, Hiroshi

2015-01-01

Background/Objectives: Targeted kinase inhibitors are an important class of agents in anticancer therapeutics, but their limited tolerability hampers their clinical performance. Identification of the molecular mechanisms underlying the development of adverse reactions will be helpful in establishing a rational method for the management of clinically adverse reactions. Here, we selected sunitinib as a model and demonstrated that the molecular mechanisms underlying the adverse reactions associated with kinase inhibitors can efficiently be identified using a systems toxicological approach. Methods: First, toxicological target candidates were short-listed by comparing the human kinase occupancy profiles of sunitinib and sorafenib, and the molecular mechanisms underlying adverse reactions were predicted by sequential simulations using publicly available mathematical models. Next, to evaluate the probability of these predictions, a clinical observation study was conducted in six patients treated with sunitinib. Finally, mouse experiments were performed for detailed confirmation of the hypothesized molecular mechanisms and to evaluate the efficacy of a proposed countermeasure against adverse reactions to sunitinib. Results: In silico simulations indicated the possibility that sunitinib-mediated off-target inhibition of phosphorylase kinase leads to the generation of oxidative stress in various tissues. Clinical observations of patients and mouse experiments confirmed the validity of this prediction. The simulation further suggested that concomitant use of an antioxidant may prevent sunitinib-mediated adverse reactions, which was confirmed in mouse experiments. Conclusions: A systems toxicological approach successfully predicted the molecular mechanisms underlying clinically adverse reactions associated with sunitinib and was used to plan a rational method for the management of these adverse reactions. PMID:28725458

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

PubMed Central

Santio, Niina M.; Arnaudova, Ralica; Eerola, Sini K.; Rainio, Eeva-Marja; Aumüller, Ingo B.; Yli-Kauhaluoma, Jari; Koskinen, Päivi J.

2013-01-01

Oncogenic Pim family kinases are often overexpressed in human hematopoietic malignancies as well as in solid tumours. These kinases contribute to tumorigenesis by promoting cell survival and by enhancing resistance against chemotherapy and radiation therapy. Furthermore, we have recently shown that they increase the metastatic potential of adherent cancer cells. Here we describe identification of tricyclic benzo[cd]azulenes and their derivatives as effective and selective inhibitors of Pim kinases. These compounds inhibit Pim autophosphorylation and abrogate the anti-apoptotic effects of Pim kinases. They also reduce cancer cell motility and suppress proliferation of lymphoblastoid cell lines infected and immortalized by the Epstein-Barr virus. Thus, these novel Pim-selective inhibitors provide promising compounds for both research and therapeutic purposes. PMID:23405147

Constitutive Activation of PINK1 Protein Leads to Proteasome-mediated and Non-apoptotic Cell Death Independently of Mitochondrial Autophagy*

PubMed Central

Akabane, Shiori; Matsuzaki, Kohei; Yamashita, Shun-ichi; Arai, Kana; Okatsu, Kei; Kanki, Tomotake; Matsuda, Noriyuki; Oka, Toshihiko

2016-01-01

Phosphatase and tensin homolog-induced putative kinase 1 (PINK1), a Ser/Thr kinase, and PARKIN, a ubiquitin ligase, are causal genes for autosomal recessive early-onset parkinsonism. Multiple lines of evidence indicate that PINK1 and PARKIN cooperatively control the quality of the mitochondrial population via selective degradation of damaged mitochondria by autophagy. Here, we report that PINK1 and PARKIN induce cell death with a 12-h delay after mitochondrial depolarization, which differs from the time profile of selective autophagy of mitochondria. This type of cell death exhibited definite morphologic features such as plasma membrane rupture, was insensitive to a pan-caspase inhibitor, and did not involve mitochondrial permeability transition. Expression of a constitutively active form of PINK1 caused cell death in the presence of a pan-caspase inhibitor, irrespective of the mitochondrial membrane potential. PINK1-mediated cell death depended on the activities of PARKIN and proteasomes, but it was not affected by disruption of the genes required for autophagy. Furthermore, fluorescence and electron microscopic analyses revealed that mitochondria were still retained in the dead cells, indicating that PINK1-mediated cell death is not caused by mitochondrial loss. Our findings suggest that PINK1 and PARKIN play critical roles in selective cell death in which damaged mitochondria are retained, independent of mitochondrial autophagy. PMID:27302064

IKAP: A heuristic framework for inference of kinase activities from Phosphoproteomics data.

PubMed

Mischnik, Marcel; Sacco, Francesca; Cox, Jürgen; Schneider, Hans-Christoph; Schäfer, Matthias; Hendlich, Manfred; Crowther, Daniel; Mann, Matthias; Klabunde, Thomas

2016-02-01

Phosphoproteomics measurements are widely applied in cellular biology to detect changes in signalling dynamics. However, due to the inherent complexity of phosphorylation patterns and the lack of knowledge on how phosphorylations are related to functions, it is often not possible to directly deduce protein activities from those measurements. Here, we present a heuristic machine learning algorithm that infers the activities of kinases from Phosphoproteomics data using kinase-target information from the PhosphoSitePlus database. By comparing the estimated kinase activity profiles to the measured phosphosite profiles, it is furthermore possible to derive the kinases that are most likely to phosphorylate the respective phosphosite. We apply our approach to published datasets of the human cell cycle generated from HeLaS3 cells, and insulin signalling dynamics in mouse hepatocytes. In the first case, we estimate the activities of 118 at six cell cycle stages and derive 94 new kinase-phosphosite links that can be validated through either database or motif information. In the second case, the activities of 143 kinases at eight time points are estimated and 49 new kinase-target links are derived. The algorithm is implemented in Matlab and be downloaded from github. It makes use of the Optimization and Statistics toolboxes. https://github.com/marcel-mischnik/IKAP.git. marcel.mischnik@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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.

Generation of tricyclic imidazo[1,2-a]pyrazines as novel PI3K inhibitors by application of a conformational restriction strategy.

PubMed

Martínez González, Sonia; Rodríguez-Arístegui, Sonsoles; Hernández, Ana Isabel; Varela, Carmen; González Cantalapiedra, Esther; Álvarez, Rosa María; Rodríguez Hergueta, Antonio; Bischoff, James R; Albarrán, María Isabel; Cebriá, Antonio; Cendón, Elena; Cebrián, David; Alfonso, Patricia; Pastor, Joaquín

2017-06-01

The involvement of the phosphoinositide 3-kinases (PI3Ks) in several diseases, especially in the oncology area, has singled it as one of the most explored therapeutic targets in the last two decades. Many different inhibitor classes have been developed by the industry and academia with a diverse selectivity profile within the PI3K family. In the present manuscript we report a further exploration of our lead PI3K inhibitor ETP-46321 (Martínez González et al., 2012) 1 by the application of a conformational restriction strategy. For that purpose we have successfully synthesized novel tricyclic imidazo[1,2-a]pyrazine derivatives as PI3K inhibitors. This new class of compounds had enable the exploration of the solvent-accessible region within PI3K and resulted in the identification of molecule 8q with the best selectivity PI3Kα/δ isoform profile in vitro, and promising in vivo PK data. Copyright © 2017 Elsevier Ltd. All rights reserved.

An integrated bioinformatics analysis to dissect kinase dependency in triple negative breast cancer.

PubMed

Ryall, Karen A; Kim, Jihye; Klauck, Peter J; Shin, Jimin; Yoo, Minjae; Ionkina, Anastasia; Pitts, Todd M; Tentler, John J; Diamond, Jennifer R; Eckhardt, S Gail; Heasley, Lynn E; Kang, Jaewoo; Tan, Aik Choon

2015-01-01

Triple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis. We integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. We validated our predictions using published and new experimental data. In summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

Contribution of Rho-kinase to membrane excitability of murine colonic smooth muscle.

PubMed

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.

“Addition” and “Subtraction”: Selectivity Design for Type II Maternal Embryonic Leucine Zipper Kinase Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Xin; Giraldes, John; Sprague, Elizabeth R.

2017-02-17

While adding the structural features that are more favored by on-target activity is the more common strategy in selectivity optimization, the opposite strategy of subtracting the structural features that contribute more to off-target activity can also be very effective. Reported here is our successful effort of improving the kinase selectivity of type II maternal embryonic leucine zipper kinase inhibitors by applying these two complementary approaches together, which clearly demonstrates the powerful synergy between them.

Structure of the β-form of human MK2 in complex with the non-selective kinase inhibitor TEI-L03090

PubMed Central

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

Loss of mutL homolog-1 (MLH1) expression promotes acquisition of oncogenic and inhibitor-resistant point mutations in tyrosine kinases.

PubMed

Springuel, Lorraine; Losdyck, Elisabeth; Saussoy, Pascale; Turcq, Béatrice; Mahon, François-Xavier; Knoops, Laurent; Renauld, Jean-Christophe

2016-12-01

Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

PubMed

Zabriskie, Matthew S; Eide, Christopher A; Tantravahi, Srinivas K; Vellore, Nadeem A; Estrada, Johanna; Nicolini, Franck E; Khoury, Hanna J; Larson, Richard A; Konopleva, Marina; Cortes, Jorge E; Kantarjian, Hagop; Jabbour, Elias J; Kornblau, Steven M; Lipton, Jeffrey H; Rea, Delphine; Stenke, Leif; Barbany, Gisela; Lange, Thoralf; Hernández-Boluda, Juan-Carlos; Ossenkoppele, Gert J; Press, Richard D; Chuah, Charles; Goldberg, Stuart L; Wetzler, Meir; Mahon, Francois-Xavier; Etienne, Gabriel; Baccarani, Michele; Soverini, Simona; Rosti, Gianantonio; Rousselot, Philippe; Friedman, Ran; Deininger, Marie; Reynolds, Kimberly R; Heaton, William L; Eiring, Anna M; Pomicter, Anthony D; Khorashad, Jamshid S; Kelley, Todd W; Baron, Riccardo; Druker, Brian J; Deininger, Michael W; O'Hare, Thomas

2014-09-08

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome. Copyright © 2014 Elsevier Inc. All rights reserved.

Using 2nd generation tyrosine kinase inhibitors in frontline management of chronic phase chronic myeloid leukemia

PubMed Central

Jayakar, Vishal

2014-01-01

Choices in medicine come with responsibility. With several TKI's (Tyrosine kinase inhibitors) available for front-line management of CML (Chronic Myeloid Leukemia), an astute clinician has to personalise, rationalise and take a pragmatic approach towards selection of the best drug for the ‘patient in question’. Though it is hotly debated as to which TKI will triumph, the truth of this debate lies in individualising treatment rather than a general ‘all size fits all’ approach with imatinib. I personally believe that the second generation TKI's will suit most patient clinical profiles rather than prescribing imatinib to all and I have strived to make a strong case for them in front line treatment of CML. Though Imatinib may remain the first line choice for some patients, my efforts in this debate are mainly geared towards breaking the myth that imatinib is the sole ‘block buster’ on the CML landscape PMID:24665456

Correlation between the Stereochemistry and Bioactivity in Octahedral Rhodium Prolinato Complexes.

PubMed

Rajaratnam, Rajathees; Martin, Elisabeth K; Dörr, Markus; Harms, Klaus; Casini, Angela; Meggers, Eric

2015-08-17

Controlling the relative and absolute configuration of octahedral metal complexes constitutes a key challenge that needs to be overcome in order to fully exploit the structural properties of octahedral metal complexes for applications in the fields of catalysis, materials sciences, and life sciences. Herein, we describe the application of a proline-based chiral tridentate ligand to decisively control the coordination mode of an octahedral rhodium(III) complex. We demonstrate the mirror-like relationship of synthesized enantiomers and differences between diastereomers. Further, we demonstrate, using the established pyridocarbazole pharmacophore ligand as part of the organometallic complexes, the importance of the relative and absolute stereochemistry at the metal toward chiral environments like protein kinases. Protein kinase profiling and inhibition data confirm that the proline-based enantiopure rhodium(III) complexes, despite having all of the same constitution, differ strongly in their selectivity properties despite their unmistakably mutual origin. Moreover, two exemplary compounds have been shown to induce different toxic effects in an ex vivo rat liver model.

p21-activated kinase inhibitors.

PubMed

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.

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

PubMed

Arora, Shilpi; Gonzales, Irma M; Hagelstrom, R Tanner; Beaudry, Christian; Choudhary, Ashish; Sima, Chao; Tibes, Raoul; Mousses, Spyro; Azorsa, David O

2010-08-18

Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

Conformation-selective inhibitors reveal differences in the activation and phosphate-binding loops of the tyrosine kinases Abl and Src

PubMed Central

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

A hit to lead discovery of novel N-methylated imidazolo-, pyrrolo-, and pyrazolo-pyrimidines as potent and selective mTOR inhibitors.

PubMed

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.

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

Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia | Office of Cancer Genomics

Cancer.gov

Publication Abstract:  Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL.

Identifying protein kinase target preferences using mass spectrometry

PubMed Central

Douglass, Jacqueline; Gunaratne, Ruwan; Bradford, Davis; Saeed, Fahad; Hoffert, Jason D.; Steinbach, Peter J.; Pisitkun, Trairak

2012-01-01

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called “PhosphoLogo,” uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions −2 and −3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3β, Wnk1, and Wnk4. PMID:22723110

Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis.

PubMed

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.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines.

PubMed

Weigelt, Britta; Warne, Patricia H; Lambros, Maryou B; Reis-Filho, Jorge S; Downward, Julian

2013-07-01

Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival. Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells. EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed. Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required. ©2013 AACR.

Application of a phenotypic drug discovery strategy to identify biological and chemical starting points for inhibition of TSLP production in lung epithelial cells

PubMed Central

Orellana, Adelina; García-González, Vicente; López, Rosa; Pascual-Guiral, Sonia; Lozoya, Estrella; Díaz, Julia; Casals, Daniel; Barrena, Antolín; Paris, Stephane; Andrés, Miriam; Segarra, Victor; Vilella, Dolors; Malhotra, Rajneesh; Eastwood, Paul; Planagumà, Anna; Miralpeix, Montserrat

2018-01-01

Thymic stromal lymphopoietin (TSLP) is a cytokine released by human lung epithelium in response to external insult. Considered as a master switch in T helper 2 lymphocyte (Th2) mediated responses, TSLP is believed to play a key role in allergic diseases including asthma. The aim of this study was to use a phenotypic approach to identify new biological and chemical starting points for inhibition of TSLP production in human bronchial epithelial cells (NHBE), with the objective of reducing Th2-mediated airway inflammation. To this end, a phenotypic screen was performed using poly I:C / IL-4 stimulated NHBE cells interrogated with a 44,974 compound library. As a result, 85 hits which downregulated TSLP protein and mRNA levels were identified and a representative subset of 7 hits was selected for further characterization. These molecules inhibited the activity of several members of the MAPK, PI3K and tyrosine kinase families and some of them have been reported as modulators of cellular phenotypic endpoints like cell-cell contacts, microtubule polymerization and caspase activation. Characterization of the biological profile of the hits suggested that mTOR could be a key activity involved in the regulation of TSLP production in NHBE cells. Among other targeted kinases, inhibition of p38 MAPK and JAK kinases showed different degrees of correlation with TSLP downregulation, while Syk kinase did not seem to be related. Overall, inhibition of TSLP production by the selected hits, rather than resulting from inhibition of single isolated targets, appeared to be due to a combination of activities with different levels of relevance. Finally, a hit expansion exercise yielded additional active compounds that could be amenable to further optimization, providing an opportunity to dissociate TSLP inhibition from other non-desired activities. This study illustrates the potential of phenotypic drug discovery to complement target based approaches by providing new chemistry and biology leads. PMID:29320511

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

PubMed Central

Xiong, Jimin; Menicanin, Danijela; Marino, Victor

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Combinatorial Drug Testing in 3D Microtumors Derived from GBM Patient-Derived Xenografts Reveals Cytotoxic Synergy in Pharmacokinomics-informed Pathway Interactions.

PubMed

Gilbert, Ashley N; Anderson, Joshua C; Duarte, Christine W; Shevin, Rachael S; Langford, Catherine P; Singh, Raj; Gillespie, G Yancey; Willey, Christopher D

2018-05-30

Glioblastoma multiforme (GBM), the most common form of primary malignant brain cancer in adults, is a devastating disease for which effective treatment has remained elusive for over 75 years. One reason for the minimal progress during this time is the lack of accurate preclinical models to represent the patient's tumor's in vivo environment, causing a disconnect in drug therapy effectiveness between the laboratory and clinic. While patient-derived xenografts (PDX's or xenolines) are excellent human tumor representations, they are not amenable to high throughput testing. Therefore, we developed a miniaturized xenoline system (microtumors) for drug testing. Nineteen GBM xenolines were profiled for global kinase (kinomic) activity revealing actionable kinase targets associated with intracranial tumor growth rate. Kinase inhibitors for these targets (WP1066, selumetinib, crizotinib, and cediranib) were selected for single and combination therapy using a fully human-derived three-dimensional (3D) microtumor model of GBM xenoline cells embedded in HuBiogel for subsequent molecular and phenotype assays. GBM microtumors closely resembled orthotopically-implanted tumors based on immunohistochemical analysis and displayed kinomic and morphological diversity. Drug response testing could be reproducibly performed in a 96-well format identifying several synergistic combinations. Our findings indicate that 3D microtumors can provide a suitable high-throughput model for combination drug testing.

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

PubMed

Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

2016-10-26

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene.

PubMed

Lupton, S D; Brunton, L L; Kalberg, V A; Overell, R W

1991-06-01

The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.

Discovery of a series of dihydroquinoxalin-2(1H)-ones as selective BET inhibitors from a dual PLK1-BRD4 inhibitor.

PubMed

Hu, Jianping; Wang, Yingqing; Li, Yanlian; Xu, Lin; Cao, Danyan; Song, ShanShan; Damaneh, Mohammadali Soleimani; Wang, Xin; Meng, Tao; Chen, Yue-Lei; Shen, Jingkang; Miao, Zehong; Xiong, Bing

2017-09-08

Recent years have seen much effort to discover new chemotypes of BRD4 inhibitors. Interestingly, some kinase inhibitors have been demonstrated to be potent bromodomain inhibitors, especially the PLK1 inhibitor BI-2536 and the JAK2 inhibitor TG101209, which can bind to BRD4 with IC 50 values of 0.025 μM and 0.13 μM, respectively. Although the concept of dual inhibition is intriguing, selective BRD4 inhibitors are preferred as they may diminish off-target effects and provide more flexibility in anticancer drug combination therapy. Inspired by BI-2536, we designed and prepared a series of dihydroquinoxalin-2(1H)-one derivatives as selective bromodomain inhibitors. We found compound 54 had slightly higher activity than (+)-JQ1 in the fluorescence anisotropy assay and potent antiproliferative cellular activity in the MM.1S cell line. We have successfully solved the cocrystal structure of 52 in complex with BRD4-BD1, providing a solid structural basis for the binding mode of compounds of this series. Compound 54 exhibited high selectivity over most non-BET subfamily members and did not show bioactivity towards the PLK1 kinase at 10 or 1 μM. From in vivo studies, compound 54 demonstrated a good PK profile, and the results from in vivo pharmacological studies clearly showed the efficacy of 54 in the mouse MM.1S xenograft model. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Tyrosine kinase receptor status in endometrial stromal sarcoma: an immunohistochemical and genetic-molecular analysis.

PubMed

Cossu-Rocca, Paolo; Contini, Marcella; Uras, Maria Gabriela; Muroni, Maria Rosaria; Pili, Francesca; Carru, Ciriaco; Bosincu, Luisanna; Massarelli, Giovannino; Nogales, Francisco F; De Miglio, Maria Rosaria

2012-11-01

Endometrial stromal sarcomas (ESS) are rare uterine malignant mesenchymal neoplasms, which are currently treated by surgery, as effective adjuvant therapies have not yet been established. Tyrosine kinase inhibitors have rarely been applied in ESS therapy, with few reports describing imatinib responsivity. The aim of this study was to analyze the status of different tyrosine kinase receptors in an ESS series, in order to evaluate their potential role as molecular targets. Immunohistochemistry was performed for EGFR, c-KIT, PDGFR-α, PDGFR-β, and ABL on 28 ESS. EGFR, PDGFR-α, and PDGFR-β gene expression was investigated by real-time polymerase chain reaction (qRT-PCR) on selected cases. "Hot-spot" mutations were screened for on EGFR, c-KIT, PDGFR-α, and PDGFR-β genes, by sequencing. All analysis was executed from formalin-fixed, paraffin-embedded specimens. Immunohistochemical overexpression of 2 or more tyrosine kinase receptors was observed in 18 of 28 tumors (64%), whereas only 5 tumors were consistently negative. Gene expression profiles were concordant with immunohistochemical overexpression in only 1 tumor, which displayed both high mRNA levels and specific immunoreactivity for PDGFR-α, and PDGFR-β. No activating mutations were found on the tumors included in the study. This study confirms that TKRs expression is frequently observed in ESS. Considering that the responsiveness to tyrosine kinase inhibitors is known to be related to the presence of specific activating mutations or gene over-expression, which are not detectable in ESS, TKRs immunohistochemical over-expression alone should not be considered as a reliable marker for targeted therapies in ESS. Specific post-translational abnormalities, responsible for activation of TKRs, should be further investigated.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis.

PubMed

Katewa, Arna; Wang, Yugang; Hackney, Jason A; Huang, Tao; Suto, Eric; Ramamoorthi, Nandhini; Austin, Cary D; Bremer, Meire; Chen, Jacob Zhi; Crawford, James J; Currie, Kevin S; Blomgren, Peter; DeVoss, Jason; DiPaolo, Julie A; Hau, Jonathan; Johnson, Adam; Lesch, Justin; DeForge, Laura E; Lin, Zhonghua; Liimatta, Marya; Lubach, Joseph W; McVay, Sami; Modrusan, Zora; Nguyen, Allen; Poon, Chungkee; Wang, Jianyong; Liu, Lichuan; Lee, Wyne P; Wong, Harvey; Young, Wendy B; Townsend, Michael J; Reif, Karin

2017-04-06

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Cell- and Tissue-based Proteome Profiling and Bioimaging with the Probes Derived from a Potent AXL Kinase Inhibitor.

PubMed

Li, Zhengqiu; Zheng, Binbin; Guo, Haijun; Xu, Jiaqian; Ma, Nan; Ni, Yun; Li, Lin; Hao, Piliang; Ding, Ke

2018-06-25

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Our group has developed a lead compound, 9im, capable of excellent inhibition against AXL. With the aim of understanding its cellular and tissue mechanism of actions and direct subsequent structure optimization, a study on competitive affinity-based proteome profiling and bioimaging was carried out. A series of unknown cellular and tissue targets, including RYK, PCK, ATP1A3, EIF4A, Ptprn and Cox5b were discovered. In addition, trans-cyclooctene (TCO) and acedan-containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of expression and activity of AXL. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The selectivity and promiscuity of brain-neuroregenerative inhibitors between ROCK1 and ROCK2 isoforms: An integration of SB-QSSR modelling, QM/MM analysis and in vitro kinase assay.

PubMed

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.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

PubMed

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.

Discovery of novel BTK inhibitors with carboxylic acids.

PubMed

Gao, Xiaolei; Wang, James; Liu, Jian; Guiadeen, Deodial; Krikorian, Arto; Boga, Sobhana Babu; Alhassan, Abdul-Basit; Selyutin, Oleg; Yu, Wensheng; Yu, Younong; Anand, Rajan; Liu, Shilan; Yang, Chundao; Wu, Hao; Cai, Jiaqiang; Cooper, Alan; Zhu, Hugh; Maloney, Kevin; Gao, Ying-Duo; Fischmann, Thierry O; Presland, Jeremy; Mansueto, My; Xu, Zangwei; Leccese, Erica; Zhang-Hoover, Jie; Knemeyer, Ian; Garlisi, Charles G; Bays, Nathan; Stivers, Peter; Brandish, Philip E; Hicks, Alexandra; Kim, Ronald; Kozlowski, Joeseph A

2017-03-15

We report the design and synthesis of a series of novel Bruton's Tyrosine Kinase (BTK) inhibitors with a carboxylic acid moiety in the ribose pocket. This series of compounds has demonstrated much improved off-target selectivities including adenosine uptake (AdU) inhibition compared to the piperidine amide series. Optimization of the initial lead compound 4 based on BTK enzyme inhibition, and human peripheral blood mononuclear cell (hPBMC) and human whole blood (hWB) activity led to the discovery of compound 40, with potent BTK inhibition, reduced off target activities, as well as favorable pharmacokinetic profile in both rat and dog. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

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

Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia

PubMed Central

Roberts, Kathryn G.; Morin, Ryan D.; Zhang, Jinghui; Hirst, Martin; Zhao, Yongjun; Su, Xiaoping; Chen, Shann-Ching; Payne-Turner, Debbie; Churchman, Michelle; Harvey, Richard C.; Chen, Xiang; Kasap, Corynn; Yan, Chunhua; Becksfort, Jared; Finney, Richard P.; Teachey, David T.; Maude, Shannon L.; Tse, Kane; Moore, Richard; Jones, Steven; Mungall, Karen; Birol, Inanc; Edmonson, Michael N.; Hu, Ying; Buetow, Kenneth E.; Chen, I-Ming; Carroll, William L.; Wei, Lei; Ma, Jing; Kleppe, Maria; Levine, Ross L.; Garcia-Manero, Guillermo; Larsen, Eric; Shah, Neil P.; Devidas, Meenakshi; Reaman, Gregory; Smith, Malcolm; Paugh, Steven W.; Evans, William E.; Grupp, Stephan A.; Jeha, Sima; Pui, Ching-Hon; Gerhard, Daniela S.; Downing, James R.; Willman, Cheryl L.; Loh, Mignon; Hunger, Stephen P.; Marra, Marco; Mullighan, Charles G.

2012-01-01

SUMMARY Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL, and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2 and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2 negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy. PMID:22897847

Virtual screening of selective multitarget kinase inhibitors by combinatorial support vector machines.

PubMed

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.

GNQ-209P Mutation in Metastatic Uveal Melanoma and Treatment Outcome.

PubMed

Abdel Karim, Nagla; Eldessouki, Ihab; Taftaf, Ahmad; Ayham, Deeb; Gaber, Ola; Makramalla, Abouelmagd; Correa, Zelia M

2018-01-01

Metastatic prognosis in uveal melanoma is assessed by gene expression profiling (GEP) testing of the tumor cells, usually obtained by fine needle aspiration (FNA). GEP has demonstrated high accuracy in distinguishing class I and II tumors, both having different metastatic potential. Transcriptomic studies identified distinct mutations including somatic mutations in GNAQ and GNA11 , detected in more than 80%, and contribute to the upregulation of the mitogen-activated protein kinase (MAPK) pathway and the development of uveal melanoma (UM). The role of these mutations in treatment selection and possible benefit from targeted therapy are somewhat unclear. However, until the discovery of novel agents, local versus systemic therapies remain options for treatment that can still be considered for disease control in certain cases. We report a series of patients with metastatic UM with distinct mutational profiles. One had significant liver metastases with proven GNQ-209P mutation on tissue biopsy while peripheral blood molecular profiling did not show these mutations. The other three cases had no GNQ-209P mutation. All cases received nab-paclitaxel (Abraxane) as a treatment drug, and we record their responses to treatment and their molecular-profiling results.

Dual kinase-bromodomain inhibitors for rationally designed polypharmacology

PubMed Central

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

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Discovery of a Diaminopyrimidine FLT3 Inhibitor Active against Acute Myeloid Leukemia

PubMed Central

2017-01-01

Profiling of the kinase-binding capabilities of an aminopyrimidine analogue detected in a cellular screen of the St. Jude small-molecule collection led to the identification of a novel series of FMS-like tyrosine kinase 3 (FLT3) inhibitors. Structure–activity relationship studies led to the development of compounds exhibiting good potency against MV4-11 and MOLM13 acute myelogenous leukemia cells driven by FLT3, regardless of their FLT3 mutation status. In vitro pharmacological profiling demonstrated that compound 5e shows characteristics suitable for further preclinical development. PMID:28580438

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

PubMed

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Profiling the changes in signaling pathways in ascorbic acid/β-glycerophosphate-induced osteoblastic differentiation.

PubMed

Chaves Neto, Antonio Hernandes; Queiroz, Karla Cristiana; Milani, Renato; Paredes-Gamero, Edgar Julian; Justo, Giselle Zenker; Peppelenbosch, Maikel P; Ferreira, Carmen Veríssima

2011-01-01

Despite numerous reports on the ability of ascorbic acid and β-glycerophosphate (AA/β-GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β-GP-induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β-GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR-1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood.

Identification of critical chemical features for Aurora kinase-B inhibitors using Hip-Hop, virtual screening and molecular docking

NASA Astrophysics Data System (ADS)

Sakkiah, Sugunadevi; Thangapandian, Sundarapandian; John, Shalini; Lee, Keun Woo

2011-01-01

This study was performed to find the selective chemical features for Aurora kinase-B inhibitors using the potent methods like Hip-Hop, virtual screening, homology modeling, molecular dynamics and docking. The best hypothesis, Hypo1 was validated toward a wide range of test set containing the selective inhibitors of Aurora kinase-B. Homology modeling and molecular dynamics studies were carried out to perform the molecular docking studies. The best hypothesis Hypo1 was used as a 3D query to screen the chemical databases. The screened molecules from the databases were sorted based on ADME and drug like properties. The selective hit compounds were docked and the hydrogen bond interactions with the critical amino acids present in Aurora kinase-B were compared with the chemical features present in the Hypo1. Finally, we suggest that the chemical features present in the Hypo1 are vital for a molecule to inhibit the Aurora kinase-B activity.

Phosphatidylinositol 4-Kinase III Beta Is Essential for Replication of Human Rhinovirus and Its Inhibition Causes a Lethal Phenotype In Vivo

PubMed Central

Spickler, Catherine; Lippens, Julie; Laberge, Marie-Kristine; Desmeules, Sophie; Bellavance, Édith; Garneau, Michel; Guo, Tim; Hucke, Oliver; Leyssen, Pieter; Neyts, Johan; Vaillancourt, Fréderic H.; Décor, Anne; O'Meara, Jeff; Franti, Michael

2013-01-01

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ). A good correlation between PI4KIIIβ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIβ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIβ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIβ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIβ is deleterious. PMID:23650168

Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype.

PubMed

Hirt, Ulrich A; Waizenegger, Irene C; Schweifer, Norbert; Haslinger, Christian; Gerlach, Daniel; Braunger, Jürgen; Weyer-Czernilofsky, Ulrike; Stadtmüller, Heinz; Sapountzis, Ioannis; Bader, Gerd; Zoephel, Andreas; Bister, Bojan; Baum, Anke; Quant, Jens; Kraut, Norbert; Garin-Chesa, Pilar; Adolf, Günther R

2018-02-23

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has attracted interest as a target for pharmacological intervention in malignant diseases. Here, we describe BI 853520, a novel ATP-competitive inhibitor distinguished by high potency and selectivity. In vitro, the compound inhibits FAK autophosphorylation in PC-3 prostate carcinoma cells with an IC 50 of 1 nmol/L and blocks anchorage-independent proliferation of PC-3 cells with an EC 50 of 3 nmol/L, whereas cells grown in conventional surface culture are 1000-fold less sensitive. In mice, the compound shows long half-life, high volume of distribution and high oral bioavailability; oral dosing of immunodeficient mice bearing subcutaneous PC-3 prostate adenocarcinoma xenografts resulted in rapid, long-lasting repression of FAK autophosphorylation in tumor tissue. Daily oral administration of BI 853520 to nude mice at doses of 50 mg/kg was well tolerated for prolonged periods of time. In a diverse panel of 16 subcutaneous adenocarcinoma xenograft models in nude mice, drug treatment resulted in a broad spectrum of outcomes, ranging from group median tumor growth inhibition values >100% and tumor regression in subsets of animals to complete lack of sensitivity. Biomarker analysis indicated that high sensitivity is linked to a mesenchymal tumor phenotype, initially defined by loss of E-cadherin expression and subsequently substantiated by gene set enrichment analysis. Further, we obtained microRNA expression profiles for 13 models and observed that hsa-miR-200c-3p expression is strongly correlated with efficacy (R 2  = 0.889). BI 853520 is undergoing evaluation in early clinical trials.

Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

PubMed

Wengner, Antje M; Siemeister, Gerhard; Koppitz, Marcus; Schulze, Volker; Kosemund, Dirk; Klar, Ulrich; Stoeckigt, Detlef; Neuhaus, Roland; Lienau, Philip; Bader, Benjamin; Prechtl, Stefan; Raschke, Marian; Frisk, Anna-Lena; von Ahsen, Oliver; Michels, Martin; Kreft, Bertolt; von Nussbaum, Franz; Brands, Michael; Mumberg, Dominik; Ziegelbauer, Karl

2016-04-01

Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR. ©2016 American Association for Cancer Research.

Efficacy and gastrointestinal tolerability of ML3403, a selective inhibitor of p38 MAP kinase and CBS-3595, a dual inhibitor of p38 MAP kinase and phosphodiesterase 4 in CFA-induced arthritis in rats.

PubMed

Koch, Diana A; Silva, Rodrigo B M; de Souza, Alessandra H; Leite, Carlos E; Nicoletti, Natália F; Campos, Maria M; Laufer, Stefan; Morrone, Fernanda B

2014-03-01

Mitogen-activated protein kinase (MAPK) p38 inhibitors have entered the clinical phase, although many of them have failed due to high toxicity and lack of efficacy. In the present study we compared the effects of the selective p38 inhibitor ML3403 and the dual p38-PDE4 inhibitor CBS-3595, on inflammatory and nociceptive parameters in a model of polyarthritis in rats. Male Wistar rats (180-200 g) were used for the complete Freund's adjuvant (CFA)-induced arthritis model and they were evaluated at 14-21 days. We also analysed the effects of these pharmacological tools on liver and gastrointestinal toxicity and on cytokine levels. Repeated CBS-3595 (3 mg/kg) or ML3403 (10 mg/kg) administration produced significant anti-inflammatory actions in the chronic arthritis model induced by CFA. CBS-3595 and ML3403 treatment also markedly reduced the production of the proinflammatory cytokine IL-6 in the paw tissue, whereas it widely increased the levels of the anti-inflammatory cytokine IL-10. Moreover, CBS-3595 produced partial anti-allodynic effects in the CFA model at 4 and 8 days after treatment. Notably, ML3403 and CBS-3595 did not show marked signs of hepatoxicity, as supported by unaltered histological observations in the liver sections. Finally, both compounds were safe in the gastrointestinal tract, according to evaluation of intestinal biopsies. CBS-3595 displayed a superior profile regarding its anti-inflammatory effects. Thus p38 MAPK/PDE4 blocking might well constitute a relevant strategy for the treatment of RA.

Contrasting cDNA-AFLP profiles between crown and leaf tissues of cold-acclimated wheat plants indicate differing regulatory circuitries for low temperature tolerance.

PubMed

Ganeshan, Seedhabadee; Sharma, Pallavi; Young, Lester; Kumar, Ashwani; Fowler, D Brian; Chibbar, Ravindra N

2011-03-01

Low-temperature (LT) tolerance in winter wheat (Triticum aestivum L.) is an economically important but complex trait. Four selected wheat genotypes, a winter hardy cultivar, Norstar, a tender spring cultivar, Manitou and two near-isogenic lines with Vrn-A1 (spring Norstar) and vrn-A1 (winter Manitou) alleles of Manitou and Norstar were cold-acclimated at 6°C and crown and leaf tissues were collected at 0, 2, 14, 21, 35, 42, 56 and 70 days of cold acclimation. cDNA-AFLP profiling was used to determine temporal expression profiles of transcripts during cold-acclimation in crown and leaf tissues, separately to determine if LT regulatory circuitries in crown and leaf tissues could be delineated using this approach. Screening 64 primer combinations identified 4,074 and 2,757 differentially expressed transcript-derived fragments (TDFs) out of which 38 and 16% were up-regulated as compared to 3 and 6% that were down-regulated in crown and leaf tissues, respectively. DNA sequencing of TDFs revealed sequences common to both tissues including genes coding for DEAD-box RNA helicase, choline-phosphate cytidylyltransferase and delta-1-pyrroline carboxylate synthetase. TDF specific to crown tissues included genes coding for phospahtidylinositol kinase, auxin response factor protein and brassinosteroid insensitive 1-associated receptor kinase. In leaf, genes such as methylene tetrahydrofolate reductase, NADH-cytochrome b5 reductase and malate dehydrogenase were identified. However, 30 and 14% of the DNA sequences from the crown and leaf tissues, respectively, were hypothetical or unknown proteins. Cluster analysis of up-, down-regulated and unique TDFs, DNA sequence and real-time PCR validation, infer that mechanisms operating in crown and leaf tissue in response to LT are differently regulated and warrant further studies.

The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation.

PubMed

Machado, Luciana E S F; Shen, Tun-Li; Page, Rebecca; Peti, Wolfgang

2017-05-26

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H 2 O 2 , which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H 2 O 2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation.

PubMed

Van Slambrouck, Séverine; Parmar, Virinder S; Sharma, Sunil K; De Bondt, Bart; Foré, Fleur; Coopman, Peter; Vanhoecke, Barbara W; Boterberg, Tom; Depypere, Herman T; Leclercq, Guy; Bracke, Marc E

2005-03-14

Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoid's action mechanism, we found that it inhibited extracellular-signal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile.

Efficacy and safety of tofacitinib in patients with active rheumatoid arthritis: review of key Phase 2 studies.

PubMed

Fleischmann, Roy; Kremer, Joel; Tanaka, Yoshiya; Gruben, David; Kanik, Keith; Koncz, Tamas; Krishnaswami, Sriram; Wallenstein, Gene; Wilkinson, Bethanie; Zwillich, Samuel H; Keystone, Edward

2016-12-01

Tofacitinib is an oral Janus kinase inhibitor for the treatment of rheumatoid arthritis (RA). Here, the safety and efficacy data from five Phase 2 studies of tofacitinib in patients with RA are summarized. Tofacitinib 1-30 mg twice daily was investigated, as monotherapy and in combination with methotrexate, in patients with RA. Tofacitinib 20 mg once daily was investigated in one study. Tofacitinib 5 and 10 mg twice daily were selected for investigation in Phase 3 studies; therefore, the efficacy and safety of tofacitinib 5 and 10 mg twice daily in Phase 2 studies are the focus of this review. Tofacitinib ≥ 5 mg twice daily was efficacious in a dose-dependent manner, with statistically significant and clinically meaningful reductions in the signs and symptoms of RA and patient-reported outcomes. The safety profile was consistent across studies. The efficacy and safety profile of tofacitinib in Phase 2 studies supported its further investigation and the selection of tofacitinib 5 mg twice daily and tofacitinib 10 mg twice daily for evaluation in Phase 3 studies. © 2016 The Authors. International Journal of Rheumatic Diseases published by Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases.

PubMed

Isozaki, Hideko; Ichihara, Eiki; Takigawa, Nagio; Ohashi, Kadoaki; Ochi, Nobuaki; Yasugi, Masayuki; Ninomiya, Takashi; Yamane, Hiromichi; Hotta, Katsuyuki; Sakai, Katsuya; Matsumoto, Kunio; Hosokawa, Shinobu; Bessho, Akihiro; Sendo, Toshiaki; Tanimoto, Mitsune; Kiura, Katsuyuki

2016-03-15

Crizotinib is the standard of care for advanced non-small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. ©2015 American Association for Cancer Research.

Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase.

PubMed

Mao, Yu-Ting; Zhu, Julia X; Hanamura, Kenji; Iurilli, Giuliano; Datta, Sandeep Robert; Dalva, Matthew B

2018-05-16

Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle. Copyright © 2018 Elsevier Inc. All rights reserved.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

PubMed

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Innovative computer-aided methods for the discovery of new kinase ligands.

PubMed

Abuhammad, Areej; Taha, Mutasem

2016-04-01

Recent evidence points to significant roles played by protein kinases in cell signaling and cellular proliferation. Faulty protein kinases are involved in cancer, diabetes and chronic inflammation. Efforts are continuously carried out to discover new inhibitors for selected protein kinases. In this review, we discuss two new computer-aided methodologies we developed to mine virtual databases for new bioactive compounds. One method is ligand-based exploration of the pharmacophoric space of inhibitors of any particular biotarget followed by quantitative structure-activity relationship-based selection of the best pharmacophore(s). The second approach is structure-based assuming that potent ligands come into contact with binding site spots distinct from those contacted by weakly potent ligands. Both approaches yield pharmacophores useful as 3D search queries for the discovery of new bioactive (kinase) inhibitors.

Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

PubMed

Ho, J C S; Nadeem, A; Rydström, A; Puthia, M; Svanborg, C

2016-02-18

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

Expression and activity of the 5'-AMP-activated protein kinase pathway in selected tissues during chicken embryonic development.

USDA-ARS?s Scientific Manuscript database

The 5’-AMP-activated protein kinase (AMPK) is a highly conserved serine/threonine protein kinase and a key part of a kinase signaling cascade that senses cellular energy status (AMP/ATP ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating m...

The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

PubMed Central

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

Definition of the binding mode of phosphoinositide 3-kinase α-selective inhibitor A-66S through molecular dynamics simulation.

PubMed

Bian, Xiaoli; Dong, Wangqing; Zhao, Yang; Sun, Rui; Kong, Wanjun; Li, Yiping

2014-04-01

Activation of the phosphatidylinositol 3-kinase α (PI3Kα) is commonly observed in human cancer and is critical for tumor progression, which has made PI3Kα an attractive target for anticancer drug discovery. To systematically investigate the binding mode of A-66S, a new selective PI3Kα inhibitor for PI3Kα, molecular docking, molecular dynamics simulation and ensuing energetic analysis were performed. The binding free energy between PI3Kα and A-66S is -11.27 kcal•mol⁻¹ using MMPBSA method, while -14.67 kcal•mol⁻¹ using MMGBSA method, which is beneficial for the binding, and the van der Waals/hydrophobic and electrostatic interactions are critical for the binding. The conserved hydrophobic adenine region of PI3Kα made up of Met772, Pro778, Ile800, Tyr836, Ile848, Val850, Val851, Met922, Phe930 and Ile932 accommodates the flat 2-tert-butyl-4'-methyl-4,5'-bithiazol moiety of A-66S, and the NH of Val851 forms a hydrogen with the nitrogen atom embedded in the aminothiazole ring of A-66S. The (S)-pyrrolidine carboxamide urea moiety especially extends toward the region of the binding site wall (Ser854-Gln859) defined by the C-terminal lobe, and has three hydrogen-bond arms with the backbone of Ser854 and the side chain of Gln859. Notably the interaction between the non-conserved residue Gln859 and A-66S is responsible for the selectivity profile of A-66S. The binding mode of A-66S for PI3Kα presented in this study should aid in the design of a new highly selective PI3Kα inhibitor.

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

PubMed Central

Fenouille, Nina; Bassil, Christopher F.; Ben-Sahra, Issam; Benajiba, Lina; Alexe, Gabriela; Ramos, Azucena; Pikman, Yana; Conway, Amy S.; Burgess, Michael R.; Li, Qing; Luciano, Frédéric; Auberger, Patrick; Galinsky, Ilene; DeAngelo, Daniel J.; Stone, Richard M.; Zhang, Yi; Perkins, Archibald S.; Shannon, Kevin; Hemann, Michael T.; Puissant, Alexandre; Stegmaier, Kimberly

2017-01-01

Expression of the EVI1 proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A pooled shRNA screen identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a metabolic dependency in EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted cell cycle arrest and apoptosis of human EVI1-positive AML cells, and prolonged survival in human orthotopic and mouse primary AML models. CKMT1 inhibition alters mitochondrial respiration and ATP production, an effect that is abrogated by phospho-creatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. PMID:28191887

Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity☆

PubMed Central

Marzano, Valeria; Santini, Simonetta; Rossi, Claudia; Zucchelli, Mirco; D'Alessandro, Annamaria; Marchetti, Carlo; Mingardi, Michele; Stagni, Venturina; Barilà, Daniela; Urbani, Andrea

2012-01-01

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics. PMID:22641158

Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

PubMed

Lamarche, M J; Borawski, J; Bose, A; Capacci-Daniel, C; Colvin, R; Dennehy, M; Ding, J; Dobler, M; Drumm, J; Gaither, L A; Gao, J; Jiang, X; Lin, K; McKeever, U; Puyang, X; Raman, P; Thohan, S; Tommasi, R; Wagner, K; Xiong, X; Zabawa, T; Zhu, S; Wiedmann, B

2012-10-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Anti-Hepatitis C Virus Activity and Toxicity of Type III Phosphatidylinositol-4-Kinase Beta Inhibitors

PubMed Central

LaMarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.

2012-01-01

Type III phosphatidylinositol-4-kinase beta (PI4KIIIβ) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIβ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIβ in lymphocyte proliferation. Previously proposed functions of PI4KIIIβ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIβ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. PMID:22825118

Discovery of 4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a Clinical p38[alpha] MAP Kinase Inhibitor for the Treatment of Inflammatory Diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chunjian; Lin, James; Wrobleski, Stephen T.

The discovery and characterization of 7k (BMS-582949), a highly selective p38{alpha} MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38{alpha} inhibitor. Unlike alkyl and other cycloalkyls, the sp{sup 2} character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38{alpha} enzymatic assay but displays a superior pharmacokinetic profile and, as such, was moremore » effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38{alpha} was confirmed by X-ray crystallographic analysis.« less

An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

PubMed Central

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

A strategy to analyse activity-based profiling of tyrosine kinase substrates in OCT-embedded lung cancer tissue.

PubMed

Arni, Stephan; de Wijn, Rik; Garcia-Villegas, Refugio; Bitanihirwe, Byron K Y; Caviezel, Claudio; Weder, Walter; Hillinger, Sven

2018-04-15

The use of optimal cutting temperature (OCT) medium has served to improve the long-term preservation of surgical tissue specimens. Unfortunately, the presence of polymers in OCT has been found to generate signal interference in proteomic-based techniques. Indeed the presence of OCT medium in tissue lysates precludes the analysis of activity based proteomic profiles obtained from lung adenocarcinoma (LuAdCa) resection specimens. In order to probe this question further tissue lysates were prepared from 47 lung non-neoplastic and tumour, node, metastasis (TNM) stage 1 LuAdCa resection specimens embedded with or without OCT, and data of activity based multiplex profiles of protein tyrosine kinase peptide substrates were obtained. We found that changes in overall phosphorylation level coincided with the use of OCT and subsequently developed an OCT per peptide median correcting strategy by performing median centering on the values of each peptide. Application of this post-analytical strategy not only can identify changes in kinase activity but can also assist in identifying novel targets for therapeutic intervention against LuAdCa. Copyright © 2018 Elsevier Inc. All rights reserved.

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Small-molecule WNK inhibition regulates cardiovascular and renal function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Ken; Park, Hyi-Man; Rigel, Dean F.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Comprehensive Genomic Profiling of Esthesioneuroblastoma Reveals Additional Treatment Options.

PubMed

Gay, Laurie M; Kim, Sungeun; Fedorchak, Kyle; Kundranda, Madappa; Odia, Yazmin; Nangia, Chaitali; Battiste, James; Colon-Otero, Gerardo; Powell, Steven; Russell, Jeffery; Elvin, Julia A; Vergilio, Jo-Anne; Suh, James; Ali, Siraj M; Stephens, Philip J; Miller, Vincent A; Ross, Jeffrey S

2017-07-01

Esthesioneuroblastoma (ENB), also known as olfactory neuroblastoma, is a rare malignant neoplasm of the olfactory mucosa. Despite surgical resection combined with radiotherapy and adjuvant chemotherapy, ENB often relapses with rapid progression. Current multimodality, nontargeted therapy for relapsed ENB is of limited clinical benefit. We queried whether comprehensive genomic profiling (CGP) of relapsed or refractory ENB can uncover genomic alterations (GA) that could identify potential targeted therapies for these patients. CGP was performed on formalin-fixed, paraffin-embedded sections from 41 consecutive clinical cases of ENBs using a hybrid-capture, adaptor ligation based next-generation sequencing assay to a mean coverage depth of 593X. The results were analyzed for base substitutions, insertions and deletions, select rearrangements, and copy number changes (amplifications and homozygous deletions). Clinically relevant GA (CRGA) were defined as GA linked to drugs on the market or under evaluation in clinical trials. A total of 28 ENBs harbored GA, with a mean of 1.5 GA per sample. Approximately half of the ENBs (21, 51%) featured at least one CRGA, with an average of 1 CRGA per sample. The most commonly altered gene was TP53 (17%), with GA in PIK3CA , NF1 , CDKN2A , and CDKN2C occurring in 7% of samples. We report comprehensive genomic profiles for 41 ENB tumors. CGP revealed potential new therapeutic targets, including targetable GA in the mTOR, CDK and growth factor signaling pathways, highlighting the clinical value of genomic profiling in ENB. Comprehensive genomic profiling of 41 relapsed or refractory ENBs reveals recurrent alterations or classes of mutation, including amplification of tyrosine kinases encoded on chromosome 5q and mutations affecting genes in the mTOR/PI3K pathway. Approximately half of the ENBs (21, 51%) featured at least one clinically relevant genomic alteration (CRGA), with an average of 1 CRGA per sample. The most commonly altered gene was TP53 (17%), and alterations in PIK3CA , NF1 , CDKN2A , or CDKN2C were identified in 7% of samples. Responses to treatment with the kinase inhibitors sunitinib, everolimus, and pazopanib are presented in conjunction with tumor genomics. © AlphaMed Press 2017.

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell–associated damage in IFNα-driven lupus nephritis

PubMed Central

Katewa, Arna; Wang, Yugang; Hackney, Jason A.; Huang, Tao; Suto, Eric; Ramamoorthi, Nandhini; Bremer, Meire; Chen, Jacob Zhi; Crawford, James J.; Currie, Kevin S.; Blomgren, Peter; DeVoss, Jason; DiPaolo, Julie A.; Hau, Jonathan; Lesch, Justin; DeForge, Laura E.; Lin, Zhonghua; Liimatta, Marya; Lubach, Joseph W.; McVay, Sami; Modrusan, Zora; Nguyen, Allen; Poon, Chungkee; Wang, Jianyong; Liu, Lichuan; Lee, Wyne P.; Wong, Harvey; Young, Wendy B.; Townsend, Michael J.

2017-01-01

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton’s tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and — similar to cyclophosphamide — improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell–mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE. PMID:28405610

Small molecule R1498 as a well-tolerated and orally active kinase inhibitor for hepatocellular carcinoma and gastric cancer treatment via targeting angiogenesis and mitosis pathways.

PubMed

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.

Small Molecule R1498 as a Well-Tolerated and Orally Active Kinase Inhibitor for Hepatocellular Carcinoma and Gastric Cancer Treatment via Targeting Angiogenesis and Mitosis Pathways

PubMed Central

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

Engineering A-kinase Anchoring Protein (AKAP)-selective Regulatory Subunits of Protein Kinase A (PKA) through Structure-based Phage Selection*

PubMed Central

Gold, Matthew G.; Fowler, Douglas M.; Means, Christopher K.; Pawson, Catherine T.; Stephany, Jason J.; Langeberg, Lorene K.; Fields, Stanley; Scott, John D.

2013-01-01

PKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces. PMID:23625929

2-Aminobenzimidazoles as potent Aurora kinase inhibitors.

PubMed

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.

Structural Characterization of Proline-rich Tyrosine Kinase 2 (PYK2) Reveals a Unique (DFG-out) Conformation and Enables Inhibitor Design

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

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

PubMed

Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

2016-04-12

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities.

PubMed

Lanning, Nathan J; Castle, Joshua P; Singh, Simar J; Leon, Andre N; Tovar, Elizabeth A; Sanghera, Amandeep; MacKeigan, Jeffrey P; Filipp, Fabian V; Graveel, Carrie R

2017-01-01

Among breast cancers, the triple-negative breast cancer (TNBC) subtype has the worst prognosis with no approved targeted therapies and only standard chemotherapy as the backbone of systemic therapy. Unique metabolic changes in cancer progression provide innovative therapeutic opportunities. The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), and MET receptor are highly expressed in TNBC, making both promising therapeutic targets. RTK signaling profoundly alters cellular metabolism by increasing glucose consumption and subsequently diverting glucose carbon sources into metabolic pathways necessary to support the tumorigenesis. Therefore, detailed metabolic profiles of TNBC subtypes and their response to tyrosine kinase inhibitors may identify therapeutic sensitivities. We quantified the metabolic profiles of TNBC cell lines representing multiple TNBC subtypes using gas chromatography mass spectrometry. In addition, we subjected MDA-MB-231, MDA-MB-468, Hs578T, and HCC70 cell lines to metabolic flux analysis of basal and maximal glycolytic and mitochondrial oxidative rates. Metabolic pool size and flux measurements were performed in the presence and absence of the MET inhibitor, INC280/capmatinib, and the EGFR inhibitor, erlotinib. Further, the sensitivities of these cells to modulators of core metabolic pathways were determined. In addition, we annotated a rate-limiting metabolic enzymes library and performed a siRNA screen in combination with MET or EGFR inhibitors to validate synergistic effects. TNBC cell line models displayed significant metabolic heterogeneity with respect to basal and maximal metabolic rates and responses to RTK and metabolic pathway inhibitors. Comprehensive systems biology analysis of metabolic perturbations, combined siRNA and tyrosine kinase inhibitor screens identified a core set of TCA cycle and fatty acid pathways whose perturbation sensitizes TNBC cells to small molecule targeting of receptor tyrosine kinases. Similar to the genomic heterogeneity observed in TNBC, our results reveal metabolic heterogeneity among TNBC subtypes and demonstrate that understanding metabolic profiles and drug responses may prove valuable in targeting TNBC subtypes and identifying therapeutic susceptibilities in TNBC patients. Perturbation of metabolic pathways sensitizes TNBC to inhibition of receptor tyrosine kinases. Such metabolic vulnerabilities offer promise for effective therapeutic targeting for TNBC patients.

Initiative for Molecular Profiling and Advanced Cancer Therapy (IMPACT): An MD Anderson Precision Medicine Study

PubMed Central

Tsimberidou, Apostolia-Maria; Hong, David S.; Ye, Yang; Cartwright, Carrie; Wheler, Jennifer J.; Falchook, Gerald S.; Naing, Aung; Fu, Siqing; Piha-Paul, Sarina; Janku, Filip; Meric-Bernstam, Funda; Hwu, Patrick; Kee, Bryan; Kies, Merrill S.; Broaddus, Russell; Mendelsohn, John; Hess, Kenneth R.; Kurzrock, Razelle

2017-01-01

Purpose Genomic profiling is increasingly used in the management of cancer. We have previously reported preliminary results of our precision medicine program. Here, we present response and survival outcomes for 637 additional patients who were referred for phase I trials and were treated with matched targeted therapy (MTT) when available. Patients and Methods Patients with advanced cancer who underwent tumor genomic analyses were treated with MTT when available. Results Overall, 1,179 (82.1%) of 1,436 patients had one or more alterations (median age, 59.7 years; men, 41.2%); 637 had one or more actionable aberrations and were treated with MTT (n = 390) or non-MTT (n = 247). Patients who were treated with MTT had higher rates of complete and partial response (11% v 5%; P = .0099), longer failure-free survival (FFS; 3.4 v 2.9 months; P = .0015), and longer overall survival (OS; 8.4 v 7.3 months; P = .041) than did unmatched patients. Two-month landmark analyses showed that, for MTT patients, FFS for responders versus nonresponders was 7.6 versus 4.3 months (P < .001) and OS was 23.4 versus 8.5 months (P < .001), whereas for non-MTT patients (responders v nonresponders), FFS was 6.6 versus 4.1 months (P = .001) and OS was 15.2 versus 7.5 months (P = .43). Patients with phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathway alterations matched to PI3K/Akt/mammalian target of rapamycin axis inhibitors alone demonstrated outcomes comparable to unmatched patients. Conclusion Our results support the use of genomic matching. Subset analyses indicate that matching patients who harbor a PI3K and mitogen-activated protein kinase pathway alteration to only a PI3K pathway inhibitor does not improve outcome. We have initiated IMPACT2, a randomized trial to compare treatment with and without genomic selection. PMID:29082359

Involvement of mitogen-activated protein kinase activation in the signal-transduction pathways of the soya bean oxidative burst.

PubMed Central

Taylor, A T; Kim, J; Low, P S

2001-01-01

The oxidative burst constitutes one of the most rapid defence responses characterized in the Plant Kingdom. We have observed that four distinct elicitors of the soya bean oxidative burst activate kinases of masses approximately 44 kDa and approximately 47 kDa. Evidence that these kinases regulate production of reactive oxygen species include: (i) their rapid activation by oxidative burst elicitors, (ii) their tight temporal correlation between activation/deactivation of the kinases and activation/deactivation of the oxidative burst, (iii) the identical pharmacological profile of kinase activation and oxidant production for 13 commonly used inhibitors, and (iv) the autologous activation of both kinases and oxidant production by calyculin A and cantharidin, two phosphatase inhibitors. Immunological and biochemical studies reveal that the activated 44 kDa and 47 kDa kinases are mitogen-activated protein (MAP) kinase family members. The kinases prefer myelin basic protein as a substrate, and they phosphorylate primarily on threonine residues. The kinases are themselves phosphorylated on tyrosine residues, and this phosphorylation is required for activity. Finally, both kinases are recognized by an antibody against activated MAP kinase immediately after (but not before) cell stimulation by elicitors. Based on these and other observations, a preliminary sequence of signalling steps linking elicitor stimulation, kinase activation and Ca(2+) entry, to initiation of oxidant production, is proposed. PMID:11311144

Differential expression profiles of poplar MAP kinase kinases in response to abiotic stresses and plant hormones, and overexpression of PtMKK4 improves the drought tolerance of poplar.

PubMed

Wang, Lei; Su, Hongyan; Han, Liya; Wang, Chuanqi; Sun, Yanlin; Liu, Fenghong

2014-07-15

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play essential roles in plant growth, development and stress response. MAPK kinases (MAPKKs), which link MAPKs and MAPKK kinases (MAPKKKs), are integral in mediating various stress responses in plants. However, to date few data about the roles of poplar MAPKKs in stress signal transduction are available. In this study, we performed a systemic analysis of poplar MAPKK gene family expression profiles in response to several abiotic stresses and stress-associated hormones. Furthermore, Populus trichocarpa MAPKK4 (PtMKK4) was chosen for functional characterization. Transgenic analysis showed that overexpression of the PtMKK4 gene remarkably enhanced drought stress tolerance in the transgenic poplar plants. The PtMKK4-overexpressing plants also exhibited much lower levels of H2O2 and higher antioxidant enzyme activity after exposure to drought stress compared to the wide type lines. Besides, some drought marker genes including PtP5CS, PtSUS3, PtLTP3 and PtDREB8 exhibited higher expression levels in the transgenic lines than in the wide type under drought conditions. This study provided valuable information for understanding the putative functions of poplar MAPKKs involved in important signaling pathways under different stress conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis of the highly selective p38 MAPK inhibitor UR-13756 for possible therapeutic use in Werner syndrome.

PubMed

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.

N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor.

PubMed

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.

Structural Basis for the Potent and Selective Inhibition of Casein Kinase 1 Epsilon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, Alexander M.; Zhao, Huilin; Huang, Xin

2012-10-29

Casein kinase 1 epsilon (CK1ε) and its closest homologue CK1δ are key regulators of diverse cellular processes. We report two crystal structures of PF4800567, a potent and selective inhibitor of CK1ε, bound to the kinase domains of human CK1ε and CK1δ as well as one apo CK1ε crystal structure. These structures provide a molecular basis for the strong and specific inhibitor interactions with CK1ε and suggest clues for further development of CK1δ inhibitors.

The Future of Janus Kinase Inhibitors in Inflammatory Bowel Disease

PubMed Central

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

The Future of Janus Kinase Inhibitors in Inflammatory Bowel Disease.

PubMed

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.

First-in-human Phase I study of Pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors

PubMed Central

Baird, Richard; Kristeleit, Rebecca; Shah, Krunal; Moreno, Victor; Clarke, Paul A.; Raynaud, Florence I.; Levy, Gallia; Ware, Joseph A; Mazina, Kathryn; Lin, Ray; Wu, Jenny; Fredrickson, Jill; Spoerke, Jill M; Lackner, Mark R; Yan, Yibing; Friedman, Lori S.; Kaye, Stan B.; Derynck, Mika K.; Workman, Paul; de Bono, Johann S.

2014-01-01

Purpose This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal tolerated dose (MTD), dose limiting toxicities (DLTs), pharmacokinetics, pharmacodynamics and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent and selective inhibitor of the Class I phosphatidylinositol-3-kinases (PI3K). Patients and Methods Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450mg once-daily, initially on days 1-21 every 28 days and later, utilizing continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma and tumor tissue. Results Pictilisib was well-tolerated. The most common toxicities were grade 1-2 nausea, rash and fatigue while the DLT was grade 3 maculopapular rash (450mg, 2 of 3 patients; 330mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in platelet rich plasma at 3 hours post-dose at the MTD and in tumor at pictilisib doses associated with AUC >20uM.hr. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. Conclusion Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100mg and signs of antitumor activity. The recommended Phase II dose was continuous dosing at 330mg once-daily. PMID:25370471

First-in-human phase I study of pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors.

PubMed

Sarker, Debashis; Ang, Joo Ern; Baird, Richard; Kristeleit, Rebecca; Shah, Krunal; Moreno, Victor; Clarke, Paul A; Raynaud, Florence I; Levy, Gallia; Ware, Joseph A; Mazina, Kathryn; Lin, Ray; Wu, Jenny; Fredrickson, Jill; Spoerke, Jill M; Lackner, Mark R; Yan, Yibing; Friedman, Lori S; Kaye, Stan B; Derynck, Mika K; Workman, Paul; de Bono, Johann S

2015-01-01

This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily. ©2014 American Association for Cancer Research.

Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

PubMed

Cardona-Correa, Albin; Rios-Velazquez, Carlos

2016-05-01

The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents.

Crystal Structure of Human AKT1 with an Allosteric Inhibitor Reveals a New Mode of Kinase Inhibition

PubMed Central

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

Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

PubMed

Jones, Mathew J K; Jallepalli, Prasad V

2016-01-01

During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

PubMed

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

2012-02-21

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability

PubMed Central

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D.; Silkworth, Whitney; Ross, Nathan T.; Scherer, Christina A.; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A.; Stern, Andrew M.; Schreiber, Stuart L.; Golub, Todd R.

2012-01-01

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33’s kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33’s kinase activity does not represent a promising anti-KRAS therapeutic strategy. PMID:22323609

Single cell gene expression profiling in Alzheimer's disease.

PubMed

Ginsberg, Stephen D; Che, Shaoli; Counts, Scott E; Mufson, Elliott J

2006-07-01

Development and implementation of microarray techniques to quantify expression levels of dozens to hundreds to thousands of transcripts simultaneously within select tissue samples from normal control subjects and neurodegenerative diseased brains has enabled scientists to create molecular fingerprints of vulnerable neuronal populations in Alzheimer's disease (AD) and related disorders. A goal is to sample gene expression from homogeneous cell types within a defined region without potential contamination by expression profiles of adjacent neuronal subpopulations and nonneuronal cells. The precise resolution afforded by single cell and population cell RNA analysis in combination with microarrays and real-time quantitative polymerase chain reaction (qPCR)-based analyses allows for relative gene expression level comparisons across cell types under different experimental conditions and disease progression. The ability to analyze single cells is an important distinction from global and regional assessments of mRNA expression and can be applied to optimally prepared tissues from animal models of neurodegeneration as well as postmortem human brain tissues. Gene expression analysis in postmortem AD brain regions including the hippocampal formation and neocortex reveals selectively vulnerable cell types share putative pathogenetic alterations in common classes of transcripts, for example, markers of glutamatergic neurotransmission, synaptic-related markers, protein phosphatases and kinases, and neurotrophins/neurotrophin receptors. Expression profiles of vulnerable regions and neurons may reveal important clues toward the understanding of the molecular pathogenesis of various neurological diseases and aid in identifying rational targets toward pharmacotherapeutic interventions for progressive, late-onset neurodegenerative disorders such as mild cognitive impairment (MCI) and AD.

Fragment-based drug discovery of potent and selective MKK3/6 inhibitors.

PubMed

Adams, Mark; Kobayashi, Toshitake; Lawson, J David; Saitoh, Morihisa; Shimokawa, Kenichiro; Bigi, Simone V; Hixon, Mark S; Smith, Christopher R; Tatamiya, Takayuki; Goto, Masayuki; Russo, Joseph; Grimshaw, Charles E; Swann, Steven

2016-02-01

The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38. Copyright © 2015 Elsevier Ltd. All rights reserved.

Use of tyrosine kinase inhibitors for chronic myeloid leukemia: management of patients and practical applications for pharmacy practitioners.

PubMed

Wong, Siu-Fun; Mirshahidi, Hamid

2011-06-01

To summarize the use of tyrosine kinase inhibitors (TKIs) for treatment of patients with chronic myeloid leukemia (CML) and provide practical information for patient management. Literature was retrieved from PubMed (2000-January 2011), using the search terms chronic myeloid leukemia and tyrosine kinase inhibitor. Abstracts presented at the 2008-2010 annual meetings of the American Society of Hematology and the American Society of Clinical Oncology, reference citations from identified publications, as well as the manufacturers' full prescribing information for cited drugs, also were reviewed. Articles evaluating the efficacy and safety of the TKIs imatinib, nilotinib, and dasatinib were evaluated. Focus was placed on publications supporting management of patients with CML in the chronic phase. Reports presenting clinical trial information of TKIs in development also were included. The discovery of targeted tyrosine kinase inhibition of BCR-ABL kinase dramatically changed the treatment of CML. Imatinib, the first TKI approved for treatment of patients with Philadelphia chromosome--positive CML, demonstrated significant superiority over the previous standard of care: interferon plus cytarabine. The newer, more potent TKIs, nilotinib and dasatinib, have demonstrated improved efficacy over imatinib as first-line therapy and provide an effective option for patients with resistance or intolerance to imatinib. To maximize efficacy of TKI therapy, close patient management, involving frequent monitoring of patient response, is essential. Given the importance of continuing TKI therapy, early recognition and management of adverse events are critical to optimizing outcomes in patients with CML. In addition to the safety profile and considerations of comorbidities, additional factors can affect therapeutic selection, including drug-drug and drug-food interactions. Research investigating new therapies, particularly for patients harboring the T315I mutation-which remains refractory to current TKIs-continues in the quest to improve outcomes in patients with CML.

Identification of a Bis-guanylhydrazone [4,4'-Diacetyldiphenylurea-bis(guanylhydrazone); NSC 109555] as a novel chemotype for inhibition of Chk2 kinase.

PubMed

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.

Rare Complex Mutational Profile in an ALK Inhibitor-resistant Non-small Cell Lung Cancer.

PubMed

Azzato, Elizabeth M; Deshpande, Charuhas; Aikawa, Vania; Aggarwal, Charu; Alley, Evan; Jacobs, Benjamin; Morrissette, Jennifer; Daber, Robert

2015-05-01

Testing for somatic alterations, including anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and epidermal growth factor receptor gene (EGFR) mutations, is standard practice in the diagnostic evaluation and therapeutic management of non-small cell lung cancer (NSCLC), where the results of such tests can predict response to targeted-therapy. ALK rearrangements, EGFR mutations and mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) are considered mutually exclusive in NSCLC. Herein we identified a KRAS Q22K mutation and frameshift mutations in the genes encoding serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated serine/threonine kinase (ATM) by next-generation sequencing in a patient with ALK rearrangement-positive oligo-metastatic NSCLC, whose disease progressed while on two ALK-targeted therapies. Such a complex diagnostic genetic profile has not been reported in ALK fusion-positive NSCLC. This case highlights the utility of comprehensive molecular testing in the diagnosis of NSCLC. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

A review on adverse event profiles of epidermal growth factor receptor-tyrosine kinase inhibitors in nonsmall cell lung cancer patients.

PubMed

Biswas, B; Ghadyalpatil, N; Krishna, M V; Deshmukh, J

2017-12-01

The epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of EGFR-mutant nonsmall cell lung cancer (NSCLC). These EGFR TKIs demonstrate a different adverse event (AE) profile as compared to conventional chemotherapy agents. They are more commonly associated with cutaneous AEs and diarrhea while hematological AEs occurred commonly with chemotherapy agents. These AEs are the extension of pharmacological effect and occur as a result of blockade of EGFR-regulated pathways in the skin and gastrointestinal tract. This review article sheds light on the safety profile of first-, second-, and third-generation EGFR TKIs based on data obtained from several clinical trials conducted in NSCLC patients and highlights trials comparing these agents with the conventional chemotherapy agents. The strategies to manage EGFR TKI-related AEs are also reviewed.

Type II Kinase Inhibitors Show an Unexpected Inhibition Mode against Parkinson’s Disease-Linked LRRK2 Mutant G2019S

PubMed Central

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

Nilotinib: optimal therapy for patients with chronic myeloid leukemia and resistance or intolerance to imatinib.

PubMed

Swords, Ronan; Mahalingam, Devalingam; Padmanabhan, Swaminathan; Carew, Jennifer; Giles, Francis

2009-09-21

Chronic myeloid leukemia (CML) is the consequence of a single balanced translocation that produces the BCR-ABL fusion oncogene which is detectable in over 90% of patients at presentation. The BCR-ABL inhibitor imatinib mesylate (IM) has improved survival in all phases of CML and is the standard of care for newly diagnosed patients in chronic phase. Despite the very significant therapeutic benefits of IM, a small minority of patients with early stage disease do not benefit optimally while IM therapy in patients with advanced disease is of modest benefit in many. Diverse mechanisms may be responsible for IM failures, with point mutations within the Bcr-Abl kinase domain being amongst the most common resistance mechanisms described in patients with advanced CML. The development of novel agents designed to overcome IM resistance, while still primarily targeted on BCR-ABL, led to the creation of the high affinity aminopyrimidine inhibitor, nilotinib. Nilotinib is much more potent as a BCR-ABL inhibitor than IM and inhibits both wild type and IM-resistant BCR-ABL with significant clinical activity across the entire spectrum of BCR-ABL mutants with the exception of T315I. The selection of a second generation tyrosine kinase inhibitor to rescue patients with imatinib failure will be based on several factors including age, co-morbid medical problems and ABL kinase mutational profile. It should be noted that while the use of targeted BCR-ABL kinase inhibitors in CML represents a paradigm shift in CML management these agents are not likely to have activity against the quiescent CML stem cell pool. The purpose of this review is to summarize the pre-clinical and clinical data on nilotinib in patients with CML who have failed prior therapy with IM or dasatinib.

A High-Content Small Molecule Screen Identifies Sensitivity of Glioblastoma Stem Cells to Inhibition of Polo-Like Kinase 1

PubMed Central

Danovi, Davide; Folarin, Amos; Gogolok, Sabine; Ender, Christine; Elbatsh, Ahmed M. O.; Engström, Pär G.; Stricker, Stefan H.; Gagrica, Sladjana; Georgian, Ana; Yu, Ding; U, Kin Pong; Harvey, Kevin J.; Ferretti, Patrizia; Paddison, Patrick J.; Preston, Jane E.; Abbott, N. Joan; Bertone, Paul; Smith, Austin; Pollard, Steven M.

2013-01-01

Glioblastoma multiforme (GBM) is the most common primary brain cancer in adults and there are few effective treatments. GBMs contain cells with molecular and cellular characteristics of neural stem cells that drive tumour growth. Here we compare responses of human glioblastoma-derived neural stem (GNS) cells and genetically normal neural stem (NS) cells to a panel of 160 small molecule kinase inhibitors. We used live-cell imaging and high content image analysis tools and identified JNJ-10198409 (J101) as an agent that induces mitotic arrest at prometaphase in GNS cells but not NS cells. Antibody microarrays and kinase profiling suggested that J101 responses are triggered by suppression of the active phosphorylated form of polo-like kinase 1 (Plk1) (phospho T210), with resultant spindle defects and arrest at prometaphase. We found that potent and specific Plk1 inhibitors already in clinical development (BI 2536, BI 6727 and GSK 461364) phenocopied J101 and were selective against GNS cells. Using a porcine brain endothelial cell blood-brain barrier model we also observed that these compounds exhibited greater blood-brain barrier permeability in vitro than J101. Our analysis of mouse mutant NS cells (INK4a/ARF−/−, or p53−/−), as well as the acute genetic deletion of p53 from a conditional p53 floxed NS cell line, suggests that the sensitivity of GNS cells to BI 2536 or J101 may be explained by the lack of a p53-mediated compensatory pathway. Together these data indicate that GBM stem cells are acutely susceptible to proliferative disruption by Plk1 inhibitors and that such agents may have immediate therapeutic value. PMID:24204733

Hit to lead optimization of pyrazolo[1,5-a]pyrimidines as B-Raf kinase inhibitors.

PubMed

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.

KinView: A visual comparative sequence analysis tool for integrated kinome research

PubMed Central

McSkimming, Daniel Ian; Dastgheib, Shima; Baffi, Timothy R.; Byrne, Dominic P.; Ferries, Samantha; Scott, Steven Thomas; Newton, Alexandra C.; Eyers, Claire E.; Kochut, Krzysztof J.; Eyers, Patrick A.

2017-01-01

Multiple sequence alignments (MSAs) are a fundamental analysis tool used throughout biology to investigate relationships between protein sequence, structure, function, evolutionary history, and patterns of disease-associated variants. However, their widespread application in systems biology research is currently hindered by the lack of user-friendly tools to simultaneously visualize, manipulate and query the information conceptualized in large sequence alignments, and the challenges in integrating MSAs with multiple orthogonal data such as cancer variants and post-translational modifications, which are often stored in heterogeneous data sources and formats. Here, we present the Multiple Sequence Alignment Ontology (MSAOnt), which represents a profile or consensus alignment in an ontological format. Subsets of the alignment are easily selected through the SPARQL Protocol and RDF Query Language for downstream statistical analysis or visualization. We have also created the Kinome Viewer (KinView), an interactive integrative visualization that places eukaryotic protein kinase cancer variants in the context of natural sequence variation and experimentally determined post-translational modifications, which play central roles in the regulation of cellular signaling pathways. Using KinView, we identified differential phosphorylation patterns between tyrosine and serine/threonine kinases in the activation segment, a major kinase regulatory region that is often mutated in proliferative diseases. We discuss cancer variants that disrupt phosphorylation sites in the activation segment, and show how KinView can be used as a comparative tool to identify differences and similarities in natural variation, cancer variants and post-translational modifications between kinase groups, families and subfamilies. Based on KinView comparisons, we identify and experimentally characterize a regulatory tyrosine (Y177PLK4) in the PLK4 C-terminal activation segment region termed the P+1 loop. To further demonstrate the application of KinView in hypothesis generation and testing, we formulate and validate a hypothesis explaining a novel predicted loss-of-function variant (D523NPKCβ) in the regulatory spine of PKCβ, a recently identified tumor suppressor kinase. KinView provides a novel, extensible interface for performing comparative analyses between subsets of kinases and for integrating multiple types of residue specific annotations in user friendly formats. PMID:27731453

Systematic review and meta-analysis of selected toxicities of approved ALK inhibitors in metastatic non-small cell lung cancer.

PubMed

Costa, Rubens Barros; Costa, Ricardo L B; Talamantes, Sarah M; Kaplan, Jason B; Bhave, Manali A; Rademaker, Alfred; Miller, Corinne; Carneiro, Benedito A; Mahalingam, Devalingam; Chae, Young Kwang

2018-04-24

Anaplastic lymphoma kinase ( ALK ) inhibitors are the mainstay treatment for patients with non-small cell lung carcinoma (NSCLC) harboring a rearrangement of the ALK gene or the ROS1 oncogenes. With the recent publication of pivotal trials leading to the approval of these compounds in different indications, their toxicity profile warrants an update. A systematic literature search was performed in July 2017. Studies evaluating US FDA approved doses of one of the following ALK inhibitors: Crizotinib, Ceritinib, Alectinib or Brigatinib as monotherapy were included. Data were analyzed using random effects meta-analysis for absolute risks (AR), study heterogeneity, publication bias and differences among treatments. Fifteen trials with a total of 2,005 patients with evaluable toxicity data were included in this report. There was significant heterogeneity amongst different studies. The pooled AR of death and severe adverse events were 0.5% and 34.5%, respectively. Grade 3/4 nausea, vomiting, diarrhea, and constipation were uncommon: 2.6%, 2.5%, 2.7%, 1.2%, respectively. ALK inhibitors have an acceptable safety profile with a low risk of treatment-related deaths. Important differences in toxicity profile were detected amongst the different drugs.

Sustained activation of c-Jun N-terminal and extracellular signal-regulated kinases in port-wine stain blood vessels.

PubMed

Tan, Wenbin; Chernova, Margarita; Gao, Lin; Sun, Victor; Liu, Huaxu; Jia, Wangcun; Langer, Stephanie; Wang, Gang; Mihm, Martin C; Nelson, J Stuart

2014-11-01

Port-wine stain (PWS) is a congenital, progressive vascular malformation but the pathogenesis remains incompletely understood. We sought to investigate the activation status of various kinases, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, AKT, phosphatidylinositol 3-kinase, P70 ribosomal S6 kinase, and phosphoinositide phospholipase C γ subunit, in PWS biopsy tissues. Immunohistochemistry was performed on 19 skin biopsy samples from 11 patients with PWS. c-Jun N-terminal kinase, extracellular signal-regulated kinase, and P70 ribosomal S6 kinase in pediatric and adult PWS blood vessels were consecutively activated. Activation of AKT and phosphatidylinositol 3-kinase was found in many adult hypertrophic PWS blood vessels but not in infants. Phosphoinositide phospholipase C γ subunit showed strong activation in nodular PWS blood vessels. Infantile PWS sample size was small. Our data suggest a subsequent activation profile of various kinases during different stages of PWS: (1) c-Jun N-terminal and extracellular signal-regulated kinases are firstly and consecutively activated in all PWS tissues, which may contribute to both the pathogenesis and progressive development of PWS; (2) AKT and phosphatidylinositol 3-kinase are subsequently activated, and are involved in the hypertrophic development of PWS blood vessels; and (3) phosphoinositide phospholipase C γ subunit is activated in the most advanced stage of PWS and may participate in nodular formation. Copyright © 2014 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

Structure and evolution analysis of pollen receptor-like kinase in Zea mays and Arabidopsis thaliana.

PubMed

Wang, Dongxu; Wang, He; Irfan, Muhammad; Fan, Mingxia; Lin, Feng

2014-08-01

Receptor-like kinase (RLKs) is an important member in protein kinase family which is widely involved in plant growth, development and defense responses. It is significant to analyze the kinase structure and evolution of pollen RLKs in order to study their mechanisms. In our study, 64 and 73 putative pollen RLKs were chosen from maize and Arabidopsis. Phylogenetic analysis showed that the pollen RLKs were conservative and might had existed before divergence between monocot and dicot which were mainly concentrated in RLCK-VII and LRR-III two subfamilies. Chromosomal localization and gene duplication analysis showed the expansion of pollen RLKs were mainly caused by segmental duplication. By calculating Ka/Ks value of extracellular domain, intracellular domain and kinase domain in pollen RLKs, we found that the pollen RLKs duplicated genes had mainly experienced the purifying selection, while maize might have experienced weaker purifying selection. Meanwhile, extracellular domain might have experienced stronger diversifying selection than intracellular domain in both species. Estimation of duplication time showed that the duplication events of Arabidopsis have occurred approximately between 18 and 69 million years ago, compared to 0.67-170 million years ago of maize. Copyright © 2014 Elsevier Ltd. All rights reserved.

Selectivity Mechanism of ATP-Competitive Inhibitors for PKB and PKA.

PubMed

Wu, Ke; Pang, Jingzhi; Song, Dong; Zhu, Ying; Wu, Congwen; Shao, Tianqu; Chen, Haifeng

2015-07-01

Protein kinase B (PKB) acts as a central node on the PI3K kinase pathway. Constitutive activation and overexpression of PKB have been identified to involve in various cancers. However, protein kinase A (PKA) sharing high homology with PKB is essential for metabolic regulation. Therefore, specific targeting on PKB is crucial strategy in drug design and development for antitumor. Here, we had revealed the selectivity mechanism for PKB inhibitors with molecular dynamics simulation and 3D-QSAR methods. Selective inhibitors of PKB could form more hydrogen bonds and hydrophobic contacts with PKB than those with PKA. This could explain that selective inhibitor M128 is more potent to PKB than to PKA. Then, 3D-QSAR models were constructed for these selective inhibitors and evaluated by test set compounds. 3D-QSAR model comparison of PKB inhibitors and PKA inhibitors reveals possible methods to improve the selectivity of inhibitors. These models can be used to design new chemical entities and make quantitative prediction of the specific selective inhibitors before resorting to in vitro and in vivo experiment. © 2014 John Wiley & Sons A/S.

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

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.

Structural Basis of Wee Kinases Functionality and Inactivation by Diverse Small Molecule Inhibitors.

PubMed

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.

Anti-inflammatory properties of Gö 6850: a selective inhibitor of protein kinase C.

PubMed

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)

The PIM kinases in hematological cancers.

PubMed

Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

2012-02-01

The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

Development of a potent and selective FLT3 kinase inhibitor by systematic expansion of a non-selective fragment-screening hit.

PubMed

Nakano, Hirofumi; Hasegawa, Tsukasa; Imamura, Riyo; Saito, Nae; Kojima, Hirotatsu; Okabe, Takayoshi; Nagano, Tetsuo

2016-05-01

A non-selective inhibitor (1) of FMS-like tyrosine kinase-3 (FLT3) was identified by fragment screening and systematically modified to afford a potent and selective inhibitor 26. We confirmed that 26 inhibited the growth of FLT-3-activated human acute myeloid leukemia cell line MV4-11. Our design strategy enabled rapid development of a novel type of FLT3 inhibitor from the hit fragment in the absence of target-structural information. Copyright © 2016 Elsevier Ltd. All rights reserved.

2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase.

PubMed

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.

Structure-functional prediction and analysis of cancer mutation effects in protein kinases.

PubMed

Dixit, Anshuman; Verkhivker, Gennady M

2014-01-01

A central goal of cancer research is to discover and characterize the functional effects of mutated genes that contribute to tumorigenesis. In this study, we provide a detailed structural classification and analysis of functional dynamics for members of protein kinase families that are known to harbor cancer mutations. We also present a systematic computational analysis that combines sequence and structure-based prediction models to characterize the effect of cancer mutations in protein kinases. We focus on the differential effects of activating point mutations that increase protein kinase activity and kinase-inactivating mutations that decrease activity. Mapping of cancer mutations onto the conformational mobility profiles of known crystal structures demonstrated that activating mutations could reduce a steric barrier for the movement from the basal "low" activity state to the "active" state. According to our analysis, the mechanism of activating mutations reflects a combined effect of partial destabilization of the kinase in its inactive state and a concomitant stabilization of its active-like form, which is likely to drive tumorigenesis at some level. Ultimately, the analysis of the evolutionary and structural features of the major cancer-causing mutational hotspot in kinases can also aid in the correlation of kinase mutation effects with clinical outcomes.

The rational design of a novel potent analogue of the 5’-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

PubMed Central

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

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%).

Fragment-based approaches to the discovery of kinase inhibitors.

PubMed

Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

2014-01-01

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

PubMed

Blake, James F; Xu, Rui; Bencsik, Josef R; Xiao, Dengming; Kallan, Nicholas C; Schlachter, Stephen; Mitchell, Ian S; Spencer, Keith L; Banka, Anna L; Wallace, Eli M; Gloor, Susan L; Martinson, Matthew; Woessner, Richard D; Vigers, Guy P A; Brandhuber, Barbara J; Liang, Jun; Safina, Brian S; Li, Jun; Zhang, Birong; Chabot, Christine; Do, Steven; Lee, Leslie; Oeh, Jason; Sampath, Deepak; Lee, Brian B; Lin, Kui; Liederer, Bianca M; Skelton, Nicholas J

2012-09-27

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

Visual Snapshots of Intracellular Kinase Activity At The Onset of Mitosis

PubMed Central

Dai, Zhaohua; Dulyaninova, Natalya G.; Kumar, Sanjai; Bresnick, Anne R.; Lawrence, David S.

2007-01-01

Summary Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell division cycle. Robust protein kinase activity transpires just prior to, but not immediately following, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC β isoform with subsequent NEB. PMID:18022564

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

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

PubMed

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Biological Effects of the Pim Kinase Inhibitor, SGI-1776, in Multiple Myeloma

PubMed Central

Cervantes-Gomez, Fabiola; Chen, Lisa S.; Orlowski, Robert Z.; Gandhi, Varsha

2013-01-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138+ cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner and effect on cell death and proliferation, induction of autophagy, as wells as changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138+ cells (<10%) as assessed by Annexin-V/PI. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, as assessed by acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266 25-70%; MM.1S 8-52%) and CD138+ cells (19-21%). Immunoblot analyses of autophagy LC3b marker and translation initiation proteins (phospho p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138+ myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. PMID:23988451

Biological effects of the Pim kinase inhibitor, SGI-1776, in multiple myeloma.

PubMed

Cervantes-Gomez, Fabiola; Chen, Lisa S; Orlowski, Robert Z; Gandhi, Varsha

2013-09-01

Pim kinases are constitutively active serine/threonine/tyrosine kinases that are overexpressed in hematological malignancies such as multiple myeloma. Pim kinase substrates are involved in transcription, protein translation, cell proliferation, and apoptosis. SGI-1776 is a potent Pim kinase inhibitor that has proven to be cytotoxic to leukemia and lymphoma cells. Based on this background, we hypothesized that SGI-1776 treatment would result in myeloma cytotoxicity. To test this, myeloma cell lines and primary CD138(+) cells from myeloma patients were treated with SGI-1776 in a dose- and time-dependent manner, and effect on cell death and proliferation, induction of autophagy, and changes in cell cycle profile were measured. SGI-1776 treatment resulted in limited apoptosis in cell lines (mean 30%) and CD138(+) cells (< 10%) assessed using Annexin-V/propidium iodide. Limited effect was observed in cell cycle profile or growth in cell lines. However, DNA synthesis was decreased by 70% at 3 μM (all time points) in U266 though this was not observed in MM.1S. In accordance, immunoblot analyses revealed no change in transcription (c-Myc and H3), or apoptotic (Bad) proteins that are substrates of Pim kinases. In contrast, autophagy, assessed using acridine orange staining, was induced with SGI-1776 treatment in both cell lines (U266, 25%-70%; MM.1S, 8%-52%) and CD138(+) cells (19%-21%). Immunoblot analyses of the autophagy LC3b marker and translation initiation proteins (phospho-p70S6K and 4E-BP1) corroborated autophagy induction. These data indicate that SGI-1776 treatment in myeloma cell lines and CD138(+) myeloma cells elicits its deleterious effects through inhibition of translation and induction of autophagy. Copyright © 2013 Elsevier Inc. All rights reserved.

Cigarette smoke inhibits efferocytosis via deregulation of sphingosine kinase signaling: reversal with exogenous S1P and the S1P analogue FTY720.

PubMed

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.

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis.

PubMed

Camps, Montserrat; Rückle, Thomas; Ji, Hong; Ardissone, Vittoria; Rintelen, Felix; Shaw, Jeffrey; Ferrandi, Chiara; Chabert, Christian; Gillieron, Corine; Françon, Bernard; Martin, Thierry; Gretener, Denise; Perrin, Dominique; Leroy, Didier; Vitte, Pierre-Alain; Hirsch, Emilio; Wymann, Matthias P; Cirillo, Rocco; Schwarz, Matthias K; Rommel, Christian

2005-09-01

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.

Pharmacological and protein profiling suggest venetoclax (ABT-199) as optimal partner with ibrutinib in chronic lymphocytic leukemia

PubMed Central

Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A.; Wierda, William G.; Keating, Michael J.; Balakrishnan, Kumudha; Gandhi, Varsha

2015-01-01

Purpose Bruton’s tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Experimental design Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199) and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. Results The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted highly cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family anti-apoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Conclusions Our biological and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. PMID:25829398

Glycogen Synthase Kinase 3 Inactivation Induces Cell Senescence through Sterol Regulatory Element Binding Protein 1-Mediated Lipogenesis in Chang Cells.

PubMed

Kim, You-Mie; Song, Insun; Seo, Yong-Hak; Yoon, Gyesoon

2013-12-01

Enhanced lipogenesis plays a critical role in cell senescence via induction of expression of the mature form of sterol regulatory element binding protein 1 (SREBP1), which contributes to an increase in organellar mass, one of the indicators of senescence. We investigated the molecular mechanisms by which signaling molecules control SREBP1-mediated lipogenesis and senescence. We developed cellular models for stress-induced senescence, by exposing Chang cells, which are immortalized human liver cells, to subcytotoxic concentrations (200 µM) of deferoxamine (DFO) and H2O2. In this model of stress-induced cell senescence using DFO and H2O2, the phosphorylation profile of glycogen synthase kinase 3α (GSK3α) and β corresponded closely to the expression profile of the mature form of SREBP-1 protein. Inhibition of GSK3 with a subcytotoxic concentration of the selective GSK3 inhibitor SB415286 significantly increased mature SREBP1 expression, as well as lipogenesis and organellar mass. In addition, GSK3 inhibition was sufficient to induce senescence in Chang cells. Suppression of GSK3 expression with siRNAs specific to GSK3α and β also increased mature SREBP1 expression and induced senescence. Finally, blocking lipogenesis with fatty acid synthase inhibitors (cerulenin and C75) and siRNA-mediated silencing of SREBP1 and ATP citrate lyase (ACL) significantly attenuated GSK3 inhibition-induced senescence. GSK3 inactivation is an important upstream event that induces SREBP1-mediated lipogenesis and consequent cell senescence.

Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

PubMed

Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A; Wierda, William G; Keating, Michael J; Balakrishnan, Kumudha; Gandhi, Varsha

2015-08-15

Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. ©2015 American Association for Cancer Research.

Targeting cancer with kinase inhibitors

PubMed Central

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

Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

PubMed

Velagapudi, Sai Pradeep; Luo, Yiling; Tran, Tuan; Haniff, Hafeez S; Nakai, Yoshio; Fallahi, Mohammad; Martinez, Gustavo J; Childs-Disney, Jessica L; Disney, Matthew D

2017-03-22

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

Defining RNA–Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA

PubMed Central

2017-01-01

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif–small molecule interactions identified via selection. Named High Throughput Structure–Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif–small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule–RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs. PMID:28386598

Medium Chain Fatty Acids Are Selective Peroxisome Proliferator Activated Receptor (PPAR) γ Activators and Pan-PPAR Partial Agonists

PubMed Central

Ayers, Steven D.; Lin, Jean Z.; Cvoro, Aleksandra; Silveira, Rodrigo L.; Martínez, Leandro; Souza, Paulo C. T.; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A.; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A. R.; Skaf, Munir S.; Webb, Paul; Polikarpov, Igor

2012-01-01

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8–C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products. PMID:22649490

Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists.

PubMed

Liberato, Marcelo Vizoná; Nascimento, Alessandro S; Ayers, Steven D; Lin, Jean Z; Cvoro, Aleksandra; Silveira, Rodrigo L; Martínez, Leandro; Souza, Paulo C T; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A R; Skaf, Munir S; Webb, Paul; Polikarpov, Igor

2012-01-01

Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of PPARγ ligand binding domain (LBD) and found that the ligand binding pocket (LBP) is occupied by bacterial medium chain fatty acids (MCFAs). We verified that MCFAs (C8-C10) bind the PPARγ LBD in vitro and showed that they are low-potency partial agonists that display assay-specific actions relative to TZDs; they act as very weak partial agonists in transfections with PPARγ LBD, stronger partial agonists with full length PPARγ and exhibit full blockade of PPARγ phosphorylation by cyclin-dependent kinase 5 (cdk5), linked to reversal of adipose tissue insulin resistance. MCFAs that bind PPARγ also antagonize TZD-dependent adipogenesis in vitro. X-ray structure B-factor analysis and molecular dynamics (MD) simulations suggest that MCFAs weakly stabilize C-terminal activation helix (H) 12 relative to TZDs and this effect is highly dependent on chain length. By contrast, MCFAs preferentially stabilize the H2-H3/β-sheet region and the helix (H) 11-H12 loop relative to TZDs and we propose that MCFA assay-specific actions are linked to their unique binding mode and suggest that it may be possible to identify selective PPARγ modulators with useful clinical profiles among natural products.

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer.

PubMed

Rankin, Andrew; Klempner, Samuel J; Erlich, Rachel; Sun, James X; Grothey, Axel; Fakih, Marwan; George, Thomas J; Lee, Jeeyun; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M; Schrock, Alexa B

2016-09-28

A KRAS mutation represented the first genomic biomarker to predict lack of benefit from anti-epidermal growth factor receptor (EGFR) antibody therapy in advanced colorectal cancer (CRC). Expanded RAS testing has further refined the treatment approach, but understanding of genomic alterations underlying primary and acquired resistance is limited and further study is needed. We prospectively analyzed 4,422 clinical samples from patients with advanced CRC, using hybrid-capture based comprehensive genomic profiling (CGP) at the request of the individual treating physicians. Comparison with prior molecular testing results, when available, was performed to assess concordance. We identified a RAS/RAF pathway mutation or amplification in 62% of cases, including samples harboring KRAS mutations outside of the codon 12/13 hotspot region in 6.4% of cases. Among cases with KRAS non-codon 12/13 alterations for which prior test results were available, 79 of 90 (88%) were not identified by focused testing. Of 1,644 RAS/RAF wild-type cases analyzed by CGP, 31% harbored a genomic alteration (GA) associated with resistance to anti-EGFR therapy in advanced CRC including mutations in PIK3CA, PTEN, EGFR, and ERBB2. We also identified other targetable GA, including novel kinase fusions, receptor tyrosine kinase amplification, activating point mutations, as well as microsatellite instability. Extended genomic profiling reliably detects alterations associated with lack of benefit to anti-EGFR therapy in advanced CRC, while simultaneously identifying alterations potentially important in guiding treatment. The use of CGP during the course of clinical care allows for the refined selection of appropriate targeted therapies and clinical trials, increasing the chance of clinical benefit and avoiding therapeutic futility. Comprehensive genomic profiling (CGP) detects diverse genomic alterations associated with lack of benefit to anti-epidermal growth factor receptor therapy in advanced colorectal cancer (CRC), as well as targetable alterations in many other genes. This includes detection of a broad spectrum of activating KRAS alterations frequently missed by focused molecular hotspot testing, as well as other RAS/RAF pathway alterations, mutations shown to disrupt antibody binding, RTK activating point mutations, amplifications, and rearrangements, and activating alterations in downstream effectors including PI3K and MEK1. The use of CGP in clinical practice is critical to guide appropriate selection of targeted therapies for patients with advanced CRC. ©AlphaMed Press.

Discovery of Selective and Noncovalent Diaminopyrimidine-Based Inhibitors of Epidermal Growth Factor Receptor Containing the T790M Resistance Mutation

PubMed Central

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

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK2 Inhibitor for Treatment of Triple-negative Breast Cancer with a Compromised G1/S Checkpoint.

PubMed

Zhu, Dan; Xu, Shuichan; Deyanat-Yazdi, Gordafaried; Peng, Sophie X; Barnes, Leo A; Narla, Rama Krishna; Tran, Tam; Mikolon, David; Ning, Yuhong; Shi, Tao; Jiang, Ning; Raymon, Heather K; Riggs, Jennifer R; Boylan, John F

2018-06-04

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases-TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1/S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1/S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line-derived and one patient tumor-derived xenograft models of TNBC following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1/S checkpoint in breast cancer cell lines. Based on these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of TNBC patients. Copyright ©2018, American Association for Cancer Research.

Tofacitinib, an Oral Janus Kinase Inhibitor: Perspectives in Dermatology.

PubMed

Kostovic, Kresimir; Gulin, Sandra J; Mokos, Zrinka B; Ceovic, Romana

2017-05-31

Tofacitinib (formerly known as CP-690,550, CP690550, tasocitinib), a novel selective immunosuppressant, is a small molecule classified as Janus kinase inhibitor. The aim of this review article is to present updated data summary on the tofacitinib in the field of dermatology. We undertook a structured search of bibliographic databases for peer-reviewed scientific articles, including review articles, original research articles as well as case report articles based on inclusion/exclusion criteria. Technical reports on tofacitinib from U.S. Food and Drug Administration and European Medical Agency were also included. Forty-three papers were included in this review. We report current data on tofacitinib chemical properties, pharmacology, non-clinical toxicity, as well as efficacy and safety in potential new indications in dermatology: psoriasis, alopecia areata, vitiligo, atopic dermatitis and nail dystrophy associated with alopecia areata. JAK/STAT pathway has an important role in the pathogenesis of psoriasis, alopecia areata, atopic dermatitis, and vitiligo. Despite encouraging efficacy, due to concerns about the overall safety profile of tofacitinib, additional studies will have to determine the adequate risk-to-benefit ratio. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

PIM1 kinase regulates cell death, tumor growth and chemotherapy response in triple-negative breast cancer.

PubMed

Brasó-Maristany, Fara; Filosto, Simone; Catchpole, Steven; Marlow, Rebecca; Quist, Jelmar; Francesch-Domenech, Erika; Plumb, Darren A; Zakka, Leila; Gazinska, Patrycja; Liccardi, Gianmaria; Meier, Pascal; Gris-Oliver, Albert; Cheang, Maggie Chon U; Perdrix-Rosell, Anna; Shafat, Manar; Noël, Elodie; Patel, Nirmesh; McEachern, Kristen; Scaltriti, Maurizio; Castel, Pau; Noor, Farzana; Buus, Richard; Mathew, Sumi; Watkins, Johnathan; Serra, Violeta; Marra, Pierfrancesco; Grigoriadis, Anita; Tutt, Andrew N

2016-11-01

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Here we identified increased copy number and expression of the PIM1 proto-oncogene in genomic data sets of patients with TNBC. TNBC cells, but not nonmalignant mammary epithelial cells, were dependent on PIM1 for proliferation and protection from apoptosis. PIM1 knockdown reduced expression of the anti-apoptotic factor BCL2, and dynamic BH3 profiling of apoptotic priming revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. In TNBC tumors and their cellular models, PIM1 expression was associated with several transcriptional signatures involving the transcription factor MYC, and PIM1 depletion in TNBC cell lines decreased, in a MYC-dependent manner, cell population growth and expression of the MYC target gene MCL1. Treatment with the pan-PIM kinase inhibitor AZD1208 impaired the growth of both cell line and patient-derived xenografts and sensitized them to standard-of-care chemotherapy. This work identifies PIM1 as a malignant-cell-selective target in TNBC and the potential use of PIM1 inhibitors for sensitizing TNBC to chemotherapy-induced apoptotic cell death.

Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders.

PubMed

Furlotti, Guido; Alisi, Maria Alessandra; Cazzolla, Nicola; Dragone, Patrizia; Durando, Lucia; Magarò, Gabriele; Mancini, Francesca; Mangano, Giorgina; Ombrato, Rosella; Vitiello, Marco; Armirotti, Andrea; Capurro, Valeria; Lanfranco, Massimiliano; Ottonello, Giuliana; Summa, Maria; Reggiani, Angelo

2015-11-25

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

Novel 3-Substituted 7-Phenylpyrrolo[3,2-f]quinolin-9(6H)-ones as Single Entities with Multitarget Antiproliferative Activity.

PubMed

Carta, Davide; Bortolozzi, Roberta; Hamel, Ernest; Basso, Giuseppe; Moro, Stefano; Viola, Giampietro; Ferlin, Maria Grazia

2015-10-22

A series of chemically modified 7-phenylpyrrolo[3,2-f]quinolinones was synthesized and evaluated as anticancer agents. Among them, the most cytotoxic (subnanomolar GI50 values) amidic derivative 5f was shown to act as an inhibitor of tubulin polymerization (IC50, 0.99 μM) by binding to the colchicine site with high affinity. Moreover, 5f induced cell cycle arrest in the G2/M phase of the cell cycle in a concentration dependent manner, followed by caspase-dependent apoptotic cell death. Compound 5f also showed lower toxicity in nontumoral cells, suggesting selectivity toward cancer cells. Additional experiments revealed that 5f inhibited the enzymatic activity of multiple kinases, including AURKA, FLT3, GSK3A, MAP3K, MEK, RSK2, RSK4, PLK4, ULK1, and JAK1. Computational studies showed that 5f can be properly accommodated in the colchicine binding site of tubulin as well as in the ATP binding clefts of all examined kinases. Our data indicate that the excellent antiproliferative profile of 5f may be derived from its interactions with multiple cellular targets.

Novel VEGF signalling inhibitors: how helpful are biomarkers in their early development?

PubMed

Wood, Joanna; Scott, Edwina; Thomas, Anne L

2009-11-01

The development of vascular endothelial growth factor (VEGF) inhibitors of tumour angiogenesis can only be described as prolific. It is therefore interesting to speculate which will reach the clinic. Of course, the most effective agents will succeed, but how is effectiveness measured? When presented with a summary of competitive compounds, it can be difficult to discriminate between their potency on target, toxicity and response rates. A comparison was undertaken between new small-molecule tyrosine kinase inhibitors with vascular endothelial growth factor receptor as one of their targets. Factors considered included mode of action (targets), toxicity and usefulness of biomarker data. We carried out a systematic review using PubMed, MEDLINE and American Society of Clinical Oncologist (ASCO) databases for articles (including abstracts) presented in 2007 - 2009. Search terms included 'angiogenesis inhibitors', 'tyrosine kinase inhibitors', 'VEGF' and 'biomarkers'. Nine compounds were selected for detailed comparison. The toxicity profiles of the compounds were similar. Many exposure biomarkers have been identified that have informed the dose and scheduling of these compounds in clinical trials. Progress has also been made in identifying potential efficacy and predictive biomarkers for these new agents; however, these are yet to be validated.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages.

PubMed

Söderholm, Sandra; Kainov, Denis E; Öhman, Tiina; Denisova, Oxana V; Schepens, Bert; Kulesskiy, Evgeny; Imanishi, Susumu Y; Corthals, Garry; Hintsanen, Petteri; Aittokallio, Tero; Saelens, Xavier; Matikainen, Sampsa; Nyman, Tuula A

2016-10-01

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Rapid synthesis of VX-745: p38 MAP kinase inhibition in Werner syndrome cells.

PubMed

Bagley, Mark C; Davis, Terence; Dix, Matthew C; Rokicki, Michal J; Kipling, David

2007-09-15

The p38 mitogen-activated protein kinase inhibitor VX-745 is prepared rapidly and efficiently in a four-step sequence using a combination of conductive heating and microwave-mediated steps. Its inhibitory activity was confirmed in hTERT immortalized HCA2 and WS dermal fibroblasts at 0.5-1.0 microM concentration by ELISA and immunoblot assay, and displays excellent kinase selectivity over the related stress-activated kinase JNK.

Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

PubMed Central

Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

2015-01-01

TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

Molecular structures of cdc2-like kinases in complex with a new inhibitor chemotype

PubMed Central

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

Monocarbonyl Curcumin Analogs: Heterocyclic Pleiotropic Kinase Inhibitors that Mediate Anti-Cancer Properties

PubMed Central

Brown, Andrew; Shi, Qi; Moore, Terry W.; Yoon, Younghyoun; Prussia, Andrew; Maddox, Clinton; Liotta, Dennis C.; Shim*, Hyunsuk; Snyder*, James P.

2014-01-01

Curcumin is a biologically active component of curry powder. A structurally-related class of mimetics possesses similar anti-inflammatory and anticancer properties. Mechanism has been examined by exploring kinase inhibition trends. In a screen of 50 kinases relevant to many forms of cancer, one member of the series (4, EF31) showed ≥85% inhibition for ten of the enzymes at 5 μM, while twenty-two of the proteins were blocked at ≥40%. IC50’s for an expanded set of curcumin analogs established a rank order of potencies, and analyses of IKKβ and AKT2 enzyme kinetics for 4 revealed a mixed inhibition model, ATP competition dominating. Our curcumin mimetics are generally selective for Ser/Thr kinases. Both selectivity and potency trends are compatible with protein sequence comparisons, while modeled kinase binding site geometries deliver a reasonable correlation with mixed inhibition. Overall, these analogs are shown to be pleiotropic inhibitors that operate at multiple points along cell signaling pathways. PMID:23550937

Monocarbonyl curcumin analogues: heterocyclic pleiotropic kinase inhibitors that mediate anticancer properties.

PubMed

Brown, Andrew; Shi, Qi; Moore, Terry W; Yoon, Younghyoun; Prussia, Andrew; Maddox, Clinton; Liotta, Dennis C; Shim, Hyunsuk; Snyder, James P

2013-05-09

Curcumin is a biologically active component of curry powder. A structurally related class of mimetics possesses similar anti-inflammatory and anticancer properties. Mechanism has been examined by exploring kinase inhibition trends. In a screen of 50 kinases relevant to many forms of cancer, one member of the series (4, EF31) showed ≥85% inhibition for 10 of the enzymes at 5 μM, while 22 of the proteins were blocked at ≥40%. IC50 values for an expanded set of curcumin analogues established a rank order of potencies, and analyses of IKKβ and AKT2 enzyme kinetics for 4 revealed a mixed inhibition model, ATP competition dominating. Our curcumin mimetics are generally selective for Ser/Thr kinases. Both selectivity and potency trends are compatible with protein sequence comparisons, while modeled kinase binding site geometries deliver a reasonable correlation with mixed inhibition. Overall, these analogues are shown to be pleiotropic inhibitors that operate at multiple points along cell signaling pathways.

Exploiting an Asp-Glu "switch" in glycogen synthase kinase 3 to design paralog-selective inhibitors for use in acute myeloid leukemia.

PubMed

Wagner, Florence F; Benajiba, Lina; Campbell, Arthur J; Weïwer, Michel; Sacher, Joshua R; Gale, Jennifer P; Ross, Linda; Puissant, Alexandre; Alexe, Gabriela; Conway, Amy; Back, Morgan; Pikman, Yana; Galinsky, Ilene; DeAngelo, Daniel J; Stone, Richard M; Kaya, Taner; Shi, Xi; Robers, Matthew B; Machleidt, Thomas; Wilkinson, Jennifer; Hermine, Olivier; Kung, Andrew; Stein, Adam J; Lakshminarasimhan, Damodharan; Hemann, Michael T; Scolnick, Edward; Zhang, Yan-Ling; Pan, Jen Q; Stegmaier, Kimberly; Holson, Edward B

2018-03-07

Glycogen synthase kinase 3 (GSK3), a key regulatory kinase in the wingless-type MMTV integration site family (WNT) pathway, is a therapeutic target of interest in many diseases. Although dual GSK3α/β inhibitors have entered clinical trials, none has successfully translated to clinical application. Mechanism-based toxicities, driven in part by the inhibition of both GSK3 paralogs and subsequent β-catenin stabilization, are a concern in the translation of this target class because mutations and overexpression of β-catenin are associated with many cancers. Knockdown of GSK3α or GSK3β individually does not increase β-catenin and offers a conceptual resolution to targeting GSK3: paralog-selective inhibition. However, inadequate chemical tools exist. The design of selective adenosine triphosphate (ATP)-competitive inhibitors poses a drug discovery challenge due to the high homology (95% identity and 100% similarity) in this binding domain. Taking advantage of an Asp 133 →Glu 196 "switch" in their kinase hinge, we present a rational design strategy toward the discovery of paralog-selective GSK3 inhibitors. These GSK3α- and GSK3β-selective inhibitors provide insights into GSK3 targeting in acute myeloid leukemia (AML), where GSK3α was identified as a therapeutic target using genetic approaches. The GSK3α-selective compound BRD0705 inhibits kinase function and does not stabilize β-catenin, mitigating potential neoplastic concerns. BRD0705 induces myeloid differentiation and impairs colony formation in AML cells, with no apparent effect on normal hematopoietic cells. Moreover, BRD0705 impairs leukemia initiation and prolongs survival in AML mouse models. These studies demonstrate feasibility of paralog-selective GSK3α inhibition, offering a promising therapeutic approach in AML. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39.

PubMed

Kong, Dexin; Dan, Shingo; Yamazaki, Kanami; Yamori, Takao

2010-04-01

As accumulating evidences suggest close involvement of phosphatidylinositol 3-kinase (PI3K) in various diseases particularly cancer, considerable competition occurs in development of PI3K inhibitors. Consequently, novel PI3K inhibitors such as ZSTK474, GDC-0941 and NVP-BEZ235 have been developed. Even though all these inhibitors were reported to inhibit class I PI3K but not dozens of protein kinases, whether they have different molecular targets remained unknown. To investigate such molecular target specificity, we have determined the inhibitory effects of these novel inhibitors together with classical PI3K inhibitor LY294002 on PI3K superfamily (including classes I, II, and III PI3Ks, PI4K and PI3K-related kinases) by using several novel non-radioactive biochemical assays. As a result, ZSTK474 and GDC-0941 indicated highly similar inhibition profiles for PI3K superfamily, with class I PI3K specificity much higher than NVP-BEZ235 and LY294002. We further investigated their growth inhibition effects on JFCR39, a human cancer cell line panel which we established for molecular target identification, and analysed their cell growth inhibition profiles (fingerprints) by using COMPARE analysis programme. Interestingly, we found ZSTK474 exhibited a highly similar fingerprint with GDC-0941 (r=0.863), more similar than with that of either NVP-BEZ235 or LY294002, suggesting that ZSTK474 shares more in molecular targets with GDC-0941 than with either of the other two PI3K inhibitors, consistent with the biochemical assay result. The biological implication of the difference in molecular target specificity of these PI3K inhibitors is under investigation. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Serum Amino Acids Profile and the Beneficial Effects of L-Arginine or L-Glutamine Supplementation in Dextran Sulfate Sodium Colitis

PubMed Central

Wu, Miaomiao; Liu, Gang; Yang, Guan; Xion, Yan; Su, Dingding; Wu, Li; Li, Tiejun; Chen, Shuai; Duan, Jielin; Yin, Yulong; Wu, Guoyao

2014-01-01

This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS)-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment). Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5%) or glutamine (0.5%, 1.0% and 2.0%) supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px)], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1β), IL-6, IL-17 and tumor necrosis factor alpha (TNF-α)] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt), and myosin light chain kinase (MLCK)- myosin light chain (MLC20), were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (P<0.05). Dietary arginine or glutamine supplementation had significant (P<0.05) influence on the clinical and biochemical parameters (T-SOD, IL-17 and TNF-α) in colitis model. These results were associated with colonic NF-κB, PI3K-Akt and MLCK signaling pathways. In conclusion, arginine or glutamine could be a potential therapy for intestinal inflammatory diseases. PMID:24505477

Serum amino acids profile and the beneficial effects of L-arginine or L-glutamine supplementation in dextran sulfate sodium colitis.

PubMed

Ren, Wenkai; Yin, Jie; Wu, Miaomiao; Liu, Gang; Yang, Guan; Xion, Yan; Su, Dingding; Wu, Li; Li, Tiejun; Chen, Shuai; Duan, Jielin; Yin, Yulong; Wu, Guoyao

2014-01-01

This study was conducted to investigate serum amino acids profile in dextran sulfate sodium (DSS)-induced colitis, and impacts of graded dose of arginine or glutamine supplementation on the colitis. Using DSS-induced colitis model, which is similar to human ulcerative colitis, we determined serum profile of amino acids at day 3, 7, 10 and 12 (5 days post DSS treatment). Meanwhile, effects of graded dose of arginine (0.4%, 0.8%, and 1.5%) or glutamine (0.5%, 1.0% and 2.0%) supplementation on clinical parameters, serum amino acids, colonic tight junction proteins, colonic anti-oxidative indicators [catalase, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px)], colonic pro-inflammatory cytokines [interleukin-1 beta (IL-1β), IL-6, IL-17 and tumor necrosis factor alpha (TNF-α)] in DSS-induced colitis were fully analyzed at day 7 and 12. Additionally, the activation of signal transduction pathways, including nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt), and myosin light chain kinase (MLCK)-myosin light chain (MLC20), were analyzed using immunoblotting. Serum amino acids analysis showed that DSS treatment changed the serum contents of amino acids, such as Trp, Glu, and Gln (P<0.05). Dietary arginine or glutamine supplementation had significant (P<0.05) influence on the clinical and biochemical parameters (T-SOD, IL-17 and TNF-α) in colitis model. These results were associated with colonic NF-κB, PI3K-Akt and MLCK signaling pathways. In conclusion, arginine or glutamine could be a potential therapy for intestinal inflammatory diseases.

Mitogen- and Stress-Activated Protein Kinase 1 Regulates Status Epilepticus-Evoked Cell Death in the Hippocampus

PubMed Central

Choi, Yun-Sik; Horning, Paul; Aten, Sydney; Karelina, Kate; Alzate-Correa, Diego; Arthur, J. Simon C.; Hoyt, Kari R.; Obrietan, Karl

2017-01-01

Mitogen-activated protein kinase (MAPK) signaling has been implicated in a wide range of neuronal processes, including development, plasticity, and viability. One of the principal downstream targets of both the extracellular signal-regulated kinase/MAPK pathway and the p38 MAPK pathway is Mitogen- and Stress-activated protein Kinase 1 (MSK1). Here, we sought to understand the role that MSK1 plays in neuroprotection against excitotoxic stimulation in the hippocampus. To this end, we utilized immunohistochemical labeling, a MSK1 null mouse line, cell viability assays, and array-based profiling approaches. Initially, we show that MSK1 is broadly expressed within the major neuronal cell layers of the hippocampus and that status epilepticus drives acute induction of MSK1 activation. In response to the status epilepticus paradigm, MSK1 KO mice exhibited a striking increase in vulnerability to pilocarpine-evoked cell death within the CA1 and CA3 cell layers. Further, cultured MSK1 null neurons exhibited a heighted level of N-methyl-D-aspartate-evoked excitotoxicity relative to wild-type neurons, as assessed using the lactate dehydrogenase assay. Given these findings, we examined the hippocampal transcriptional profile of MSK1 null mice. Affymetrix array profiling revealed that MSK1 deletion led to the significant (>1.25-fold) downregulation of 130 genes and an upregulation of 145 genes. Notably, functional analysis indicated that a subset of these genes contribute to neuroprotective signaling networks. Together, these data provide important new insights into the mechanism by which the MAPK/MSK1 signaling cassette confers neuroprotection against excitotoxic insults. Approaches designed to upregulate or mimic the functional effects of MSK1 may prove beneficial against an array of degenerative processes resulting from excitotoxic insults. PMID:28870089

Combined metabolic and transcriptional profiling identifies pentose phosphate pathway activation by HSP27 phosphorylation during cerebral ischemia.

PubMed

Imahori, Taichiro; Hosoda, Kohkichi; Nakai, Tomoaki; Yamamoto, Yusuke; Irino, Yasuhiro; Shinohara, Masakazu; Sato, Naoko; Sasayama, Takashi; Tanaka, Kazuhiro; Nagashima, Hiroaki; Kohta, Masaaki; Kohmura, Eiji

2017-05-04

The metabolic pathophysiology underlying ischemic stroke remains poorly understood. To gain insight into these mechanisms, we performed a comparative metabolic and transcriptional analysis of the effects of cerebral ischemia on the metabolism of the cerebral cortex using middle cerebral artery occlusion (MCAO) rat model. Metabolic profiling by gas-chromatography/mass-spectrometry analysis showed clear separation between the ischemia and control group. The decreases of fructose 6-phosphate and ribulose 5-phosphate suggested enhancement of the pentose phosphate pathway (PPP) during cerebral ischemia (120-min MCAO) without reperfusion. Transcriptional profiling by microarray hybridization indicated that the Toll-like receptor and mitogen-activated protein kinase (MAPK) signaling pathways were upregulated during cerebral ischemia without reperfusion. In relation to the PPP, upregulation of heat shock protein 27 (HSP27) was observed in the MAPK signaling pathway and was confirmed through real-time polymerase chain reaction. Immunoblotting showed a slight increase in HSP27 protein expression and a marked increase in HSP27 phosphorylation at serine 85 after 60-min and 120-min MCAO without reperfusion. Corresponding upregulation of glucose 6-phosphate dehydrogenase (G6PD) activity and an increase in the NADPH/NAD + ratio were also observed after 120-min MCAO. Furthermore, intracerebroventricular injection of ataxia telangiectasia mutated (ATM) kinase inhibitor (KU-55933) significantly reduced HSP27 phosphorylation and G6PD upregulation after MCAO, but that of protein kinase D inhibitor (CID755673) did not affect HSP27 phosphorylation. Consequently, G6PD activation via ischemia-induced HSP27 phosphorylation by ATM kinase may be part of an endogenous antioxidant defense neuroprotection mechanism during the earliest stages of ischemia. These findings have important therapeutic implications for the treatment of stroke. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Assessment of tris (1, 3-dichloro-2-propyl) phosphate toxicology in PC12 cells by using digital gene expression profiling.

PubMed

Li, Li; Jiang, Shuai; Li, Kang; Lin, Bencheng; Wang, Ziyu; Zhang, Zhiqing; Fang, Yanjun

2017-09-01

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP), one of the most universally used organophosphate flame retardants (OPFRs), is an environmental pollutant. However, limited information is available regarding its toxicity and environmental health risk. In the present study, PC12 cells provided a useful model for the evaluation of the toxic effects of TDCIPP. Exposure to 7.5, 15, 30, or 60 μM TDCIPP for 72 h inhibited cell viability, and enhanced cellular apoptosis and oxidative stress. To further explore the underlying mechanisms, digital gene expression (DGE) technology was used to identify early transcriptional changes following TDCIPP exposure. Expression of the transcripts of 161 genes was significantly altered upon treatment with TDCIPP. Functional and pathway analysis of the transcriptional profile demonstrated that genes showing significant TDCIPP-associated changes in expression were involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, extracellular matrix-receptor interactions, protein digestion and absorption, and microRNAs in cancer. Using quantitative real-time PCR, we validated the differential expression of selected genes. These results showed that the expression profiles of cells exposed to 60 μM TDCIPP were consistent with the DGE data. Furthermore, western blotting showed that treatment with TDCIPP reduced the Bcl-2/Bax ratio and attenuated PI3K/Akt/Myc signaling. Taken together, these data suggest that TDCIPP exposure can reduce cell viability and induce apoptosis in PC12 cells by inhibiting activation of the PI3K/Akt/Myc signaling pathway. These observations provide valuable preliminary information regarding the mechanisms of TDCIPP-induced toxicity in PC12 cells and indicate that further study of the toxicity of other environmental OPFRs is warranted. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative transcriptome profiling of resistant and susceptible rice genotypes in response to the seedborne pathogen Fusarium fujikuroi.

PubMed

Matić, Slavica; Bagnaresi, Paolo; Biselli, Chiara; Orru', Luigi; Amaral Carneiro, Greice; Siciliano, Ilenia; Valé, Giampiero; Gullino, Maria Lodovica; Spadaro, Davide

2016-08-11

Fusarium fujikuroi is the causal agent of bakanae, the most significant seed-borne disease of rice. Molecular mechanisms regulating defence responses of rice towards this fungus are not yet fully known. To identify transcriptional mechanisms underpinning rice resistance, a RNA-seq comparative transcriptome profiling was conducted on infected seedlings of selected rice genotypes at one and three weeks post germination (wpg). Twelve rice genotypes were screened against bakanae disease leading to the identification of Selenio and Dorella as the most resistant and susceptible cultivars, respectively. Transcriptional changes were more appreciable at 3 wpg, suggesting that this infection stage is essential to study the resistance mechanisms: 3,119 DEGs were found in Selenio and 5,095 in Dorella. PR1, germin-like proteins, glycoside hydrolases, MAP kinases, and WRKY transcriptional factors were up-regulated in the resistant genotype upon infection with F. fujikuroi. Up-regulation of chitinases and down-regulation of MAP kinases and WRKY transcriptional factors were observed in the susceptible genotype. Gene ontology (GO) enrichment analyses detected in Selenio GO terms specific to response to F. fujikuroi: 'response to chitin', 'jasmonic acid biosynthetic process', and 'plant-type hypersensitive response', while Dorella activated different mechanisms, such as 'response to salicylic acid stimulus' and 'gibberellin metabolic process', which was in agreement with the production of gibberellin A3 in Dorella plants. RNA-seq profiling was performed for the first time to analyse response of rice to F. fujikuroi infection. Our findings allowed the identification of genes activated in one- and three- week-old rice seedlings of two genotypes infected with F. fujikuroi. Furthermore, we found the pathways involved in bakanae resistance, such as response to chitin, JA-dependent signalling and hypersensitive response. Collectively, this provides important information to elucidate the molecular and cellular processes occurring in rice during F. fujikuroi infection and to develop bakanae resistant rice germplasm.

Kinase Pathway Dependence in Primary Human Leukemias Determined by Rapid Inhibitor Screening

PubMed Central

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

Cumulus cells surrounding oocytes with high developmental competence exhibit down-regulation of phosphoinositol 1,3 kinase/protein kinase B (PI3K/AKT) signalling genes involved in proliferation and survival

PubMed Central

Artini, P G; Tatone, C; Sperduti, S; D’Aurora, M; Franchi, S; Di Emidio, G; Ciriminna, R; Vento, M; Di Pietro, C; Stuppia, L; Gatta, V

2017-01-01

Abstract STUDY QUESTION Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome? SUMMARY ANSWER Eleven genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome. WHAT IS KNOWN ALREADY The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs. STUDY DESIGN SIZE, DURATION The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile. PARTICIPANTS/MATERIALS, SETTING, METHODS Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERβ, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples. MAIN RESULTS AND THE ROLE OF CHANCE Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway. LIMITATIONS REASONS FOR CAUTION Small sample size and retrospective design. WIDER IMPLICATIONS OF THE FINDINGS The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals. STUDY FUNDING/COMPETING INTEREST(S) Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest. PMID:29087515

Structural characterization of nonactive site, TrkA-selective kinase inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Hua-Poo; Rickert, Keith; Burlein, Christine

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residuesmore » from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of—but adjacent to—the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.« less

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies.

PubMed

Patel, Dhilon S; Bharatam, Prasad V

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies

NASA Astrophysics Data System (ADS)

Patel, Dhilon S.; Bharatam, Prasad V.

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256.

PubMed

Bradford, Davis; Raghuram, Viswanathan; Wilson, Justin L L; Chou, Chung-Lin; Hoffert, Jason D; Knepper, Mark A; Pisitkun, Trairak

2014-07-15

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroupmore » (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.« less

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

PubMed

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.

Anaplastic lymphoma kinase inhibition in metastatic non-small cell lung cancer: clinical impact of alectinib

PubMed Central

Muller, Ittai B; de Langen, Adrianus J; Giovannetti, Elisa; Peters, Godefridus J

2017-01-01

A subset of non-small cell lung cancer (NSCLC) tumors (5%) harbors an anaplastic lymphoma kinase (ALK) translocation that drives tumorigenesis. The clinically approved first-line treatment crizotinib specifically inhibits ALK and improves progression-free survival (PFS) in treated and untreated patients by 4 months compared to standard chemotherapy. While some patients relapse after crizotinib treatment due to resistance mutations in ALK, second-generation ALK inhibitors effectively induce tumor response and prolong PFS. Alectinib, a second-generation ALK inhibitor, has recently been approved for ALK-rearranged NSCLC after patients progressed on crizotinib. Alectinib is able to inhibit several crizotinib- and ceritinib-resistant ALK mutations in vitro. Furthermore, alectinib is a more potent tyrosine kinase inhibitor (TKI), with favorable safety profile, and has increased penetration into the central nervous system, inhibiting crizotinib-resistant brain metastases. The discovery of effective personalized therapies to combat ALK-rearranged NSCLC such as alectinib is an example of the importance of genomic profiling of NSCLC and provides an excellent template for future discoveries in managing these tumors. PMID:28979145

Anaplastic lymphoma kinase inhibition in metastatic non-small cell lung cancer: clinical impact of alectinib.

PubMed

Muller, Ittai B; de Langen, Adrianus J; Giovannetti, Elisa; Peters, Godefridus J

2017-01-01

A subset of non-small cell lung cancer (NSCLC) tumors (5%) harbors an anaplastic lymphoma kinase (ALK) translocation that drives tumorigenesis. The clinically approved first-line treatment crizotinib specifically inhibits ALK and improves progression-free survival (PFS) in treated and untreated patients by 4 months compared to standard chemotherapy. While some patients relapse after crizotinib treatment due to resistance mutations in ALK, second-generation ALK inhibitors effectively induce tumor response and prolong PFS. Alectinib, a second-generation ALK inhibitor, has recently been approved for ALK-rearranged NSCLC after patients progressed on crizotinib. Alectinib is able to inhibit several crizotinib- and ceritinib-resistant ALK mutations in vitro. Furthermore, alectinib is a more potent tyrosine kinase inhibitor (TKI), with favorable safety profile, and has increased penetration into the central nervous system, inhibiting crizotinib-resistant brain metastases. The discovery of effective personalized therapies to combat ALK-rearranged NSCLC such as alectinib is an example of the importance of genomic profiling of NSCLC and provides an excellent template for future discoveries in managing these tumors.

The 'retro-design' concept for novel kinase inhibitors.

PubMed

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.

Computational study of Gleevec and G6G reveals molecular determinants of kinase inhibitor selectivity

DOE PAGES

Lin, Yen -Lin; Meng, Yilin; Huang, Lei; ...

2014-10-22

Gleevec is a potent inhibitor of Abl tyrosine kinase but not of the highly homologous c-Src kinase. Because the ligand binds to an inactive form of the protein in which an Asp-Phe-Gly structural motif along the activation loop adopts a so-called DFG-out conformation, it was suggested that binding specificity was controlled by a “conformational selection” mechanism. In this context, the binding affinity displayed by the kinase inhibitor G6G poses an intriguing challenge. Although it possesses a chemical core very similar to that of Gleevec, G6G is a potent inhibitor of both Abl and c-Src kinases. Both inhibitors bind to themore » DFG-out conformation of the kinases, which seems to be in contradiction with the conformational selection mechanism. To address this issue and display the hidden thermodynamic contributions affecting the binding selectivity, molecular dynamics free energy simulations with explicit solvent molecules were carried out. Relative to Gleevec, G6G forms highly favorable van der Waals dispersive interactions upon binding to the kinases via its triazine functional group, which is considerably larger than the corresponding pyridine moiety in Gleevec. Upon binding of G6G to c-Src, these interactions offset the unfavorable free energy cost of the DFG-out conformation. When binding to Abl, however, G6G experiences an unfavorable free energy penalty due to steric clashes with the phosphate-binding loop, yielding an overall binding affinity that is similar to that of Gleevec. Such steric clashes are absent when G6G binds to c-Src, due to the extended conformation of the phosphate-binding loop.« less

Structure–Activity Relationship in a Purine-Scaffold Compound Series with Selectivity for the Endoplasmic Reticulum Hsp90 Paralog Grp94

PubMed Central

Patel, Hardik J.; Patel, Pallav D.; Ochiana, Stefan O.; Yan, Pengrong; Sun, Weilin; Patel, Maulik R.; Shah, Smit K.; Tramentozzi, Elisa; Brooks, James; Bolaender, Alexander; Shrestha, Liza; Stephani, Ralph; Finotti, Paola; Leifer, Cynthia; Li, Zihai; Gewirth, Daniel T.; Taldone, Tony; Chiosis, Gabriela

2015-01-01

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 μM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/β and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor. PMID:25901531

Quantitative Mass Spectrometry by Isotope Dilution and Multiple Reaction Monitoring (MRM).

PubMed

Russo, Paul; Hood, Brian L; Bateman, Nicholas W; Conrads, Thomas P

2017-01-01

Selected reaction monitoring (SRM) is used in molecular profiling to detect and quantify specific known proteins in complex mixtures. Using isotope dilution (Barnidge et al., Anal Chem 75(3):445-451, 2003) methodologies, peptides can be quantified without the need for an antibody-based method. Selected reaction monitoring assays employ electrospray ionization mass spectrometry (ESI-MS) followed by two stages of mass selection: a first stage where the mass of the peptide ion is selected and, after fragmentation by collision-induced dissociation (CID), a second stage (tandem MS) where either a single (e.g., SRM) or multiple (multiple reaction monitoring, MRM) specific peptide fragment ions are transmitted for detection. The MRM experiment is accomplished by specifying the parent masses of the selected endogenous and isotope-labeled peptides for MS/MS fragmentation and then monitoring fragment ions of interest, using their intensities/abundances and relative ratios to quantify the parent protein of interest. In this example protocol, we will utilize isotope dilution MRM-MS to quantify in absolute terms the total levels of the protein of interest, ataxia telangiectasia mutated (ATM) serine/threonine protein kinase. Ataxia telangiectasia mutated (ATM) phosphorylates several key proteins that initiate activation of the DNA damage checkpoint leading to cell cycle arrest.

Structure-Functional Prediction and Analysis of Cancer Mutation Effects in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2014-01-01

A central goal of cancer research is to discover and characterize the functional effects of mutated genes that contribute to tumorigenesis. In this study, we provide a detailed structural classification and analysis of functional dynamics for members of protein kinase families that are known to harbor cancer mutations. We also present a systematic computational analysis that combines sequence and structure-based prediction models to characterize the effect of cancer mutations in protein kinases. We focus on the differential effects of activating point mutations that increase protein kinase activity and kinase-inactivating mutations that decrease activity. Mapping of cancer mutations onto the conformational mobility profiles of known crystal structures demonstrated that activating mutations could reduce a steric barrier for the movement from the basal “low” activity state to the “active” state. According to our analysis, the mechanism of activating mutations reflects a combined effect of partial destabilization of the kinase in its inactive state and a concomitant stabilization of its active-like form, which is likely to drive tumorigenesis at some level. Ultimately, the analysis of the evolutionary and structural features of the major cancer-causing mutational hotspot in kinases can also aid in the correlation of kinase mutation effects with clinical outcomes. PMID:24817905

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

[Management of the cardiovascular disease risk during nilotinib treatment in chronic myeloid leukemia: 2015 recommendations from the France Intergroupe des Leucémies Myéloïdes Chroniques].

PubMed

Rea, Delphine; Ame, Shanti; Charbonnier, Aude; Coiteux, Valérie; Cony-Makhoul, Pascale; Escoffre-Barbe, Martine; Etienne, Gabriel; Gardembas, Martine; Guerci-Bresler, Agnès; Legros, Laurence; Nicolini, Franck; Tulliez, Michel; Hermet, Eric; Huguet, Françoise; Johnson-Ansah, Hyacinthe; Lapusan, Simona; Quittet, Philippe; Rousselot, Philippe; Mahon, François-Xavier; Messas, Emmanuel

2016-02-01

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein represent an outstanding progress in chronic myeloid leukemia and long-term progression-free survival has become a reality for a majority of patients. However, tyrosine kinase inhibitors may at best chronicize rather than cure the disease thus current recommendation is to pursue treatment indefinitely. As a consequence, high quality treatment and care must integrate optimal disease control and treatment tolerability. Tyrosine kinase inhibitors have an overall favorable safety profile in clinical practice since most adverse events are mild to moderate in intensity. However, recent evidence has emerged that new generation tyrosine kinase inhibitors may sometimes damage vital organs and if not adequately managed, morbidity and mortality may increase. The 2nd generation tyrosine kinase inhibitor nilotinib is licensed for the treatment of chronic myeloid leukemia with resistance or intolerance to imatinib and newly diagnosed chronic phase-chronic myeloid leukemia. Nilotinib represents an important therapeutic option but it is associated with an increased risk of cardiovascular events. The purpose of this article by the France Intergroupe des Leucémies Myéloïdes Chroniques is to provide an overview of nilotinib efficacy and cardiovascular safety profile and to propose practical recommendations with the goal to minimize the risk and severity of cardiovascular events in nilotinib-treated patients. Copyright © 2015 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Recent Progress on Liver Kinase B1 (LKB1): Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

PubMed Central

Gan, Ren-You; Li, Hua-Bin

2014-01-01

Liver kinase B1 (LKB1), known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK)-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK), salt-inducible kinase (SIK), sucrose non-fermenting protein-related kinase (SNRK) and brain selective kinase (BRSK) signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers. PMID:25244018

A highly selective, orally active inhibitor of Janus kinase 2, CEP-33779, ablates disease in two mouse models of rheumatoid arthritis

PubMed Central

2011-01-01

Introduction Janus kinase 2 (JAK2) is involved in the downstream activation of signal transducer and activator of transcription 3 (STAT3) and STAT5 and is responsible for transducing signals for several proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), including interleukin (IL)-6, interferon γ (IFNγ) and IL-12. In this paper, we describe the efficacy profile of CEP-33779, a highly selective, orally active, small-molecule inhibitor of JAK2 evaluated in two mouse models of RA. Methods Collagen antibody-induced arthritis (CAIA) and collagen type II (CII)-induced arthritis (CIA) were established before the oral administration of a small-molecule JAK2 inhibitor, CEP-33779, twice daily at 10 mg/kg, 30 mg/kg, 55 mg/kg or 100 mg/kg over a period of 4 to 8 weeks. Results Pharmacodynamic inhibition of JAK2 reduced mean paw edema and clinical scores in both CIA and CAIA models of arthritis. Reduction in paw cytokines (IL-12, IFNγ and tumor necrosis factor α) and serum cytokines (IL-12 and IL-2) correlated with reduced spleen CII-specific T helper 1 cell frequencies as measured by ex vivo IFNγ enzyme-linked immunosorbent spot assay. Both models demonstrated histological evidence of disease amelioration upon treatment (for example, reduced matrix erosion, subchondral osteolysis, pannus formation and synovial inflammation) and reduced paw phosphorylated STAT3 levels. No changes in body weight or serum anti-CII autoantibody titers were observed in either RA model. Conclusions This study demonstrates the utility of using a potent and highly selective, orally bioavailable JAK2 inhibitor for the treatment of RA. Using a selective inhibitor of JAK2 rather than pan-JAK inhibitors avoids the potential complication of immunosuppression while targeting critical signaling pathways involved in autoimmune disease progression. PMID:21510883

Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

PubMed

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.

Design and Discovery of N -(2-Methyl-5'-morpholino-6'-((tetrahydro-2 H -pyran-4-yl)oxy)-[3,3'-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

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

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

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

X-Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muckelbauer, Jodi; Sack, John S.; Ahmed, Nazia

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 {angstrom} resolution and PP2 at 1.9 {angstrom} resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-orderedmore » protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.« less

Group II p21-activated kinases as therapeutic targets in gastrointestinal cancer.

PubMed

Shao, Yang-Guang; Ning, Ke; Li, Feng

2016-01-21

P21-activated kinases (PAKs) are central players in various oncogenic signaling pathways. The six PAK family members are classified into group I (PAK1-3) and group II (PAK4-6). Focus is currently shifting from group I PAKs to group II PAKs. Group II PAKs play important roles in many fundamental cellular processes, some of which have particular significance in the development and progression of cancer. Because of their important functions, group II PAKs have become popular potential drug target candidates. However, few group II PAKs inhibitors have been reported, and most do not exhibit satisfactory kinase selectivity and "drug-like" properties. Isoform- and kinase-selective PAK inhibitors remain to be developed. This review describes the biological activities of group II PAKs, the importance of group II PAKs in the development and progression of gastrointestinal cancer, and small-molecule inhibitors of group II PAKs for the treatment of cancer.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs).

PubMed

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-12-19

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC 50 : 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC 50 : 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan ™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs)

PubMed Central

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-01-01

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC50: 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC50: 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs. PMID:29340041

Discovery of Selective and Noncovalent Diaminopyrimidine-Based Inhibitors of Epidermal Growth Factor Receptor Containing the T790M Resistance Mutation

DOE PAGES

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

Safety, Efficacy, and Patient Acceptability of Everolimus in the Treatment of Breast Cancer.

PubMed

Lousberg, Laurence; Jerusalem, Guy

2016-01-01

Everolimus combined with exemestane is an important treatment option for patients suffering from estrogen receptor-positive, human epidermal growth factor receptor 2-negative, advanced breast cancer (ABC) who have been previously treated with a nonsteroidal aromatase inhibitor (NSAI). After presentation of phase III registration trial BOLERO-2, several phase IIIb trials have been started to evaluate this regimen in a more real-world setting. Here, we review the efficacy and safety data published or presented at selected international meetings. These studies confirmed the outcome observed in the BOLERO-2 trial. Patient acceptance rate is also discussed by focusing on the permanent everolimus discontinuation rate in these trials. Factors influencing the safety profile are also reported, including the impact of age. The optimal sequence of combined therapy approaches associating targeted and endocrine therapy (ET) has yet to be determined as new treatment options such as cyclin-dependent kinase inhibitors become available. However, everolimus-exemestane remains an important treatment option with a major impact on progression-free survival (PFS) and an acceptable safety profile.

Safety, Efficacy, and Patient Acceptability of Everolimus in the Treatment of Breast Cancer

PubMed Central

Lousberg, Laurence; Jerusalem, Guy

2016-01-01

Everolimus combined with exemestane is an important treatment option for patients suffering from estrogen receptor-positive, human epidermal growth factor receptor 2-negative, advanced breast cancer (ABC) who have been previously treated with a nonsteroidal aromatase inhibitor (NSAI). After presentation of phase III registration trial BOLERO-2, several phase IIIb trials have been started to evaluate this regimen in a more real-world setting. Here, we review the efficacy and safety data published or presented at selected international meetings. These studies confirmed the outcome observed in the BOLERO-2 trial. Patient acceptance rate is also discussed by focusing on the permanent everolimus discontinuation rate in these trials. Factors influencing the safety profile are also reported, including the impact of age. The optimal sequence of combined therapy approaches associating targeted and endocrine therapy (ET) has yet to be determined as new treatment options such as cyclin-dependent kinase inhibitors become available. However, everolimus–exemestane remains an important treatment option with a major impact on progression-free survival (PFS) and an acceptable safety profile. PMID:28096680

Signal transduction networks in rheumatoid arthritis

PubMed Central

Hammaker, D; Sweeney, S; Firestein, G

2003-01-01

Signal transduction pathways regulate cellular responses to stress and play a critical role in inflammation. The complexity and specificity of signalling mechanisms represent major hurdles for developing effective, safe therapeutic interventions that target specific molecules. One approach is to dissect the pathways methodically to determine their hierarchy in various cell types and diseases. This approach contributed to the identification and prioritisation of specific kinases that regulate NF-κB and the mitogen activated protein (MAP) kinase cascade as especially attractive targets. Although significant issues remain with regard to the discovery of truly selective kinase inhibitors, the risks that accompany inhibition of fundamental signal transduction mechanisms can potentially be decreased by careful dissection of the pathways and rational target selection. PMID:14532158

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

PubMed Central

Boyd, Penelope J.; Shorrock, Hannah K.; Carter, Roderick N.; Powis, Rachael A.; Thomson, Sophie R.; Thomson, Derek; Graham, Laura C.; Motyl, Anna A. L.; Highley, J. Robin; Becker, Thomas; Becker, Catherina G.; Heath, Paul R.

2017-01-01

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo. PMID:28426667

Systematic review and meta-analysis of selected toxicities of approved ALK inhibitors in metastatic non-small cell lung cancer

PubMed Central

Costa, Rubens Barros; Costa, Ricardo L.B.; Talamantes, Sarah M.; Kaplan, Jason B.; Bhave, Manali A.; Rademaker, Alfred; Miller, Corinne; Carneiro, Benedito A.; Mahalingam, Devalingam; Chae, Young Kwang

2018-01-01

Introduction Anaplastic lymphoma kinase (ALK) inhibitors are the mainstay treatment for patients with non-small cell lung carcinoma (NSCLC) harboring a rearrangement of the ALK gene or the ROS1 oncogenes. With the recent publication of pivotal trials leading to the approval of these compounds in different indications, their toxicity profile warrants an update. Materials and Methods A systematic literature search was performed in July 2017. Studies evaluating US FDA approved doses of one of the following ALK inhibitors: Crizotinib, Ceritinib, Alectinib or Brigatinib as monotherapy were included. Data were analyzed using random effects meta-analysis for absolute risks (AR), study heterogeneity, publication bias and differences among treatments. Results Fifteen trials with a total of 2,005 patients with evaluable toxicity data were included in this report. There was significant heterogeneity amongst different studies. The pooled AR of death and severe adverse events were 0.5% and 34.5%, respectively. Grade 3/4 nausea, vomiting, diarrhea, and constipation were uncommon: 2.6%, 2.5%, 2.7%, 1.2%, respectively. Conclusions ALK inhibitors have an acceptable safety profile with a low risk of treatment-related deaths. Important differences in toxicity profile were detected amongst the different drugs. PMID:29774128

Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

PubMed

Zoraghi, Roya; Campbell, Sara; Kim, Catrina; Dullaghan, Edie M; Blair, Lachlan M; Gillard, Rachel M; Reiner, Neil E; Sperry, Jonathan

2014-11-01

Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

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

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

PubMed

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

2017-05-05

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Theoretical studies on the selective mechanisms of GSK3β and CDK2 by molecular dynamics simulations and free energy calculations.

PubMed

Zhao, Sufang; Zhu, Jingyu; Xu, Lei; Jin, Jian

2017-06-01

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase which is widely involved in cell signaling and controls a broad number of cellular functions. GSK3 contains α and β isoforms, and GSK3β has received more attention and becomes an attractive drug target for the treatment of several diseases. The binding pocket of cyclin-dependent kinase 2 (CDK2) shares high sequence identity to that of GSK3β, and therefore, the design of highly selective inhibitors toward GSK3β remains a big challenge. In this study, a computational strategy, which combines molecular docking, molecular dynamics simulations, free energy calculations, and umbrella sampling simulations, was employed to explore the binding mechanisms of two selective inhibitors to GSK3β and CDK2. The simulation results highlighted the key residues critical for GSK3β selectivity. It was observed that although GSK3β and CDK2 share the conserved ATP-binding pockets, some different residues have significant contributions to protein selectivity. This study provides valuable information for understanding the GSK3β-selective binding mechanisms and the rational design of selective GSK3β inhibitors. © 2016 John Wiley & Sons A/S.

Osimertinib - effective treatment of NSCLC with activating EGFR mutations after progression on EGFR tyrosine kinase inhibitors.

PubMed

Skrzypski, Marcin; Szymanowska-Narloch, Amelia; Dziadziuszko, Rafał

2017-01-01

Non-small cell lung cancer (NSCLC) driven by activating mutations in epidermal growth factor receptor (EGFR) constitutes up to 10% of NSCLC cases. According to the NCCN recommendations, all patients (with the exception of smoking patients with squamous cell lung cancer) should be screened for the presence of activating EGFR mutations, i.e. deletion in exon 19 or point mutation L858R in exon 21, in order to select the group that benefits from EGFR tyrosine kinase inhibitors (EGFR TKIs) treatment. Among approved agents there are the 1 st generation reversible EGFR TKIs, erlotinib and gefitinib, and the 2 nd generation irreversible EGFR TKI, afatinib. The objective response rates to these drugs in randomised clinical trials were in the range of 56-74%, and median time to progression 9-13 months. The most common determinant of resistance to these drugs is the clonal expansion of cancer cells with T790M mutation (Thr790Met) in exon 20 of EGFR. Osimertinib (Tagrisso™), a 3 rd generation, irreversible EGFR tyrosine kinase inhibitor, constitutes a novel, highly efficacious treatment for NSCLC patients progressing on EGFR TKIs with T790M mutation confirmed as the resistance mechanism. Resistance mutation can be determined in tissue or liquid biopsy obtained after progression on EGFR TKIs. Osimertinib has a favourable toxicity profile, with mild rash and diarrhoea being the most common. In this article, we present three cases that were successfully treated with osimertinib after progression on 1st and 2nd generation EGFR TKIs.

Effects of angiotensin-converting enzyme inhibitor versus valsartan on cellular signaling events in heart transplant.

PubMed

White, Michel; Ross, Heather; Levesque, Sylvie; Whittom, Lucette; Pelletier, Guy B; Racine, Normand; Meloche, Sylvain; Voisin, Laure

2009-05-01

Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) provide similar biologic effects in model systems and similar clinical impacts in humans. The changes in the cardiac angiotensin system signaling pathways in the human heart in response to ACE inhibitors versus ARBs have been incompletely studied. To investigate the effects of ACE inhibitors versus valsartan on the angiotensin II signal transduction pathways in the transplanted human heart. Twenty-seven stable cardiac transplant recipients were randomized to remain on ACE inhibitor therapy (n = 8) or to receive valsartan (n = 19). Two additional endomyocardial biopsy samples were obtained at baseline and after 9 months of therapy. The expression of cardiac angiotensin type I and II receptors and atrial natriuretic factor (ANF) was measured by quantitative polymerase chain reaction. The expression and phosphorylation levels of selected signal transduction pathways were analyzed by immunoblotting. The mean dose of valsartan was 114 +/- 41 mg/day. The use of valsartan resulted in a similar impact on blood pressure and biochemistry profile. There were no significant changes in the expression of angiotensin type I and II receptors and ANF with valsartan. Similarly, no significant changes in the expression and phosphorylation of Jun N-terminal kinase, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinases or AKT, and mammalian target of rapamycin was observed in the valsartan-treated group. Valsartan use is associated with similar clinical and molecular cardiac effects as ACE inhibitor therapy in stable long-term cardiac transplant recipients.

τ kinases in the rat heat shock model: Possible implications for Alzheimer disease

PubMed Central

Shanavas, Alikunju; Papasozomenos, Sozos Ch.

2000-01-01

We have previously shown, by using the phosphate-dependent anti-τ antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of τ followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague–Dawley rats the activities of extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at 0 (n = 5), 3 (n = 4), 6 (n = 5), and 12 (n = 5) h after heat shock and in non-heat-shocked controls (n = 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3β, which showed increased activity. At 3–6 h, the activities of ERK1/2, JNK, Cdk5, and GSK-3β toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3β but not ERK1/2 were overactivated toward purified bovine τ. At 3–6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either τ or selective substrates. All of eight anti-τ antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3–6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1/2, JNK, and GSK-3β confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because τ is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3β, and Cdk5 may play a role in its pathogenesis. PMID:11121021

Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia.

PubMed

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.

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

PubMed

Chong, Irene Yushing; Aronson, Lauren; Bryant, Hanna; Gulati, Aditi; Campbell, James; Elliott, Richard; Pettitt, Stephen; Wilkerson, Paul; Lambros, Maryou B; Reis-Filho, Jorge S; Ramessur, Anisha; Davidson, Michael; Chau, Ian; Cunningham, David; Ashworth, Alan; Lord, Christopher J

2017-08-22

Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G 1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). NCT02884453; Pre-results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Synergistic interactions of the anti-casein kinase 2 CIGB-300 peptide and chemotherapeutic agents in lung and cervical preclinical cancer models.

PubMed

Perera, Yasser; Toro, Neylen Del; Gorovaya, Larisa; Fernandez-DE-Cossio, Jorge; Farina, Hernan G; Perea, Silvio E

2014-11-01

CIGB-300 is a novel clinical-stage synthetic peptide that impairs the casein kinase 2 (CK2)-mediated phosphorylation of B23/nucleophosmin in different experimental settings and cancer models. As a single agent, CIGB-300 induces apoptosis in vitro and in vivo and modulates an array of proteins that are mainly involved in drug resistance, cell proliferation and apoptosis, as determined by proteomic analysis. However, the clinical oncology practice and cumulative knowledge on tumor biology suggest that drug combinations are more likely to cope with tumor complexity compared to single agents. In this study, we investigated the antiproliferative effect of CIGB-300 when combined with different anticancer drugs, such as cisplatin (alkylating), paclitaxel (antimitotic), doxorubicin (antitopoisomerase II) or 5-fluorouracil (DNA/RNA antimetabolite) in cell lines derived from lung and cervical cancer. Of note, using a Latin square design and subsequent analysis by CalcuSyn software, we observed that paclitaxel and cisplatin exhibited the best synergistic/additive profile when combined with CIGB-300, according to the combination and dose reduction indices. Such therapeutically favorable profiles may be explained by a direct cytotoxic effect and also by the observed cell cycle impairment following incubation of tumor cells with selected drug combinations. Importantly, on in vivo dose-finding schedules in human cervical tumors xenografted in nude mice, we observed that concomitant administration of CIGB-300 and cisplatin increased mice survival compared to single-agent treatment. Collectively, these findings provide a rationale for combining the anti-CK2 CIGB-300 peptide with currently available anticancer agents in the clinical setting and indicate platins and taxanes as compounds with major perspectives.

Synergistic interactions of the anti-casein kinase 2 CIGB-300 peptide and chemotherapeutic agents in lung and cervical preclinical cancer models

PubMed Central

PERERA, YASSER; TORO, NEYLEN DEL; GOROVAYA, LARISA; FERNANDEZ-DE-COSSIO, JORGE; FARINA, HERNAN G.; PEREA, SILVIO E.

2014-01-01

CIGB-300 is a novel clinical-stage synthetic peptide that impairs the casein kinase 2 (CK2)-mediated phosphorylation of B23/nucleophosmin in different experimental settings and cancer models. As a single agent, CIGB-300 induces apoptosis in vitro and in vivo and modulates an array of proteins that are mainly involved in drug resistance, cell proliferation and apoptosis, as determined by proteomic analysis. However, the clinical oncology practice and cumulative knowledge on tumor biology suggest that drug combinations are more likely to cope with tumor complexity compared to single agents. In this study, we investigated the antiproliferative effect of CIGB-300 when combined with different anticancer drugs, such as cisplatin (alkylating), paclitaxel (antimitotic), doxorubicin (antitopoisomerase II) or 5-fluorouracil (DNA/RNA antimetabolite) in cell lines derived from lung and cervical cancer. Of note, using a Latin square design and subsequent analysis by CalcuSyn software, we observed that paclitaxel and cisplatin exhibited the best synergistic/additive profile when combined with CIGB-300, according to the combination and dose reduction indices. Such therapeutically favorable profiles may be explained by a direct cytotoxic effect and also by the observed cell cycle impairment following incubation of tumor cells with selected drug combinations. Importantly, on in vivo dose-finding schedules in human cervical tumors xenografted in nude mice, we observed that concomitant administration of CIGB-300 and cisplatin increased mice survival compared to single-agent treatment. Collectively, these findings provide a rationale for combining the anti-CK2 CIGB-300 peptide with currently available anticancer agents in the clinical setting and indicate platins and taxanes as compounds with major perspectives. PMID:25279177

Distinct TrkA and Ret modulated negative and positive neuropathic behaviors in a mouse model of resiniferatoxin-induced small fiber neuropathy.

PubMed

Hsieh, Yu-Lin; Kan, Hung-Wei; Chiang, Hao; Lee, Yi-Chen; Hsieh, Sung-Tsang

2018-02-01

Neurotrophic factors and their corresponding receptors play key roles in the maintenance of different phenotypic dorsal root ganglion (DRG) neurons, the axons of which degenerate in small fiber neuropathy, leading to various neuropathic manifestations. Mechanisms underlying positive and negative symptoms of small fiber neuropathy have not been systematically explored. This study investigated the molecular basis of these seemingly paradoxical neuropathic behaviors according to the profiles of TrkA and Ret with immunohistochemical and pharmacological interventions in a mouse model of resiniferatoxin (RTX)-induced small fiber neuropathy. Mice with RTX neuropathy exhibited thermal hypoalgesia and mechanical allodynia, reduced skin innervation, and altered DRG expression profiles with decreased TrkA(+) neurons and increased Ret(+) neurons. RTX neuropathy induced the expression of activating transcription factor 3 (ATF3), and ATF3(+) neurons were colocalized with Ret but not with TrkA (P<0.001). As a neuroprotectant, 4-Methylcatechol (4MC) promoted skin reinnervation partially with correlated reversal of the neuropathic behaviors and altered neurochemical expression. Gambogic amide, a selective TrkA agonist, normalized thermal hypoalgesia, and GW441756, a TrkA kinase inhibitor, induced thermal hypoalgesia, which was already reversed by 4MC. Mechanical allodynia was reversed by a Ret kinase inhibitor, AST487, which induced thermal hyperalgesia in naïve mice. The activation of Ret signaling by XIB4035 induced mechanical allodynia and thermal hypoalgesia in RTX neuropathy mice in which the neuropathic behaviors were previously normalized by 4MC. Distinct neurotrophic factor receptors, TrkA and Ret, accounted for negative and positive neuropathic behaviors in RTX-induced small fiber neuropathy, respectively: TrkA for thermal hypoalgesia and Ret for mechanical allodynia and thermal hypoalgesia. Copyright © 2017 Elsevier Inc. All rights reserved.

Gastrointestinal adverse effects of cyclin-dependent kinase 4 and 6 inhibitors in breast cancer patients: a systematic review and meta-analysis

PubMed Central

Shohdy, Kyrillus S.; Lasheen, Shaimaa; Kassem, Loay; Abdel-Rahman, Omar

2017-01-01

Background: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors show promising results in metastatic breast cancer. However, an increased incidence of adverse events is remarkable. Among others, gastrointestinal (GI) involvement is of momentous impact on patients and their quality of life. Methods: Our search included PubMed, ASCO, ESMO and SABCS databases. Randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors were identified and considered relevant based on providing a sufficient safety profile on the incidence of adverse GI effects. Results: Of the 999 records initially screened for relevance, 33 articles were found relevant and 4 studies were finally eligible for meta-analysis with a total of 2007 patients. The relative risk (RR) for all-grade nausea was 1.48 [95% confidence interval (CI): 1.12–1.93, p = 0.005], vomiting was 1.74 (95% CI: 1.09–2.76, p = 0.02), decreased appetite was 1.42 (95% CI: 1.07–1.88, p = 0.02), and for diarrhea it was 1.44 (95% CI: 1.19–1.74, p = 0.0002). Meanwhile, the RR for high-grade nausea was 1.10 (95% CI: 0.29–4.13, p = 0.89), vomiting was 1.38 (95% CI: 0.25–7.75, p = 0.72), decreased appetite was 4.00 (95% CI: 0.87–18.37, p = 0.07), and high-grade diarrhea was 1.19 (95% CI: 0.44–3.21, p = 0.73). Conclusion: Selective CDK4/6 inhibitors were not associated with higher-grade GI toxicities reflecting a well-tolerated safety profile. Regarding the increase in all-grade GI toxicities, it needs further caution with addition of cytotoxic chemotherapy. PMID:29090083

Gastrointestinal adverse effects of cyclin-dependent kinase 4 and 6 inhibitors in breast cancer patients: a systematic review and meta-analysis.

PubMed

Shohdy, Kyrillus S; Lasheen, Shaimaa; Kassem, Loay; Abdel-Rahman, Omar

2017-11-01

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors show promising results in metastatic breast cancer. However, an increased incidence of adverse events is remarkable. Among others, gastrointestinal (GI) involvement is of momentous impact on patients and their quality of life. Our search included PubMed, ASCO, ESMO and SABCS databases. Randomized phase II/III trials in metastatic breast cancer receiving CDK4/6 inhibitors were identified and considered relevant based on providing a sufficient safety profile on the incidence of adverse GI effects. Of the 999 records initially screened for relevance, 33 articles were found relevant and 4 studies were finally eligible for meta-analysis with a total of 2007 patients. The relative risk (RR) for all-grade nausea was 1.48 [95% confidence interval (CI): 1.12-1.93, p = 0.005], vomiting was 1.74 (95% CI: 1.09-2.76, p = 0.02), decreased appetite was 1.42 (95% CI: 1.07-1.88, p = 0.02), and for diarrhea it was 1.44 (95% CI: 1.19-1.74, p = 0.0002). Meanwhile, the RR for high-grade nausea was 1.10 (95% CI: 0.29-4.13, p = 0.89), vomiting was 1.38 (95% CI: 0.25-7.75, p = 0.72), decreased appetite was 4.00 (95% CI: 0.87-18.37, p = 0.07), and high-grade diarrhea was 1.19 (95% CI: 0.44-3.21, p = 0.73). Selective CDK4/6 inhibitors were not associated with higher-grade GI toxicities reflecting a well-tolerated safety profile. Regarding the increase in all-grade GI toxicities, it needs further caution with addition of cytotoxic chemotherapy.

Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study.

PubMed

Hauer, J A; Taylor, S S; Johnson, D A

1999-05-25

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.

cDNA identification, comparison and phylogenetic aspects of lombricine kinase from two oligochaete species.

PubMed

Doumen, Chris

2010-06-01

Creatine kinase and arginine kinase are the typical representatives of an eight-member phosphagen kinase family, which play important roles in the cellular energy metabolism of animals. The phylum Annelida underwent a series of evolutionary processes that resulted in rapid divergence and radiation of these enzymes, producing the greatest diversity of the phosphagen kinases within this phylum. Lombricine kinase (EC 2.7.3.5) is one of such enzymes and sequence information is rather limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two oligochaete species, the California blackworm (Lumbriculus variegatus) and the sludge worm (Tubifex tubifex). The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases, including two additional lombricine kinase sequences extracted from DNA databases and provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. The data confirms the presence of a deleted region within the flexible loop (the GS region) of all six examined lombricine kinases. A phylogenetic analysis of these six lombricine kinases clearly positions the enzymes together in a small subcluster within the larger creatine kinase (EC 2.7.3.2) clade. 2010. Published by Elsevier Inc.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.« less

Protein kinase C negatively regulates Akt activity and modifies UVC-induced apoptosis in mouse keratinocytes.

PubMed

Li, Luowei; Sampat, Keeran; Hu, Nancy; Zakari, Julia; Yuspa, Stuart H

2006-02-10

Skin keratinocytes are subject to frequent chemical and physical injury and have developed elaborate cell survival mechanisms to compensate. Among these, the Akt/protein kinase B (PKB) pathway protects keratinocytes from the toxic effects of ultraviolet light (UV). In contrast, the protein kinase C (PKC) family is involved in several keratinocyte death pathways. During an examination of potential interactions among these two pathways, we found that the insulin-like growth factor (IGF-1) activates both the PKC and the Akt signaling pathways in cultured primary mouse keratinocytes as indicated by increased phospho-PKC and phospho-Ser-473-Akt. IGF-1 also selectively induced translocation of PKCdelta and PKCepsilon from soluble to particulate fractions in mouse keratinocytes. Furthermore, the PKC-specific inhibitor, GF109203X, increased IGF-1-induced phospho-Ser-473-Akt and Akt kinase activity and enhanced IGF-1 protection from UVC-induced apoptosis. Selective activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced phospho-Ser-473-Akt, suggesting that activation of PKC inhibits Akt activity. TPA also attenuated IGF-1 and epidermal growth factor-induced phospho-Ser-473-Akt, reduced Akt kinase activity, and blocked IGF-1 protection from UVC-induced apoptosis. The inhibition of Akt activity by TPA was reduced by inhibitors of protein phosphatase 2A, and TPA stimulated the association of phosphatase 2A with Akt. Individual PKC isoforms were overexpressed in cultured keratinocytes by transduction with adenoviral vectors or inhibited with PKC-selective inhibitors. These studies indicated that PKCdelta and PKCepsilon were selectively potent at causing dephosphorylation of Akt and modifying cell survival, whereas PKCalpha enhanced phosphorylation of Akt on Ser-473. Our results suggested that activation of PKCdelta and PKCepsilon provide a negative regulation for Akt phosphorylation and kinase activity in mouse keratinocytes and serve as modulators of cell survival pathways in response to external stimuli.

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

PubMed

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.

Involvement of the Tyr kinase/JNK pathway in carbachol-induced bronchial smooth muscle contraction in the rat.

PubMed

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.

Giant hub Src and Syk tyrosine kinase thermodynamic profiles recapitulate evolution

NASA Astrophysics Data System (ADS)

Phillips, J. C.

2017-10-01

Thermodynamic scaling theory, previously applied mainly to small proteins, here analyzes quantitative evolution of the titled functional network giant hub enzymes. The broad domain structure identified homologically is confirmed hydropathically using amino acid sequences only. The most surprising results concern the evolution of the tyrosine kinase globular surface roughness from avians to mammals, which is first order, compared to the evolution within mammals from rodents to humans, which is second order. The mystery of the unique amide terminal region of proto oncogene tyrosine protein kinase is resolved by the discovery there of a rare hydroneutral septad targeting cluster, which is paralleled by an equally rare octad catalytic cluster in tyrosine kinase in humans and a few other species (cat and dog). These results, which go far towards explaining why these proteins are among the largest giant hubs in protein interaction networks, use no adjustable parameters.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

PubMed

Kwiatkowski, Jacek; Liu, Boping; Tee, Doris Hui Ying; Chen, Guoying; Ahmad, Nur Huda Binte; Wong, Yun Xuan; Poh, Zhi Ying; Ang, Shi Hua; Tan, Eldwin Sum Wai; Ong, Esther Hq; Nurul Dinie; Poulsen, Anders; Pendharkar, Vishal; Sangthongpitag, Kanda; Lee, May Ann; Sepramaniam, Sugunavathi; Ho, Soo Yei; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Hung, Alvin W

2018-05-24

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases

PubMed Central

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

Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases.

PubMed

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.

Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages*

PubMed Central

Söderholm, Sandra; Kainov, Denis E.; Öhman, Tiina; Denisova, Oxana V.; Schepens, Bert; Kulesskiy, Evgeny; Imanishi, Susumu Y.; Corthals, Garry; Hintsanen, Petteri; Aittokallio, Tero; Saelens, Xavier; Matikainen, Sampsa; Nyman, Tuula A.

2016-01-01

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase–, mitogen-activated protein kinase–, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections. PMID:27486199

Computational synchronization of microarray data with application to Plasmodium falciparum.

PubMed

Zhao, Wei; Dauwels, Justin; Niles, Jacquin C; Cao, Jianshu

2012-06-21

Microarrays are widely used to investigate the blood stage of Plasmodium falciparum infection. Starting with synchronized cells, gene expression levels are continually measured over the 48-hour intra-erythrocytic cycle (IDC). However, the cell population gradually loses synchrony during the experiment. As a result, the microarray measurements are blurred. In this paper, we propose a generalized deconvolution approach to reconstruct the intrinsic expression pattern, and apply it to P. falciparum IDC microarray data. We develop a statistical model for the decay of synchrony among cells, and reconstruct the expression pattern through statistical inference. The proposed method can handle microarray measurements with noise and missing data. The original gene expression patterns become more apparent in the reconstructed profiles, making it easier to analyze and interpret the data. We hypothesize that reconstructed gene expression patterns represent better temporally resolved expression profiles that can be probabilistically modeled to match changes in expression level to IDC transitions. In particular, we identify transcriptionally regulated protein kinases putatively involved in regulating the P. falciparum IDC. By analyzing publicly available microarray data sets for the P. falciparum IDC, protein kinases are ranked in terms of their likelihood to be involved in regulating transitions between the ring, trophozoite and schizont developmental stages of the P. falciparum IDC. In our theoretical framework, a few protein kinases have high probability rankings, and could potentially be involved in regulating these developmental transitions. This study proposes a new methodology for extracting intrinsic expression patterns from microarray data. By applying this method to P. falciparum microarray data, several protein kinases are predicted to play a significant role in the P. falciparum IDC. Earlier experiments have indeed confirmed that several of these kinases are involved in this process. Overall, these results indicate that further functional analysis of these additional putative protein kinases may reveal new insights into how the P. falciparum IDC is regulated.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Nancy L., E-mail: nlcho@partners.org; Lin, Chi-Iou; Du, Jinyan

Highlights: Black-Right-Pointing-Pointer Kinome profiling is a novel technique for identifying activated kinases in human cancers. Black-Right-Pointing-Pointer Src activity is increased in invasive thyroid cancers. Black-Right-Pointing-Pointer Inhibition of Src activity decreased proliferation and invasion in vitro. Black-Right-Pointing-Pointer Further investigation of Src targeted therapies in thyroid cancer is warranted. -- Abstract: Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using amore » bead-based, high-throughput system. Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation. Conclusion: Global kinome analysis enables the discovery of novel targets for thyroid cancer therapy. Further investigation of Src targeted therapy for advanced thyroid cancer is warranted.« less

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics

PubMed Central

Haake, Scott M.; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio; Chen, Yian Ann; Koomen, John; Rathmell, W. Kimryn; Fishman, Mayer; Haura, Eric B.

2016-01-01

Purpose Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Experimental Design Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. In order to describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Results Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, while DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Conclusions Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology amongst kidney cancer subtypes. PMID:27220961

Application of adenosine triphosphate affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment.

PubMed

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2014-11-04

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli.

Application of Adenosine Triphosphate Affinity Probe and Scheduled Multiple-Reaction Monitoring Analysis for Profiling Global Kinome in Human Cells in Response to Arsenite Treatment

PubMed Central

2015-01-01

Phosphorylation of cellular components catalyzed by kinases plays important roles in cell signaling and proliferation. Quantitative assessment of perturbation in global kinome may provide crucial knowledge for elucidating the mechanisms underlying the cytotoxic effects of environmental toxicants. Here, we utilized an adenosine triphosphate (ATP) affinity probe coupled with stable isotope labeling by amino acids in cell culture (SILAC) to assess quantitatively the arsenite-induced alteration of global kinome in human cells. We constructed a SILAC-compatible kinome library for scheduled multiple-reaction monitoring (MRM) analysis and adopted on-the-fly recalibration of retention time shift, which provided better throughput of the analytical method and enabled the simultaneous quantification of the expression of ∼300 kinases in two LC-MRM runs. With this improved analytical method, we conducted an in-depth quantitative analysis of the perturbation of kinome of GM00637 human skin fibroblast cells induced by arsenite exposure. Several kinases involved in cell cycle progression, including cyclin-dependent kinases (CDK1 and CDK4) and Aurora kinases A, B, and C, were found to be hyperactivated, and the altered expression of CDK1 was further validated by Western analysis. In addition, treatment with a CDK inhibitor, flavopiridol, partially restored the arsenite-induced growth inhibition of human skin fibroblast cells. Thus, sodium arsenite may confer its cytotoxic effect partly through the aberrant activation of CDKs and the resultant perturbation of cell cycle progression. Together, we developed a high-throughput, SILAC-compatible, and MRM-based kinome profiling method and demonstrated that the method is powerful in deciphering the molecular modes of action of a widespread environmental toxicant. The method should be generally applicable for uncovering the cellular pathways triggered by other extracellular stimuli. PMID:25301106

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

PubMed

Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

2017-09-22

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases

PubMed Central

2010-01-01

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation. PMID:24900195

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.

PubMed

Paruch, Kamil; Dwyer, Michael P; Alvarez, Carmen; Brown, Courtney; Chan, Tin-Yau; Doll, Ronald J; Keertikar, Kerry; Knutson, Chad; McKittrick, Brian; Rivera, Jocelyn; Rossman, Randall; Tucker, Greg; Fischmann, Thierry; Hruza, Alan; Madison, Vincent; Nomeir, Amin A; Wang, Yaolin; Kirschmeier, Paul; Lees, Emma; Parry, David; Sgambellone, Nicole; Seghezzi, Wolfgang; Schultz, Lesley; Shanahan, Frances; Wiswell, Derek; Xu, Xiaoying; Zhou, Quiao; James, Ray A; Paradkar, Vidyadhar M; Park, Haengsoon; Rokosz, Laura R; Stauffer, Tara M; Guzi, Timothy J

2010-08-12

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Development of Heat Shock Protein (Hsp90) Inhibitors To Combat Resistance to Tyrosine Kinase Inhibitors through Hsp90-Kinase Interactions.

PubMed

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.

Identification of a Novel and Selective Series of Itk Inhibitors via a Template-Hopping Strategy

PubMed Central

2013-01-01

Inhibition of Itk potentially constitutes a novel, nonsteroidal treatment for asthma and other T-cell mediated diseases. In-house kinase cross-screening resulted in the identification of an aminopyrazole-based series of Itk inhibitors. Initial work on this series highlighted selectivity issues with several other kinases, particularly AurA and AurB. A template-hopping strategy was used to identify a series of aminobenzothiazole Itk inhibitors, which utilized an inherently more selective hinge binding motif. Crystallography and modeling were used to rationalize the observed selectivity. Initial exploration of the SAR around this series identified potent Itk inhibitors in both enzyme and cellular assays. PMID:24900590

Indolinone based LRRK2 kinase inhibitors with a key hydrogen bond.

PubMed

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.

A bioinformatics approach for precision medicine off-label drug drug selection among triple negative breast cancer patients

PubMed Central

Cheng, Lijun; Schneider, Bryan P

2016-01-01

Background Cancer has been extensively characterized on the basis of genomics. The integration of genetic information about cancers with data on how the cancers respond to target based therapy to help to optimum cancer treatment. Objective The increasing usage of sequencing technology in cancer research and clinical practice has enormously advanced our understanding of cancer mechanisms. The cancer precision medicine is becoming a reality. Although off-label drug usage is a common practice in treating cancer, it suffers from the lack of knowledge base for proper cancer drug selections. This eminent need has become even more apparent considering the upcoming genomics data. Methods In this paper, a personalized medicine knowledge base is constructed by integrating various cancer drugs, drug-target database, and knowledge sources for the proper cancer drugs and their target selections. Based on the knowledge base, a bioinformatics approach for cancer drugs selection in precision medicine is developed. It integrates personal molecular profile data, including copy number variation, mutation, and gene expression. Results By analyzing the 85 triple negative breast cancer (TNBC) patient data in the Cancer Genome Altar, we have shown that 71.7% of the TNBC patients have FDA approved drug targets, and 51.7% of the patients have more than one drug target. Sixty-five drug targets are identified as TNBC treatment targets and 85 candidate drugs are recommended. Many existing TNBC candidate targets, such as Poly (ADP-Ribose) Polymerase 1 (PARP1), Cell division protein kinase 6 (CDK6), epidermal growth factor receptor, etc., were identified. On the other hand, we found some additional targets that are not yet fully investigated in the TNBC, such as Gamma-Glutamyl Hydrolase (GGH), Thymidylate Synthetase (TYMS), Protein Tyrosine Kinase 6 (PTK6), Topoisomerase (DNA) I, Mitochondrial (TOP1MT), Smoothened, Frizzled Class Receptor (SMO), etc. Our additional analysis of target and drug selection strategy is also fully supported by the drug screening data on TNBC cell lines in the Cancer Cell Line Encyclopedia. Conclusions The proposed bioinformatics approach lays a foundation for cancer precision medicine. It supplies much needed knowledge base for the off-label cancer drug usage in clinics. PMID:27107440

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

PubMed

Stockwell, Simon R; Platt, Georgina; Barrie, S Elaine; Zoumpoulidou, Georgia; Te Poele, Robert H; Aherne, G Wynne; Wilson, Stuart C; Sheldrake, Peter; McDonald, Edward; Venet, Mathilde; Soudy, Christelle; Elustondo, Frédéric; Rigoreau, Laurent; Blagg, Julian; Workman, Paul; Garrett, Michelle D; Mittnacht, Sibylle

2012-01-01

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes.

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

DOEpatents

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.

Neurofibromin Deficiency-Associated Transcriptional Dysregulation Suggests a Novel Therapy for Tibial Pseudoarthrosis in NF1

PubMed Central

Paria, Nandina; Cho, Tae-Joon; Choi, In Ho; Kamiya, Nobuhiro; Kayembe, Kay; Mao, Rong; Margraf, Rebecca L.; Obermosser, Gerlinde; Oxendine, Ila; Sant, David W.; Song, Mi Hyun; Stevenson, David A.; Viskochil, David H.; Wise, Carol A.; Kim, Harry K.W.; Rios, Jonathan J

2014-01-01

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic bi-allelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinosital-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant over-expression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing. PMID:24932921

Interplay between Leucine-Rich Repeat Kinase 2 (LRRK2) and p62/SQSTM-1 in Selective Autophagy.

PubMed

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.

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970

PubMed Central

Boucher, Diane M.; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A.; Milton, Sean; Murphy, Cheryl E.; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M.; Pollard, John R.

2014-01-01

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients. PMID:25010037

Potentiation of tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

PubMed

Hall, Amy B; Newsome, Dave; Wang, Yuxin; Boucher, Diane M; Eustace, Brenda; Gu, Yong; Hare, Brian; Johnson, Mac A; Milton, Sean; Murphy, Cheryl E; Takemoto, Darin; Tolman, Crystal; Wood, Mark; Charlton, Peter; Charrier, Jean-Damien; Furey, Brinley; Golec, Julian; Reaper, Philip M; Pollard, John R

2014-07-30

Platinum-based DNA-damaging chemotherapy is standard-of-care for most patients with lung cancer but outcomes remain poor. This has been attributed, in part, to the highly effective repair network known as the DNA-damage response (DDR). ATR kinase is a critical regulator of this pathway, and its inhibition has been shown to sensitize some cancer, but not normal, cells in vitro to DNA damaging agents. However, there are limited in vivo proof-of-concept data for ATR inhibition. To address this we profiled VX-970, the first clinical ATR inhibitor, in a series of in vitro and in vivo lung cancer models and compared it with an inhibitor of the downstream kinase Chk1. VX-970 markedly sensitized a large proportion of a lung cancer cell line and primary tumor panel in vitro to multiple DNA damaging drugs with clear differences to Chk1 inhibition observed. In vivo VX-970 blocked ATR activity in tumors and dramatically enhanced the efficacy of cisplatin across a panel of patient derived primary lung xenografts. The combination led to complete tumor growth inhibition in three cisplatin-insensitive models and durable tumor regression in a cisplatin-sensitive model. These data provide a strong rationale for the clinical evaluation of VX-970 in lung cancer patients.

Regulatory Efficacy of Brown Seaweed Lessonia nigrescens Extract on the Gene Expression Profile and Intestinal Microflora in Type 2 Diabetic Mice.

PubMed

Zhao, Chao; Yang, Chengfeng; Chen, Mingjun; Lv, Xucong; Liu, Bin; Yi, Lunzhao; Cornara, Laura; Wei, Ming-Chi; Yang, Yu-Chiao; Tundis, Rosa; Xiao, Jianbo

2018-02-01

In this study, the antidiabetic activity of Lessonia nigrescens ethanolic extract (LNE) is investigated in streptozotocin (SZT)-induced type 2 diabetic mice fed with a high-sucrose/high-fat diet. Ultra high performance liquid chromatography coupled with photo-DAD and electospray ionization-mass spectrometry (ESI-MS) is employed to analyze the major compounds in LNE. The components of the intestinal microflora in type 2 diabetic mice are analyzed by high-throughput next-generation 16S rRNA gene sequencing. Fasting blood glucose levels in diabetic mice are significantly decreased after LNE administration. The histology reveals that LNE could protect the cellular architecture of liver and kidney. LNE treatment significantly increases Bacteroidetes and decreases Firmicutes populations in intestinal microflora. Specifically, It could selectively enrich the amounts of beneficial bacteria, Barnesiella, as well as reduce the abundances of Clostridium and Alistipes. The increased gene and protein expression levels of phosphatidylinositol 3-kinase (PI3K) in the liver are observed in LNE treatment groups, while the expressions of c-Jun N-terminal kinase (JNK) are significantly downregulated. The above findings suggest that LNE could be considered as a functional food for reducing blood glucose and regulating intestinal microflora. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Practical management of everolimus-related toxicities in patients with advanced solid tumors.

PubMed

Grünwald, Viktor; Weikert, Steffen; Pavel, Marianne E; Hörsch, Dieter; Lüftner, Diana; Janni, Wolfgang; Geberth, Matthias; Weber, Matthias M

2013-01-01

Everolimus is an orally administered inhibitor of the mammalian target of rapamycin (mTOR), an intracellular protein kinase downstream of the phosphatidylinositol 3-kinase/AKT pathway involved in key components of tumorigenesis, including cell growth, proliferation, and angiogenesis. In the advanced cancer setting, based on favorable results from phase III trials, everolimus is indicated for the treatment of advanced renal cell carcinoma, advanced neuroendocrine tumors of pancreatic origin, and advanced hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. Additional oncology indications for everolimus include renal angiomyolipoma with tuberous sclerosis complex and subependymal giant-cell astrocytoma. Although it is generally well tolerated, with most adverse events of mild to moderate severity and manageable, everolimus exhibits a distinct adverse event profile that warrants guidance for proper diagnostic and medical management. This guidance is particularly important given the potential for widespread long-term use of everolimus. This review will focus on the most relevant toxicities associated with mTOR inhibitors and on their management. Practical treatment recommendations are presented for stomatitis, noninfectious pneumonitis, rash, selected metabolic abnormalities, and infections. Provided these events are rapidly identified and treated, the vast majority should resolve with minimal effect on treatment outcomes and patients' quality of life. Copyright © 2013 S. Karger AG, Basel.

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

PubMed Central

Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

2012-01-01

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics.

PubMed

Law, Mary E; Corsino, Patrick E; Narayan, Satya; Law, Brian K

2015-11-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non-ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non-ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Cyclin-Dependent Kinase Inhibitors as Anticancer Therapeutics

PubMed Central

Corsino, Patrick E.; Narayan, Satya

2015-01-01

Cyclin-dependent kinases (CDKs) have been considered promising drug targets for a number of years, but most CDK inhibitors have failed rigorous clinical testing. Recent studies demonstrating clear anticancer efficacy and reduced toxicity of CDK4/6 inhibitors such as palbociclib and multi-CDK inhibitors such as dinaciclib have rejuvenated the field. Favorable results with palbociclib and its recent U.S. Food and Drug Administration approval demonstrate that CDK inhibitors with narrow selectivity profiles can have clinical utility for therapy based on individual tumor genetics. A brief overview of results obtained with ATP-competitive inhibitors such as palbociclib and dinaciclib is presented, followed by a compilation of new avenues that have been pursued toward the development of novel, non–ATP-competitive CDK inhibitors. These creative ways to develop CDK inhibitors are presented along with crystal structures of these agents complexed with CDK2 to highlight differences in their binding sites and mechanisms of action. The recent successes of CDK inhibitors in the clinic, combined with the potential for structure-based routes to the development of non–ATP-competitive CDK inhibitors, and evidence that CDK inhibitors may have use in suppressing chromosomal instability and in synthetic lethal drug combinations inspire optimism that CDK inhibitors will become important weapons in the fight against cancer. PMID:26018905

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

PubMed

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.

Autogrid-based clustering of kinases: selection of representative conformations for docking purposes.

PubMed

Marzaro, Giovanni; Ferrarese, Alessandro; Chilin, Adriana

2014-08-01

The selection of the most appropriate protein conformation is a crucial aspect in molecular docking experiments. In order to reduce the errors arising from the use of a single protein conformation, several authors suggest the use of several tridimensional structures for the target. However, the selection of the most appropriate protein conformations still remains a challenging goal. The protein 3D-structures selection is mainly performed based on pairwise root-mean-square-deviation (RMSD) values computation, followed by hierarchical clustering. Herein we report an alternative strategy, based on the computation of only two atom affinity map for each protein conformation, followed by multivariate analysis and hierarchical clustering. This methodology was applied on seven different kinases of pharmaceutical interest. The comparison with the classical RMSD-based strategy was based on cross-docking of co-crystallized ligands. In the case of epidermal growth factor receptor kinase, also the docking performance on 220 known ligands were evaluated, followed by 3D-QSAR studies. In all the cases, the herein proposed methodology outperformed the RMSD-based one.

Discovery of Selective, Substrate-Competitive, and Passive Membrane Permeable Glycogen Synthase Kinase-3β Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling of New C-Glycosylflavones.

PubMed

Liang, Zhibin; Li, Qing X

2018-05-16

Glycogen synthase kinase-3β (GSK-3β) is a key enzyme responsible for tau hyperphosphorylation and is a viable therapeutic target of Alzheimer's disease (AD). We developed a new class of GSK-3β inhibitors based on the 6- C-glycosylflavone isoorientin (1). The new inhibitors are passive membrane permeable and constitutively attenuate GSK-3β mediated tau hyperphosphorylation and amyloid neurotoxicity in an AD cellular model. Enzymatic assays and kinetic studies demonstrated that compound 30 is a GSK-3β substrate-competitive inhibitor with distinct kinase selectivity, isoform-selectivity and over 310-fold increased potency as compared to 1. Structure-activity relationship analyses and in silico modeling suggest the mechanism of actions by which the hydrophobic, π-cation, and orthogonal multipolar interactions of 30 with the substrate site are critical for the GSK-3β inhibition and selectivity. The results provide new insights into GSK-3β drug discovery. The new inhibitors are valuable chemical probes and drug leads with therapeutic potential to tackle AD and other GSK-3β relevant diseases.

Wall-associated kinase-like polypeptide mediates nutritional status perception and response

DOEpatents

Yang, Zhenbiao; Karr, Stephen

2014-02-11

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C.

PubMed Central

Maizels, E T; Peters, C A; Kline, M; Cutler, R E; Shanmugam, M; Hunzicker-Dunn, M

1998-01-01

Small heat-shock proteins (sHSPs) are widely expressed 25-28 kDa proteins whose functions are dynamically regulated by phosphorylation. While recent efforts have clearly delineated a stress-responsive p38 mitogen-activated protein-kinase (MAPK)-dependent kinase pathway culminating in activation of the heat-shock (HSP)-kinases, mitogen-activated protein-kinase-activated protein kinase-2 and -3, not all sHSP phosphorylation events can be explained by the p38 MAPK-dependent pathway. The contribution of protein kinase C (PKC) to sHSP phosphorylation was suggested by early studies but later questioned on the basis of the reported poor ability of purified PKC to phosphorylate sHSP in vitro. The current study re-evaluates the role of PKC in sHSP phosphorylation in the light of the isoform complexity of the PKC family. We evaluated the sHSP phosphorylation status in rat corpora lutea obtained from two stages of pregnancy, mid-pregnancy and late-pregnancy, which express different levels of the novel PKC isoform, PKC-delta. Two-dimensional Western blot analysis showed that HSP-27 was more highly phosphorylated in vivo in corpora lutea of late pregnancy, corresponding to the developmental stage in which PKC-delta is abundant and active. Late-pregnant luteal extracts contained a lipid-sensitive HSP-kinase activity which exactly co-purified with PKC-delta using hydroxyapatite and S-Sepharose column chromatography. To determine whether there might be preferential phosphorylation of sHSP by a particular PKC isoform, purified recombinant PKC isoforms corresponding to those PKC isoforms detected in rat corpora lutea were evaluated for HSP-kinase activity in vitro. Recombinant PKC-delta effectively catalysed the phosphorylation of sHSP in vitro, and PKC-alpha was 30-50% as effective as an HSP-kinase; other PKCs tested (beta1, beta2, epsilon and zeta) were poor HSP-kinases. These results show that select PKC family members can function as direct HSP-kinases in vitro. Moreover, the observation of enhanced luteal HSP-27 phosphorylation in vivo, in late pregnancy, when PKC-delta is abundant and active, suggests that select PKC family members contribute to sHSP phosphorylation events in vivo. PMID:9620873

[Preliminary analysis of retinal gene expression profile of diabetic rat].

PubMed

Mei, Yan; Zhou, Hong-ying; Xiang, Tao; Lu, You-guang; Li, Ai-dong; Tang, En-jie; Yang, Hui-jun

2005-10-01

Establishing the retinal gene expression profiles of non-diabetic rat and diabetic rat and comparing the profiles in order to analyze the possible genes related with diabetic retinopathy. The whole retinal transcriptional fragments of non-diabetic rat and 8-week diabetic rat were obtained by restriction fragments differential display-PCR (RFDD-PCR). Bioinformatic analysis of retinal gene expression was performed using soft wares, including Fragment Analysis. After comparison of the expression profiles, the related gene fragments of diabetic retinopathy were initially selected as the target gene of further approach. A total of 3639 significant fragments were obtained. By means of more than 3-fold contrast of fluorescent intensity as the differential expression standard, the authors got 840 differential fragments, accounting for 23.08% of the expressed numbers and including 5 visual related genes, 13 excitatory neruotransmitter genes and 3 inhibitory neurotransmitter genes. At the 8th week, the expression of Rhodopsin kinase, beta-arrestin, Phosducinìrod photoreceptor cGMP-gated channel and Rpe65 as well as iGlu R1-4 were down-regulated. mGluRs and GABA-Rs were all up-regulated, whereas the expression of GlyR was unchanged. These results prompt again that the changes in retinal nervous layer of rat have occurred at an early stage of diabetes. The genes expression pattern of visual related genes and excitatory and inhibitory neurotransmitters in rat diabetic retina have been involved in neuro-dysfunctions of diabetic retina.

A chemogenomic analysis of the human proteome: application to enzyme families.

PubMed

Bernasconi, Paul; Chen, Min; Galasinski, Scott; Popa-Burke, Ioana; Bobasheva, Anna; Coudurier, Louis; Birkos, Steve; Hallam, Rhonda; Janzen, William P

2007-10-01

Sequence-based phylogenies (SBP) are well-established tools for describing relationships between proteins. They have been used extensively to predict the behavior and sensitivity toward inhibitors of enzymes within a family. The utility of this approach diminishes when comparing proteins with little sequence homology. Even within an enzyme family, SBPs must be complemented by an orthogonal method that is independent of sequence to better predict enzymatic behavior. A chemogenomic approach is demonstrated here that uses the inhibition profile of a 130,000 diverse molecule library to uncover relationships within a set of enzymes. The profile is used to construct a semimetric additive distance matrix. This matrix, in turn, defines a sequence-independent phylogeny (SIP). The method was applied to 97 enzymes (kinases, proteases, and phosphatases). SIP does not use structural information from the molecules used for establishing the profile, thus providing a more heuristic method than the current approaches, which require knowledge of the specific inhibitor's structure. Within enzyme families, SIP shows a good overall correlation with SBP. More interestingly, SIP uncovers distances within families that are not recognizable by sequence-based methods. In addition, SIP allows the determination of distance between enzymes with no sequence homology, thus uncovering novel relationships not predicted by SBP. This chemogenomic approach, used in conjunction with SBP, should prove to be a powerful tool for choosing target combinations for drug discovery programs as well as for guiding the selection of profiling and liability targets.

Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

PubMed

Azevedo, Marisa F; Camsari, Cagri; Sá, Carla M; Lima, Cristovao F; Fernandes-Ferreira, Manuel; Pereira-Wilson, Cristina

2010-06-01

In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

B-Raf kinase inhibitors: hit enrichment through scaffold hopping.

PubMed

Gopalsamy, Ariamala; Shi, Mengxiao; Hu, Yongbo; Lee, Frederick; Feldberg, Larry; Frommer, Eileen; Kim, Steven; Collins, Karen; Wojciechowicz, Donald; Mallon, Robert

2010-04-15

In continuation of our efforts toward hit identification and optimization for a B-Raf kinase project, we have employed a scaffold hopping strategy. The original HTS hit scaffold pyrazolo[1,5-a]pyrimidine was replaced with different thienopyrimidine and thienopyridine scaffolds to append the optimal pharmacophore moieties in order to generate novel B-raf kinase inhibitors with desirable potency and properties. This strategy led to the identification of additional lead compound 11b which had good enzyme and cell potency, while maintaining selectivity over a number of kinases. Copyright 2010 Elsevier Ltd. All rights reserved.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

PubMed Central

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system. PMID:26222440

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

PubMed

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important kinase system.

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Staurosporine scaffold-based rational discovery of the wild-type sparing reversible inhibitors of EGFR T790M gatekeeper mutant in lung cancer with analog-sensitive kinase technology.

PubMed

Song, Xiaoyun; Liu, Xingcai; Ding, Xi

2017-04-01

The human epidermal growth factor receptor (EGFR) has been established as an attractive target for lung cancer therapy. However, an acquired EGFR T790M gatekeeper mutation is frequently observed in patients treated with first-line anticancer agents such as gefitinib and erlotinib to cause drug resistance, largely limiting the application of small-molecule kinase inhibitors in EGFR-targeted chemotherapy. Previously, the reversible pan-kinase inhibitor staurosporine and its several analogs such as Gö6976 and K252a have been reported to selectively inhibit the EGFR T790M mutant (EGFR T790M ) over wild-type kinase (EGFR WT ), suggesting that the staurosporine scaffold is potentially to develop the wild-type sparing reversible inhibitors of EGFR T790M . Here, we systematically evaluated the inhibitor response of 28 staurosporine scaffold-based compounds to EGFR T790M mutation at structural, energetic, and molecular levels by using an integrated in silico-in vitro analog-sensitive (AS) kinase technology. With the strategy, we were able to identify 4 novel wild-type sparing inhibitors UCN-01, UCN-02, AFN941, and SB-218078 with high or moderate selectivity of 30-, 45-, 5-, and 8-fold for EGFR T790M over EGFR WT , respectively, which are comparable with or even better than that of the parent compound staurosporine (24-fold). Molecular modeling and structural analysis revealed that van der Waals contacts and hydrophobic forces can form between the side chain of mutated residue Met790 and the pyrrolidinone moiety of inhibitor ligand UCN-02, which may simultaneously improve the favorable interaction energy between the kinase and inhibitor, and reduce the unfavorable desolvation penalty upon the kinase-inhibitor binding. A hydroxyl group of UCN-02 additional to staurosporine locates at the pyrrolidinone moiety, which can largely alter the electronic distribution of pyrrolidinone moiety and thus promote the intermolecular interaction with Met790 residue. This can well explain the measured higher selectivity of UCN-02 than staurosporine for mutant over wild-type kinase. Copyright © 2016 John Wiley & Sons, Ltd.

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

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.

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A.

PubMed

Canela, Núria; Orzáez, Mar; Fucho, Raquel; Mateo, Francesca; Gutierrez, Ricardo; Pineda-Lucena, Antonio; Bachs, Oriol; Pérez-Payá, Enrique

2006-11-24

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.

Auto-phosphorylation Represses Protein Kinase R Activity.

PubMed

Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

2017-03-10

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

PubMed

Goodman, Krista B; Cui, Haifeng; Dowdell, Sarah E; Gaitanopoulos, Dimitri E; Ivy, Robert L; Sehon, Clark A; Stavenger, Robert A; Wang, Gren Z; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Semus, Simon F; Evans, Christopher; Fries, Harvey E; Jolivette, Larry J; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Bentley, Ross; Doe, Christopher P; Hu, Erding; Lee, Dennis

2007-01-11

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Evaluation of the first cytostatically active 1-aza-9-oxafluorenes as novel selective CDK1 inhibitors with P-glycoprotein modulating properties.

PubMed

Brachwitz, Kristin; Voigt, Burkhardt; Meijer, Laurent; Lozach, Olivier; Schächtele, Christoph; Molnár, Josef; Hilgeroth, Andreas

2003-02-27

The first series of synthetic 1-aza-9-oxafluorenes with cytostatic activities in the micromolar range was evaluated as cyclin-dependent kinase (CDK1) inhibitors. Activity was found to be selective in comparison to the inhibition of other kinases within the CDK family. Compounds were shown to inhibit the membrane-efflux pump P-glycoprotein responsible for multidrug resistance in cancer cells. First structure-activity relationships are discussed.

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.

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

PubMed

Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

2008-02-01

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

PubMed

Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

2003-09-18

The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

Discovery of Potent and Selective MRCK Inhibitors with Therapeutic Effect on Skin Cancer.

PubMed

Unbekandt, Mathieu; Belshaw, Simone; Bower, Justin; Clarke, Maeve; Cordes, Jacqueline; Crighton, Diane; Croft, Daniel R; Drysdale, Martin J; Garnett, Mathew J; Gill, Kathryn; Gray, Christopher; Greenhalgh, David A; Hall, James A M; Konczal, Jennifer; Lilla, Sergio; McArthur, Duncan; McConnell, Patricia; McDonald, Laura; McGarry, Lynn; McKinnon, Heather; McMenemy, Carol; Mezna, Mokdad; Morrice, Nicolas A; Munro, June; Naylor, Gregory; Rath, Nicola; Schüttelkopf, Alexander W; Sime, Mairi; Olson, Michael F

2018-04-15

The myotonic dystrophy-related Cdc42-binding kinases MRCKα and MRCKβ contribute to the regulation of actin-myosin cytoskeleton organization and dynamics, acting in concert with the Rho-associated coiled-coil kinases ROCK1 and ROCK2. The absence of highly potent and selective MRCK inhibitors has resulted in relatively little knowledge of the potential roles of these kinases in cancer. Here, we report the discovery of the azaindole compounds BDP8900 and BDP9066 as potent and selective MRCK inhibitors that reduce substrate phosphorylation, leading to morphologic changes in cancer cells along with inhibition of their motility and invasive character. In over 750 human cancer cell lines tested, BDP8900 and BDP9066 displayed consistent antiproliferative effects with greatest activity in hematologic cancer cells. Mass spectrometry identified MRCKα S1003 as an autophosphorylation site, enabling development of a phosphorylation-sensitive antibody tool to report on MRCKα status in tumor specimens. In a two-stage chemical carcinogenesis model of murine squamous cell carcinoma, topical treatments reduced MRCKα S1003 autophosphorylation and skin papilloma outgrowth. In parallel work, we validated a phospho-selective antibody with the capability to monitor drug pharmacodynamics. Taken together, our findings establish an important oncogenic role for MRCK in cancer, and they offer an initial preclinical proof of concept for MRCK inhibition as a valid therapeutic strategy. Significance: The development of selective small-molecule inhibitors of the Cdc42-binding MRCK kinases reveals their essential roles in cancer cell viability, migration, and invasive character. Cancer Res; 78(8); 2096-114. ©2018 AACR . ©2018 American Association for Cancer Research.

Cyclin-dependent kinase 8 module expression profiling reveals requirement of mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila.

PubMed

Kuuluvainen, Emilia; Hakala, Heini; Havula, Essi; Sahal Estimé, Michelle; Rämet, Mika; Hietakangas, Ville; Mäkelä, Tomi P

2014-06-06

The Cdk8 (cyclin-dependent kinase 8) module of Mediator integrates regulatory cues from transcription factors to RNA polymerase II. It consists of four subunits where Med12 and Med13 link Cdk8 and cyclin C (CycC) to core Mediator. Here we have investigated the contributions of the Cdk8 module subunits to transcriptional regulation using RNA interference in Drosophila cells. Genome-wide expression profiling demonstrated separation of Cdk8-CycC and Med12-Med13 profiles. However, transcriptional regulation by Cdk8-CycC was dependent on Med12-Med13. This observation also revealed that Cdk8-CycC and Med12-Med13 often have opposite transcriptional effects. Interestingly, Med12 and Med13 profiles overlapped significantly with that of the GATA factor Serpent. Accordingly, mutational analyses indicated that GATA sites are required for Med12-Med13 regulation of Serpent-dependent genes. Med12 and Med13 were also found to be required for Serpent-activated innate immunity genes in defense to bacterial infection. The results reveal a novel role for the Cdk8 module in Serpent-dependent transcription and innate immunity. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Gene Transcription Profile of the Detached Retina (An AOS Thesis)

PubMed Central

Zacks, David N.

2009-01-01

Purpose: Separation of the neurosensory retina from the retinal pigment epithelium (RPE) yields many morphologic and functional consequences, including death of the photoreceptor cells, Müller cell hypertrophy, and inner retinal rewiring. Many of these changes are due to the separation-induced activation of specific genes. In this work, we define the gene transcription profile within the retina as a function of time after detachment. We also define the early activation of kinases that might be responsible for the detachment-induced changes in gene transcription. Methods: Separation of the retina from the RPE was induced in Brown-Norway rats by the injection of 1% hyaluronic acid into the subretinal space. Retinas were harvested at 1, 7, and 28 days after separation. Gene transcription profiles for each time point were determined using the Affymetrix Rat 230A gene microarray chip. Transcription levels in detached retinas were compared to those of nondetached retinas with the BRB-ArrayTools Version 3.6.0 using a random variance analysis of variance (ANOVA) model. Confirmation of the significant transcriptional changes for a subset of the genes was performed using microfluidic quantitative real-time polymerase chain reaction (qRT-PCR) assays. Kinase activation was explored using Western blot analysis to look for early phosphorylation of any of the 3 main families of mitogen-activated protein kinases (MAPK): the p38 family, the Janus kinase family, and the p42/p44 family. Results: Retinas separated from the RPE showed extensive alterations in their gene transcription profile. Many of these changes were initiated as early as 1 day after separation, with significant increases by 7 days. ANOVA analysis defined 144 genes that had significantly altered transcription levels as a function of time after separation when setting a false discovery rate at ≤0.1. Confirmatory RT-PCR was performed on 51 of these 144 genes. Differential transcription detected on the microarray chip was confirmed by qRT-PCR for all 51 genes. Western blot analysis showed that the p42/p44 family of MAPK was phosphorylated within 2 hours of retinal-RPE separation. This phosphorylation was detachment-induced and could be inhibited by specific inhibitors of MAPK phosphorylation. Conclusions: Separation of the retina from the RPE induces significant alteration in the gene transcription profile within the retina. These profiles are not static, but change as a function of time after detachment. These gene transcription changes are preceded by the activation of the p42/p44 family of MAPK. This altered transcription may serve as the basis for many of the morphologic, biochemical, and functional changes seen within the detached retina. PMID:20126507

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

PubMed

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.

Expression, purification, and characterization of an enzymatically active truncated human rho-kinase I (ROCK I) domain expressed in Sf-9 insect cells.

PubMed

Khandekar, Sanjay S; Yi, Tracey; Dul, Ed; Wright, Lois L; Chen, Susan; Scott, Gilbert F; Smith, Gary K; Lee, Dennis; Hu, Erding; Kirkpatrick, Robert B

2006-01-01

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9-baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.

Approved and Experimental Small-Molecule Oncology Kinase Inhibitor Drugs: A Mid-2016 Overview.

PubMed

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.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Naphthalenesulphonamides block neutrophil superoxide production by intact cells and in a cell-free system: is myosin light chain kinase responsible for these effects?

PubMed Central

Heyworth, P G; Erickson, R W; Ding, J; Curnutte, J T; Badwey, J A

1995-01-01

Selective antagonists of myosin light chain kinase (MLCK) [e.g. ML-7; 1-(5-iodonaphthalene-1-sulphonyl)-1H-hexahydro-1,4-diazepine hydrochloride] were found to inhibit superoxide (O2-) release from stimulated neutrophils. The concentrations of ML-7 that were inhibitory were substantially lower than those reported for a selective antagonist of protein kinase C [i.e. H-7; 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride]. ML-7 also reduced the phosphorylation of the 47 kDa subunit of the NADPH-oxidase system (p47-phox) and blocked translocation of this protein to the Triton X-100-insoluble fraction in stimulated cells. Interestingly, ML-7 also inhibited O2- production in a cell-free system derived from neutrophils at concentrations similar to those that were effective in vivo. This cell-free system does not require ATP and is insensitive to all other inhibitors of protein kinases tested, including some highly effective against MLCK (i.e. staurosporine). Thus, the data suggest that ML-7 does not block O2- release by inhibiting a protein kinase but instead may interact directly with a subunit of the oxidase. The binding site for ML-7 may provide a valuable target for inhibiting the inflammatory properties of phagocytic leucocytes by naphthalenesulphonamides designed to lack activity against protein kinases. Images Figure 3 Figure 4 PMID:7575484

Tyrosine Kinase Signaling in Clear Cell and Papillary Renal Cell Carcinoma Revealed by Mass Spectrometry-Based Phosphotyrosine Proteomics.

PubMed

Haake, Scott M; Li, Jiannong; Bai, Yun; Kinose, Fumi; Fang, Bin; Welsh, Eric A; Zent, Roy; Dhillon, Jasreman; Pow-Sang, Julio M; Chen, Y Ann; Koomen, John M; Rathmell, W Kimryn; Fishman, Mayer; Haura, Eric B

2016-11-15

Targeted therapies in renal cell carcinoma (RCC) are limited by acquired resistance. Novel therapeutic targets are needed to combat resistance and, ideally, target the unique biology of RCC subtypes. Tyrosine kinases provide critical oncogenic signaling and their inhibition has significantly impacted cancer care. To describe a landscape of tyrosine kinase activity in RCC that could inform novel therapeutic strategies, we performed a mass spectrometry-based system-wide survey of tyrosine phosphorylation in 10 RCC cell lines as well as 15 clear cell and 15 papillary RCC human tumors. To prioritize identified tyrosine kinases for further analysis, a 63 tyrosine kinase inhibitor (TKI) drug screen was performed. Among the cell lines, 28 unique tyrosine phosphosites were identified across 19 kinases and phosphatases including EGFR, MET, JAK2, and FAK in nearly all samples. Multiple FAK TKIs decreased cell viability by at least 50% and inhibited RCC cell line adhesion, invasion, and proliferation. Among the tumors, 49 unique tyrosine phosphosites were identified across 44 kinases and phosphatases. FAK pY576/7 was found in all tumors and many cell lines, whereas DDR1 pY792/6 was preferentially enriched in the papillary RCC tumors. Both tyrosine kinases are capable of transmitting signals from the extracellular matrix and emerged as novel RCC therapeutic targets. Tyrosine kinase profiling informs novel therapeutic strategies in RCC and highlights the unique biology among kidney cancer subtypes. Clin Cancer Res; 22(22); 5605-16. ©2016 AACR. ©2016 American Association for Cancer Research.

KinMap: a web-based tool for interactive navigation through human kinome data.

PubMed

Eid, Sameh; Turk, Samo; Volkamer, Andrea; Rippmann, Friedrich; Fulle, Simone

2017-01-05

Annotations of the phylogenetic tree of the human kinome is an intuitive way to visualize compound profiling data, structural features of kinases or functional relationships within this important class of proteins. The increasing volume and complexity of kinase-related data underlines the need for a tool that enables complex queries pertaining to kinase disease involvement and potential therapeutic uses of kinase inhibitors. Here, we present KinMap, a user-friendly online tool that facilitates the interactive navigation through kinase knowledge by linking biochemical, structural, and disease association data to the human kinome tree. To this end, preprocessed data from freely-available sources, such as ChEMBL, the Protein Data Bank, and the Center for Therapeutic Target Validation platform are integrated into KinMap and can easily be complemented by proprietary data. The value of KinMap will be exemplarily demonstrated for uncovering new therapeutic indications of known kinase inhibitors and for prioritizing kinases for drug development efforts. KinMap represents a new generation of kinome tree viewers which facilitates interactive exploration of the human kinome. KinMap enables generation of high-quality annotated images of the human kinome tree as well as exchange of kinome-related data in scientific communications. Furthermore, KinMap supports multiple input and output formats and recognizes alternative kinase names and links them to a unified naming scheme, which makes it a useful tool across different disciplines and applications. A web-service of KinMap is freely available at http://www.kinhub.org/kinmap/ .

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870.

PubMed

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).

Discovery of 5-Amino- N -(1 H -pyrazol-4-yl)pyrazolo[1,5- a ]pyrimidine-3-carboxamide Inhibitors of IRAK4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lim, Jongwon; Altman, Michael D.; Baker, James

2015-06-11

Interleukin-1 receptor associated kinase 4 (IRAK4) is an essential signal transducer downstream of the IL-1R and TLR superfamily, and selective inhibition of the kinase activity of the protein represents an attractive target for the treatment of inflammatory diseases. A series of 5-amino-N-(1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamides was developed via sequential modifications to the 5-position of the pyrazolopyrimidine ring and the 3-position of the pyrazole ring. Replacement of substituents responsible for poor permeability and improvement of physical properties guided by cLogD led to the identification of IRAK4 inhibitors with excellent potency, kinase selectivity, and pharmacokinetic properties suitable for oral dosing.

Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

PubMed

Csukai, M; Mochly-Rosen, D

1999-04-01

Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems. Copyright 1999 The Italian Pharmacological Society.

Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

PubMed

Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

2015-01-01

Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

Omacetaxine Mepesuccinate for Chronic Myeloid Leukemia.

PubMed

Rosshandler, Yasmin; Shen, Ann Q; Cortes, Jorge; Khoury, Hanna Jean

2016-05-01

Omacetaxine mepesuccinate is approved by the Food and Drug Administration in the United States for the treatment of chronic myeloid leukemia in chronic or accelerated phase resistant to two or more tyrosine kinase inhibitors. This review summarizes the mode of action, pharmacokinetics, efficacy and safety of omacetaxine mepesuccinate. Omacetaxine mepesuccinate has activity in chronic myeloid leukemia, especially in the chronic phase, regardless of the presence of ABL1 kinase domain mutations. Omacetaxine mepesuccinate has distinct but manageable adverse events profile. Omacetaxine mepesuccinate is a treatment option for a subset of patients with refractory chronic myeloid leukemia.

Structure-based optimization of oxadiazole-based GSK-3 inhibitors.

PubMed

Lo Monte, Fabio; Kramer, Thomas; Gu, Jiamin; Brodrecht, Martin; Pilakowski, Johannes; Fuertes, Ana; Dominguez, Juan Manuel; Plotkin, Batya; Eldar-Finkelman, Hagit; Schmidt, Boris

2013-03-01

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases β-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimer's disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and β, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3α and 17 nM for GSK-3β. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3β, with an IC50 of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

PET imaging of T cells: Target identification and feasibility assessment.

PubMed

Auberson, Yves P; Briard, Emmanuelle; Rudolph, Bettina; Kaupmann, Klemen; Smith, Paul; Oberhauser, Berndt

2018-06-01

Imaging T cells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are however no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in T cells, and for which lead molecules were available: PKC , Lck, ZAP70 and Itk. Ultimately, we focused on Itk (interleukin-2-inducible T cell kinase) and identified a tool molecule with properties suitable for in vivo imaging of T cells, (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)-phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydro-cyclopropa[f]-indazole-3-carboxamide (23). While not having the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of T cells in patients. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

PubMed Central

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway.

PubMed

Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn; Mai, Chun-Wai

2017-01-01

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted.

Neutron diffraction reveals hydrogen bonds critical for cGMP-selective activation: Insights for cGMP-dependent protein kinase agonist design

DOE PAGES

Huang, Gilbert Y.; Gerlits, Oksana O.; Blakeley, Matthew P.; ...

2014-10-01

High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). Finally, the XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted,more » explaining its low affinity for cAMP.« less

Mechanisms of resistance to imatinib in CML patients: a paradigm for the advantages and pitfalls of molecularly targeted therapy.

PubMed

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.

eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

PubMed Central

Menacho-Márquez, Mauricio; Rodríguez-Hernández, Carlos J; Villaronga, M Ángeles; Pérez-Valle, Jorge; Gadea, José; Belandia, Borja; Murguía, José R

2015-01-01

β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses. PMID:25590579

The mTOR kinase inhibitor everolimus synergistically enhances the anti-tumor effect of the Bruton's tyrosine kinase (BTK) inhibitor PLS-123 on Mantle cell lymphoma.

PubMed

Li, Jiao; Wang, Xiaogan; Xie, Yan; Ying, Zhitao; Liu, Weiping; Ping, Lingyan; Zhang, Chen; Pan, Zhengying; Ding, Ning; Song, Yuqin; Zhu, Jun

2018-01-01

Mantle cell lymphoma (MCL) is an aggressive and incurable malignant disease. Despite of general chemotherapy, relapse and mortality are common, highlighting the need for the development of novel targeted drugs or combination of therapeutic regimens. Recently, several drugs that target the B-cell receptor (BCR) signaling pathway, especially the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, have demonstrated notable therapeutic effects in relapsed/refractory patients, which indicate that pharmacological inhibition of BCR pathway holds promise in MCL treatment. Here, we have developed a novel irreversible BTK inhibitor, PLS-123, that has more potent and selective anti-tumor activity than ibrutinib in vitro and in vivo. Using in vitro screening, we discovered that the combination of PLS-123 and the mammalian target of rapamycin (mTOR) inhibitor everolimus exert synergistic activity in attenuating proliferation and motility of MCL cell lines. Simultaneous inhibition of BTK and mTOR resulted in marked induction of apoptosis and cell cycle arrest in the G1 phase, which were accompanied by upregulation of pro-apoptotic proteins (cleaved Caspase-3, cleaved PARP and Bax), repression of anti-apoptotic proteins (Mcl-1, Bcl-xl and XIAP), and downregulation of regulators of the G1/S phase transition (CDK2, CDK4, CDK6 and Cyclin D1). Gene expression profile analysis revealed simultaneous treatment with these agents led to inhibition of the JAK2/STAT3, AKT/mTOR signaling pathways and SGK1 expression. Finally, the anti-tumor and pro-apoptotic activities of combination strategy have also been demonstrated using xenograft mice models. Taken together, simultaneous suppression of BTK and mTOR may be indicated as a potential therapeutic modality for the treatment of MCL. © 2017 UICC.

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.

PubMed

Stefan, E; Aquin, S; Berger, N; Landry, C R; Nyfeler, B; Bouvier, M; Michnick, S W

2007-10-23

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

Trial Watch: Targeting ATM–CHK2 and ATR–CHK1 pathways for anticancer therapy

PubMed Central

Manic, Gwenola; Obrist, Florine; Sistigu, Antonella; Vitale, Ilio

2015-01-01

The ataxia telangiectasia mutated serine/threonine kinase (ATM)/checkpoint kinase 2 (CHEK2, best known as CHK2) and the ATM and Rad3-related serine/threonine kinase (ATR)/CHEK1 (best known as CHK1) cascades are the 2 major signaling pathways driving the DNA damage response (DDR), a network of processes crucial for the preservation of genomic stability that act as a barrier against tumorigenesis and tumor progression. Mutations and/or deletions of ATM and/or CHK2 are frequently found in tumors and predispose to cancer development. In contrast, the ATR–CHK1 pathway is often upregulated in neoplasms and is believed to promote tumor growth, although some evidence indicates that ATR and CHK1 may also behave as haploinsufficient oncosuppressors, at least in a specific genetic background. Inactivation of the ATM–CHK2 and ATR–CHK1 pathways efficiently sensitizes malignant cells to radiotherapy and chemotherapy. Moreover, ATR and CHK1 inhibitors selectively kill tumor cells that present high levels of replication stress, have a deficiency in p53 (or other DDR players), or upregulate the ATR–CHK1 module. Despite promising preclinical results, the clinical activity of ATM, ATR, CHK1, and CHK2 inhibitors, alone or in combination with other therapeutics, has not yet been fully demonstrated. In this Trial Watch, we give an overview of the roles of the ATM-CHK2 and ATR-CHK1 pathways in cancer initiation and progression, and summarize the results of clinical studies aimed at assessing the safety and therapeutic profile of regimens based on inhibitors of ATR and CHK1, the only 2 classes of compounds that have so far entered clinics. PMID:27308506

SD-208, a Novel Protein Kinase D Inhibitor, Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

PubMed Central

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

Masitinib Combined with Standard Gemcitabine Chemotherapy: In Vitro and In Vivo Studies in Human Pancreatic Tumour Cell Lines and Ectopic Mouse Model

PubMed Central

Humbert, Martine; Castéran, Nathalie; Letard, Sébastien; Hanssens, Katia; Iovanna, Juan; Finetti, Pascal; Bertucci, François; Bader, Thomas; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier; Dubreuil, Patrice

2010-01-01

Background Tyrosine kinases are attractive targets for pancreatic cancer therapy because several are over-expressed, including PDGFRα/β, FAK, Src and Lyn. A critical role of mast cells in the development of pancreatic cancer has also been reported. Masitinib is a tyrosine kinase inhibitor that selectively targets c-Kit, PDGFRα/β, Lyn, and to a lesser extent the FAK pathway, without inhibiting kinases of known toxicities. Masitinib is particularly efficient in controlling the proliferation, differentiation and degranulation of mast cells. This study evaluates the therapeutic potential of masitinib in pancreatic cancer, as a single agent and in combination with gemcitabine. Methodology/Findings Proof-of-concept studies were performed in vitro on human pancreatic tumour cell lines and then in vivo using a mouse model of human pancreatic cancer. Molecular mechanisms were investigated via gene expression profiling. Masitinib as a single agent had no significant antiproliferative activity while the masitinib/gemcitabine combination showed synergy in vitro on proliferation of gemcitabine-refractory cell lines Mia Paca2 and Panc1, and to a lesser extent in vivo on Mia Paca2 cell tumour growth. Specifically, masitinib at 10 µM strongly sensitised Mia Paca2 cells to gemcitabine (>400-fold reduction in IC50); and moderately sensitised Panc1 cells (10-fold reduction). Transcriptional analysis identified the Wnt/β-catenin signalling pathway as down-regulated in the cell lines resensitised by the masitinib/gemcitabine combination. Conclusions These data establish proof-of-concept that masitinib can sensitise gemcitabine-refractory pancreatic cancer cell lines and warrant further in vivo investigation. Indeed, such an effect has been recently observed in a phase 2 clinical study of patients with pancreatic cancer who received a masitinib/gemcitabine combination. PMID:20209107

Design of Potent and Selective Inhibitors to Overcome Clinical Anaplastic Lymphoma Kinase Mutations Resistant to Crizotinib

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Qinhua; Johnson, Ted W.; Bailey, Simon

2014-02-27

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).

A Novel Recombinant Anti-CD22 Immunokinase Delivers Proapoptotic Activity of Death-Associated Protein Kinase (DAPK) and Mediates Cytotoxicity in Neoplastic B Cells.

PubMed

Lilienthal, Nils; Lohmann, Gregor; Crispatzu, Giuliano; Vasyutina, Elena; Zittrich, Stefan; Mayer, Petra; Herling, Carmen Diana; Tur, Mehmet Kemal; Hallek, Michael; Pfitzer, Gabriele; Barth, Stefan; Herling, Marco

2016-05-01

The serine/threonine death-associated protein kinases (DAPK) provide pro-death signals in response to (oncogenic) cellular stresses. Lost DAPK expression due to (epi)genetic silencing is found in a broad spectrum of cancers. Within B-cell lymphomas, deficiency of the prototypic family member DAPK1 represents a predisposing or early tumorigenic lesion and high-frequency promoter methylation marks more aggressive diseases. On the basis of protein studies and meta-analyzed gene expression profiling data, we show here that within the low-level context of B-lymphocytic DAPK, particularly CLL cells have lost DAPK1 expression. To target this potential vulnerability, we conceptualized B-cell-specific cytotoxic reconstitution of the DAPK1 tumor suppressor in the format of an immunokinase. After rounds of selections for its most potent cytolytic moiety and optimal ligand part, a DK1KD-SGIII fusion protein containing a constitutive DAPK1 mutant, DK1KD, linked to the scFv SGIII against the B-cell-exclusive endocytic glyco-receptor CD22 was created. Its high purity and large-scale recombinant production provided a stable, selectively binding, and efficiently internalizing construct with preserved robust catalytic activity. DK1KD-SGIII specifically and efficiently killed CD22-positive cells of lymphoma lines and primary CLL samples, sparing healthy donor- or CLL patient-derived non-B cells. The mode of cell death was predominantly PARP-mediated and caspase-dependent conventional apoptosis as well as triggering of an autophagic program. The notoriously high apoptotic threshold of CLL could be overcome by DK1KD-SGIII in vitro also in cases with poor prognostic features, such as therapy resistance. The manufacturing feasibility of the novel CD22-targeting DAPK immunokinase and its selective antileukemic efficiency encourage intensified studies towards specific clinical application. Mol Cancer Ther; 15(5); 971-84. ©2016 AACR. ©2016 American Association for Cancer Research.

Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study

PubMed Central

Kim, Pora; Jia, Peilin; Zhao, Zhongming

2018-01-01

Abstract Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5′-kinase fusion genes, combinatorial effects between 3′-KDR kinases and their 5′-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3′-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of ‘effective’ (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3′-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs’ clinical implications. PMID:28013235

Biomarkers for personalized oncology: recent advances and future challenges.

PubMed

Kalia, Madhu

2015-03-01

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and oncology is a branch of medicine that deals with tumors. The last decade has seen significant advances in the development of biomarkers in oncology that play a critical role in understanding molecular and cellular mechanisms which drive tumor initiation, maintenance and progression. Clinical molecular diagnostics and biomarker discoveries in oncology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one. These discoveries have fueled the development of novel drug targets and new treatment strategies. The standard of care for patients with advanced-stage cancers has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment algorithm based on the molecular profile of the tumor is used. Recent advances in multiplex genotyping technologies and high-throughput genomic profiling by next-generation sequencing make possible the rapid and comprehensive analysis of the cancer genome of individual patients even from very little tumor biopsy material. Predictive (diagnostic) biomarkers are helpful in matching targeted therapies with patients and in preventing toxicity of standard (systemic) therapies. Prognostic biomarkers identify somatic germ line mutations, changes in DNA methylation, elevated levels of microRNA (miRNA) and circulating tumor cells (CTC) in blood. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized oncotherapy for the treatment of five diseases: chronic myeloid leukemia, colon, breast, lung cancer and melanoma and these biomarkers are being used successfully to evaluate benefits that can be achieved through targeted therapy. Examples of these molecularly targeted biomarker therapies are: tyrosine kinase inhibitors in chronic myeloid leukemia and gastrointestinal tumors; anaplastic lymphoma kinase (ALK) inhibitors in lung cancer with EML4-ALk fusion; HER2/neu blockage in HER2/neu-positive breast cancer; and epidermal growth factor receptors (EGFR) inhibition in EGFR-mutated lung cancer. This review presents the current state of our knowledge of biomarkers in five selected cancers: chronic myeloid leukemia, colorectal cancer, breast cancer, non-small cell lung cancer and melanoma. Copyright © 2015 Elsevier Inc. All rights reserved.

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

PubMed

Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

2012-04-15

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division. Published by Elsevier GmbH.

Phosphorylation Reaction in cAPK Protein Kinase - Free Energy Quantum Mechanic/Molecular Mechanics Simulations.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Valiev, Marat; Yang, Jie; Adams, Joseph

2007-11-29

Protein kinases catalyze the transfer of the γ-phosphoryl group from ATP, a key regulatory process governing signalling pathways in eukaryotic cells. The structure of the active site in these enzymes is highly conserved implying common catalytic mechanism. In this work we investigate the reaction process in cAPK protein kinase (PKA) using a combined quantum mechanics and molecular mechanics approach. The novel computational features of our work include reaction pathway determination with nudged elastic band methodology and calculation of free energy profiles of the reaction process taking into account finite temperature fluctuations of the protein environment. We find that the transfermore » of the γ-phosphoryl group in the protein environment is an exothermic reaction with the reaction barrier of 15 kcal/mol.« less

Protein Phosphorylation during Coconut Zygotic Embryo Development1

PubMed Central

Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

1998-01-01

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

Tofacitinib and analogs as inhibitors of the histone kinase PRK1 (PKN1).

PubMed

Ostrovskyi, Dmytro; Rumpf, Tobias; Eib, Julia; Lumbroso, Alexandre; Slynko, Inna; Klaeger, Susan; Heinzlmeir, Stephanie; Forster, Michael; Gehringer, Matthias; Pfaffenrot, Ellen; Bauer, Silke Mona; Schmidtkunz, Karin; Wenzler, Sandra; Metzger, Eric; Kuster, Bernhard; Laufer, Stefan; Schüle, Roland; Sippl, Wolfgang; Breit, Bernhard; Jung, Manfred

2016-09-01

The histone kinase PRK1 has been identified as a potential target to combat prostate cancer but selective PRK1 inhibitors are lacking. The US FDA -approved JAK1-3 inhibitor tofacitinib also potently inhibits PRK1 in vitro. We show that tofacitinib also inhibits PRK1 in a cellular setting. Using tofacitinib as a starting point for structure-activity relationship studies, we identified a more potent and another more selective PRK1 inhibitor compared with tofacitinib. Furthermore, we found two potential PRK1/JAK3-selectivity hotspots. The identified inhibitors and the selectivity hotspots lay the basis for the development of selective PRK1 inhibitors. The identification of PRK1, but also of other cellular tofacitinib targets, has implications on its clinical use and on future development of tofacitinib-like JAK inhibitors. [Formula: see text].

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunz, Roxanne K.; Rumfelt, Shannon; Chen, Ning

2010-01-12

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.

Does the Loss of Stromal Caveolin-1 Remodel the Tumor Microenvironment by Activating Src-Mediated PEAK1 and PI3K Pathways

DTIC Science & Technology

2016-09-01

Inhibition of MAP kinase pathway prevents plasma protrusions Next we used a selective inhibitor of MAP kinases , PD98059, to address whether we can...from human patients harbor AKT1 and that AKT1 kinase activity is sustained in these particles, nominating them as active signaling platforms...with the extracellular matrix (ECM) and extracellular molecules (2). Though many classic extracellular signaling molecules (e.g., hormones, peptide

Kinase inhibition by the Jamaican ball moss, Tillandsia recurvata L.

PubMed

Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

2012-10-01

This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd(50)s were below 20 μg/ml. Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.

Meta-analysis of the relationship of mycotoxins with biochemical and hematological parameters in broilers.

PubMed

Andretta, I; Kipper, M; Lehnen, C R; Lovatto, P A

2012-02-01

A meta-analysis was carried out to study the association of mycotoxins with hematological and biochemical profiles in broilers. Ninety-eight articles published between 1980 and 2009 were used in the database, totaling 37,371 broilers. The information was selected from the Materials and Methods and Results sections in the selected articles and then tabulated in a database. Meta-analysis followed 3 sequential analyses: graphic, correlation, and variance-covariance. Mycotoxins reduced (P < 0.05) the hematocrit (-5%), hemoglobin (-15%), leukocytes (-25%), heterophils (-2%), lymphocytes (-2%), uric acid (-31%), creatine kinase (-27%), creatinine (-23%), triglycerides (-39%), albumin (-17%), globulin (-1%), total cholesterol (-14%), calcium (-5%), and inorganic phosphorus (-12%). Mycotoxins also altered (P < 0.05) the concentrations of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase. A quadratic effect was observed on the relationship between the concentration of aflatoxin in diets and the serum concentration of alkaline phosphatase, γ-glutamyl transferase, alanine aminotransferase, and aspartate aminotransferase. The total protein concentration in blood was 18% lower (P < 0.05) in broilers challenged by aflatoxins compared with that of the unchallenged ones. The inclusion of antimycotoxin additives in diets with aflatoxins altered (P < 0.05) some variables (uric acid, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyl transferase) in relation to the group that received diets with the mycotoxin and without the additive. The meta-analysis performed in this study allowed us to address and quantify systematically the relationship of mycotoxins with alterations in hematologic and biochemical profiles in broilers.

A class of selective antibacterials derived from a protein kinase inhibitor pharmacophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, J. Richard; Dunham, Steve; Mochalkin, Igor

2009-06-25

As the need for novel antibiotic classes to combat bacterial drug resistance increases, the paucity of leads resulting from target-based antibacterial screening of pharmaceutical compound libraries is of major concern. One explanation for this lack of success is that antibacterial screening efforts have not leveraged the eukaryotic bias resulting from more extensive chemistry efforts targeting eukaryotic gene families such as G protein-coupled receptors and protein kinases. Consistent with a focus on antibacterial target space resembling these eukaryotic targets, we used whole-cell screening to identify a series of antibacterial pyridopyrimidines derived from a protein kinase inhibitor pharmacophore. In bacteria, the pyridopyrimidinesmore » target the ATP-binding site of biotin carboxylase (BC), which catalyzes the first enzymatic step of fatty acid biosynthesis. These inhibitors are effective in vitro and in vivo against fastidious Gram-negative pathogens including Haemophilus influenzae. Although the BC active site has architectural similarity to those of eukaryotic protein kinases, inhibitor binding to the BC ATP-binding site is distinct from the protein kinase-binding mode, such that the inhibitors are selective for bacterial BC. In summary, we have discovered a promising class of potent antibacterials with a previously undescribed mechanism of action. In consideration of the eukaryotic bias of pharmaceutical libraries, our findings also suggest that pursuit of a novel inhibitor leads for antibacterial targets with active-site structural similarity to known human targets will likely be more fruitful than the traditional focus on unique bacterial target space, particularly when structure-based and computational methodologies are applied to ensure bacterial selectivity.« less

Targeting the RhoA-ROCK Pathway to Reverse T Cell Dysfunction in SLE

PubMed Central

Rozo, Cristina; Chinenov, Yurii; Maharaj, Reena Khianey; Gupta, Sanjay; Leuenberger, Laura; Kirou, Kyriakos A.; Bykerk, Vivian P.; Goodman, Susan M.; Salmon, Jane E.; Pernis, Alessandra B.

2018-01-01

Objectives Deregulated production of IL-17 and IL-21 contributes to the pathogenesis of autoimmune disorders like SLE and RA. Production of IL-17 and IL-21 can be regulated by ROCK2, one of the two Rho kinases. Increased ROCK activation was previously observed in an SLE cohort. Here, we evaluated ROCK activity in a new SLE cohort, an RA cohort, and assessed the ability of distinct inhibitors of the ROCK pathway to suppress production of IL-17 and IL-21 by SLE T cells or human Th17 cells. Methods ROCK activity in PBMCs from 29 SLE patients, 31 RA patients, and 28 healthy controls was determined by ELISA. SLE T cells or in vitro-differentiated Th17 cells were treated with Y27632 (a pan-ROCK inhibitor), KD025 (a selective ROCK2 inhibitor), or simvastatin (which inhibits RhoA, a major ROCK activator). ROCK activity, IL-17, and IL-21 production were assessed. The transcriptional profile altered by ROCK inhibitors was evaluated by NanoString technology. Results ROCK activity levels were significantly higher in SLE and RA patients than healthy controls. Th17 cells exhibited high ROCK activity that was inhibited by Y276327, KD025, or simvastatin; each also decreased IL-17 and IL-21 production by purified SLE T cells or Th17 cells. Immune profiling revealed both overlapping and distinct effects of the different ROCK inhibitors. Conclusions ROCK activity is elevated in PBMCs from SLE and RA patients. Production of IL-17 and IL-21 by SLE T cells or Th17 cells can furthermore be inhibited by targeting the RhoA-ROCK pathway via both non-selective and selective approaches. PMID:28283529

The Potential Role of Aurora Kinase Inhibitors in Haematological Malignancies

PubMed Central

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

Prediction model of potential hepatocarcinogenicity of rat hepatocarcinogens using a large-scale toxicogenomics database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uehara, Takeki, E-mail: takeki.uehara@shionogi.co.jp; Toxicogenomics Informatics Project, National Institute of Biomedical Innovation, 7-6-8 Asagi, Ibaraki, Osaka 567-0085; Minowa, Yohsuke

2011-09-15

The present study was performed to develop a robust gene-based prediction model for early assessment of potential hepatocarcinogenicity of chemicals in rats by using our toxicogenomics database, TG-GATEs (Genomics-Assisted Toxicity Evaluation System developed by the Toxicogenomics Project in Japan). The positive training set consisted of high- or middle-dose groups that received 6 different non-genotoxic hepatocarcinogens during a 28-day period. The negative training set consisted of high- or middle-dose groups of 54 non-carcinogens. Support vector machine combined with wrapper-type gene selection algorithms was used for modeling. Consequently, our best classifier yielded prediction accuracies for hepatocarcinogenicity of 99% sensitivity and 97% specificitymore » in the training data set, and false positive prediction was almost completely eliminated. Pathway analysis of feature genes revealed that the mitogen-activated protein kinase p38- and phosphatidylinositol-3-kinase-centered interactome and the v-myc myelocytomatosis viral oncogene homolog-centered interactome were the 2 most significant networks. The usefulness and robustness of our predictor were further confirmed in an independent validation data set obtained from the public database. Interestingly, similar positive predictions were obtained in several genotoxic hepatocarcinogens as well as non-genotoxic hepatocarcinogens. These results indicate that the expression profiles of our newly selected candidate biomarker genes might be common characteristics in the early stage of carcinogenesis for both genotoxic and non-genotoxic carcinogens in the rat liver. Our toxicogenomic model might be useful for the prospective screening of hepatocarcinogenicity of compounds and prioritization of compounds for carcinogenicity testing. - Highlights: >We developed a toxicogenomic model to predict hepatocarcinogenicity of chemicals. >The optimized model consisting of 9 probes had 99% sensitivity and 97% specificity. >This model enables us to detect genotoxic as well as non-genotoxic hepatocarcinogens.« less

Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

2016-03-21

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "COT drives resistance to RAF inhibition through MAPK pathway reactivation" by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

PubMed

Sugden, M C; Lall, H S; Harris, R A; Holness, M J

2000-11-01

The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

Development of Potent, Selective SRPK1 Inhibitors as Potential Topical Therapeutics for Neovascular Eye Disease.

PubMed

Batson, Jennifer; Toop, Hamish D; Redondo, Clara; Babaei-Jadidi, Roya; Chaikuad, Apirat; Wearmouth, Stephen F; Gibbons, Brian; Allen, Claire; Tallant, Cynthia; Zhang, Jingxue; Du, Chunyun; Hancox, Jules C; Hawtrey, Tom; Da Rocha, Joana; Griffith, Renate; Knapp, Stefan; Bates, David O; Morris, Jonathan C

2017-03-17

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

University of Texas Southwestern Medical Center: NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

University of Texas Southwestern Medical Center (UTSW): NSCLC Cell Lines with Loss of SMARCA4 Expression are Hypersensitive to Inhibitors of Aurora Kinase A | Office of Cancer Genomics

Cancer.gov

A genome-wide siRNA screen was employed to identify genes that were selectively toxic for a non-small cell lung cancer (NSCLC) cell line that lacked expression of SMARCA4, but were not toxic in non-cancerous immortalized human bronchial epithelial cells lacking SMARCA4 expression. Among the selectively toxic genes were several mapping to the molecular machinery regulating activity of Aurora kinase A on the mitotic spindle.

Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

NASA Technical Reports Server (NTRS)

Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

2002-01-01

Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

The molecular genetic makeup of acute lymphoblastic leukemia.

PubMed

Mullighan, Charles G

2012-01-01

Genomic profiling has transformed our understanding of the genetic basis of acute lymphoblastic leukemia (ALL). Recent years have seen a shift from microarray analysis and candidate gene sequencing to next-generation sequencing. Together, these approaches have shown that many ALL subtypes are characterized by constellations of structural rearrangements, submicroscopic DNA copy number alterations, and sequence mutations, several of which have clear implications for risk stratification and targeted therapeutic intervention. Mutations in genes regulating lymphoid development are a hallmark of ALL, and alterations of the lymphoid transcription factor gene IKZF1 (IKAROS) are associated with a high risk of treatment failure in B-ALL. Approximately 20% of B-ALL cases harbor genetic alterations that activate kinase signaling that may be amenable to treatment with tyrosine kinase inhibitors, including rearrangements of the cytokine receptor gene CRLF2; rearrangements of ABL1, JAK2, and PDGFRB; and mutations of JAK1 and JAK2. Whole-genome sequencing has also identified novel targets of mutation in aggressive T-lineage ALL, including hematopoietic regulators (ETV6 and RUNX1), tyrosine kinases, and epigenetic regulators. Challenges for the future are to comprehensively identify and experimentally validate all genetic alterations driving leukemogenesis and treatment failure in childhood and adult ALL and to implement genomic profiling into the clinical setting to guide risk stratification and targeted therapy.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.

PubMed

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.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

PubMed Central

Gordon, Sara; Simithy, Johayra; Goodwin, Douglas C; Calderón, Angela I

2015-01-01

Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents. PMID:25861218

Structural, biochemical, and biophysical characterization of idelalisib binding to phosphoinositide 3-kinase δ

DOE PAGES

Somoza, John R.; Koditek, David; Villaseñor, Armando G.; ...

2015-01-28

Idelalisib (also known as GS-1101, CAL-101, IC489666, and Zydelig) is a PI3Kδ inhibitor that has recently been approved for the treatment of several hematological malignancies. Given its use in human diseases, we needed a clear picture of how idelalisib binds to and inhibits PI3Kδ. Here, our data show that idelalisib is a potent and selective inhibitor of the kinase activity of PI3Kδ. A kinetic characterization clearly demonstrated ATP-competitive inhibition, and several additional biochemical and biophysical assays showed that the compound binds reversibly and noncovalently to the kinase. Lastly, a crystal structure of idelalisib bound to the p110δ subunit of PI3Kδmore » furthers our understanding of the binding interactions that confer the potency and selectivity of idelalisib.« less

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

USDA-ARS?s Scientific Manuscript database

Sarcocystis neurona is the most frequent cause of Equine Protozoal Myeloencephalitis (EPM), a debilitating neurologic 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...

Targeting Mitogen-activated Protein Kinase-activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts to Lead Small Molecule Inhibitors to Clinical Trials

PubMed Central

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

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

Discovery of novel Ponatinib analogues for reducing KDR activity as potent FGFRs inhibitors.

PubMed

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.

Mitochondrial ADCK3 employs an atypical protein kinase-like fold to enable coenzyme Q biosynthesis

DOE PAGES

Stefely, Jonathan A.; Reidenbach, Andrew G.; Ulbrich, Arne; ...

2014-12-11

The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. In this paper, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flipsmore » this coenzyme selectivity and enables autophosphorylation but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Finally, our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.« less

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

PubMed

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site prediction tools. In the independent testing, the high sensitivity and specificity of the proposed method demonstrate the predictive effectiveness of the identified substrate motifs and the importance of investigating potential kinases for viral protein phosphorylation sites.

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

PubMed

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

Insights into the conformational switching mechanism of the human vascular endothelial growth factor receptor type 2 kinase domain.

PubMed

Chioccioli, Matteo; Marsili, Simone; Bonaccini, Claudia; Procacci, Piero; Gratteri, Paola

2012-02-27

Human vascular endothelial growth factor receptor type 2 (h-VEFGR2) is a receptor tyrosine kinase involved in the angiogenesis process and regarded as an interesting target for the design of anticancer drugs. Its activation/inactivation mechanism is related to conformational changes in its cytoplasmatic kinase domain, involving first among all the αC-helix in N-lobe and the A-loop in C-lobe. Affinity of inhibitors for the active or inactive kinase form could dictate the open or closed conformation of the A-loop, thus making the different conformations of the kinase domain receptor (KDR) domain different drug targets in drug discovery. In this view, a detailed knowledge of the conformational landscape of KDR domain is of central relevance to rationalize the efficiency and selectivity of kinase inhibitors. Here, molecular dynamics simulations were used to gain insight into the conformational switching activity of the KDR domain and to identify intermediate conformations between the two limiting active and inactive conformations. Specific energy barriers have been selectively removed to induce, and hence highlight at the atomistic level, the regulation mechanism of the A-loop opening. The proposed strategy allowed to repeatedly observe the escape of the KDR domain from the DFG-out free energy basin and to identify rare intermediate conformations between the DFG-out and the DFG-in structures to be employed in a structure-based drug discovery process.

Chemical biology of natural indolocarbazole products: 30 years since the discovery of staurosporine.

PubMed

Nakano, Hirofumi; Omura, Satoshi

2009-01-01

Staurosporine was discovered at the Kitasato Institute in 1977 while screening for microbial alkaloids using chemical detection methods. It was during the same era that protein kinase C was discovered and oncogene v-src was shown to have protein kinase activity. Staurosporine was first isolated from a culture of Actinomyces that originated in a soil sample collected in Mizusawa City, Japan. Thereafter, indolocarbazole compounds have been isolated from a variety of organisms. The biosynthesis of staurosporine and related indolocarbazoles was finally elucidated during the past decade through genetic and biochemical studies. Subsequently, several novel indolocarbazoles have been produced using combinatorial biosynthesis. In 1986, 9 years since its discovery, staurosporine and related indolocarbazoles were shown to be nanomolar inhibitors of protein kinases. They can thus be viewed as forerunners of today's crop of novel anticancer drugs. The finding led many pharmaceutical companies to search for selective protein kinase inhibitors by screening natural products and through chemical synthesis. In the 1990s, imatinib, a Bcr-Abl tyrosine kinase inhibitor, was synthesized and, following human clinical trials for chronic myelogenous leukemia, it was approved for use in the USA in 2001. In 1992, mammalian topoisomerases were shown to be targets for indolocarbazoles. This opened up new possibilities in that indolocarbazole compounds could selectively interact with ATP-binding sites of not only protein kinases but also other proteins that had slight differences in ATP-binding sites. ABCG2, an ATP-binding cassette transporter, was recently identified as an important new target for indolocarbazoles.

Heterometallic titanium–gold complexes inhibit renal cancer cells in vitro and in vivo † †This paper is dedicated to Prof. Roberto Sánchez-Delgado, great mentor and excellent friend, on the occasion of his 65th birthday. ‡ ‡Electronic supplementary information (ESI) available: Stability studies of the new compounds by NMR, UV-vis spectroscopy and MS spectrometry, crystallographic data for compound 6, DFT calculations for compounds 4–7, IC50 values in human renal cells at both 24 and 72 h, details on migration studies, TrxR inhibition studies for 3, 5 and AF at different times, inhibition studies of compound 5 against a panel of 35 protein kinases, effects of AF on MAPKAPK-3 in Caki-1 cells, effects of compound 3 in Caki-1 mouse xenografts. CCDC 1400886. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc01753j Click here for additional data file. Click here for additional data file.

PubMed Central

Fernández-Gallardo, Jacob; Elie, Benelita T.; Sadhukha, Tanmoy; Prabha, Swayam; Sanaú, Mercedes; Rotenberg, Susan A.

2015-01-01

Following recent work on heterometallic titanocene–gold complexes as potential chemotherapeutics for renal cancer, we report here on the synthesis, characterization and stability studies of new titanocene complexes containing a methyl group and a carboxylate ligand (mba = S–C6H4–COO–) bound to gold(i)-phosphane fragments through a thiolate group [(η-C5H5)2TiMe(μ-mba)Au(PR3)]. The compounds are more stable in physiological media than those previously reported and are highly cytotoxic against human cancer renal cell lines. We describe here preliminary mechanistic data involving studies on the interaction of selected compounds with plasmid (pBR322) DNA used as a model nucleic acid, and with selected protein kinases from a panel of 35 protein kinases having oncological interest. Preliminary mechanistic studies in Caki-1 renal cells indicate that the cytotoxic and anti-migration effects of the most active compound 5 [(η-C5H5)2TiMe(μ-mba)Au(PPh3)] involve inhibition of thioredoxin reductase and loss of expression of protein kinases that drive cell migration (AKT, p90-RSK, and MAPKAPK3). The co-localization of both titanium and gold metals (1 : 1 ratio) in Caki-1 renal cells was demonstrated for 5 indicating the robustness of the heterometallic compound in vitro. Two compounds were selected for further in vivo studies on mice based on their selectivity in vitro against renal cancer cell lines when compared to non-tumorigenic human kidney cell lines (HEK-293T and RPTC) and the favourable preliminary toxicity profile in C57BL/6 mice. Evaluation of Caki-1 xenografts in NOD.CB17-Prkdc SCID/J mice showed an impressive tumor reduction (67%) after treatment for 28 days (3 mg per kg per every other day) with heterometallic compound 5 as compared with the previously described [(η-C5H5)2Ti{OC(O)-4-C6H4-P(Ph2)AuCl}2] 3 which was non-inhibitory. These findings indicate that structural modifications on the ligand scaffold affect the in vivo efficacy of this class of compounds. PMID:27213034

CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in oesophageal squamous cell carcinoma.

PubMed

Zhou, Jin; Wu, Zhong; Wong, Gabrielle; Pectasides, Eirini; Nagaraja, Ankur; Stachler, Matthew; Zhang, Haikuo; Chen, Ting; Zhang, Haisheng; Liu, Jie Bin; Xu, Xinsen; Sicinska, Ewa; Sanchez-Vega, Francisco; Rustgi, Anil K; Diehl, J Alan; Wong, Kwok-Kin; Bass, Adam J

2017-01-06

Oesophageal squamous cell carcinoma is a deadly disease where systemic therapy has relied upon empiric chemotherapy despite the presence of genomic alterations pointing to candidate therapeutic targets, including recurrent amplification of the gene encoding receptor tyrosine kinase epidermal growth factor receptor (EGFR). Here, we demonstrate that EGFR-targeting small-molecule inhibitors have efficacy in EGFR-amplified oesophageal squamous cell carcinoma (ESCC), but may become quickly ineffective. Resistance can occur following the emergence of epithelial-mesenchymal transition and by reactivation of the mitogen-activated protein kinase (MAPK) pathway following EGFR blockade. We demonstrate that blockade of this rebound activation with MEK (mitogen-activated protein kinase kinase) inhibition enhances EGFR inhibitor-induced apoptosis and cell cycle arrest, and delays resistance to EGFR monotherapy. Furthermore, genomic profiling shows that cell cycle regulators are altered in the majority of EGFR-amplified tumours and a combination of cyclin-dependent kinase 4/6 (CDK4/6) and EGFR inhibitors prevents the emergence of resistance in vitro and in vivo. These data suggest that upfront combination strategies targeting EGFR amplification, guided by adaptive pathway reactivation or by co-occurring genomic alterations, should be tested clinically.

Fluorophore Labeled Kinase Detects Ligands That Bind within the MAPK Insert of p38α Kinase

PubMed Central

Termathe, Martin; Grütter, Christian; Rabiller, Matthias; van Otterlo, Willem A. L.; Rauh, Daniel

2012-01-01

The vast majority of small molecules known to modulate kinase activity, target the highly conserved ATP-pocket. Consequently, such ligands are often less specific and in case of inhibitors, this leads to the inhibition of multiple kinases. Thus, selective modulation of kinase function remains a major hurdle. One of the next great challenges in kinase research is the identification of ligands which bind to less conserved sites and target the non-catalytic functions of protein kinases. However, approaches that allow for the unambiguous identification of molecules that bind to these less conserved sites are few in number. We have previously reported the use of fluorescent labels in kinases (FLiK) to develop direct kinase binding assays that exclusively detect ligands which stabilize inactive (DFG-out) kinase conformations. Here, we present the successful application of the FLiK approach to develop a high-throughput binding assay capable of directly monitoring ligand binding to a remote site within the MAPK insert of p38α mitogen-activated protein kinase (MAPK). Guided by the crystal structure of an initially identified hit molecule in complex with p38α, we developed a tight binding ligand which may serve as an ideal starting point for further investigations of the biological function of the MAPK insert in regulating the p38α signaling pathway. PMID:22768308

Redox Regulation of Protein Kinases

PubMed Central

Truong, Thu H.; Carroll, Kate S.

2015-01-01

Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin-dependent control of epithelial Na+ absorption.

PubMed

Mansley, Morag K; Wilson, Stuart M

2010-10-01

Insulin-induced Na(+) retention in the distal nephron may contribute to the development of oedema/hypertension in patients with type 2 diabetes. This response to insulin is usually attributed to phosphatidylinositol-3-kinase (PI3K)/serum and glucocorticoid-inducible kinase 1 (SGK1) but a role for protein kinase B (PKB) has been proposed. The present study therefore aimed to clarify the way in which insulin can evoke Na(+) retention. We examined the effects of nominally selective inhibitors of PI3K (wortmannin, PI103, GDC-0941), SGK1 (GSK650394A) and PKB (Akti-1/2) on Na(+) transport in hormone-deprived and insulin-stimulated cortical collecting duct (mpkCCD) cells, while PI3K, SGK1 and PKB activities were assayed by monitoring the phosphorylation of endogenous proteins. Wortmannin substantially inhibited basal Na(+) transport whereas PI103 and GDC-0941 had only very small effects. However, these PI3K inhibitors all abolished insulin-induced Na(+) absorption and inactivated PI3K, SGK1 and PKB fully. GSK650394A and Akti-1/2 also inhibited insulin-evoked Na(+) absorption and while GSK650394A inhibited SGK1 without affecting PKB, Akti-1/2 inactivated both kinases. While studies undertaken using PI103 and GDC-0941 show that hormone-deprived cells can absorb Na(+) independently of PI3K, PI3K seems to be essential for insulin induced Na(+) transport. Akti-1/2 does not act as a selective inhibitor of PKB and data obtained using this compound must therefore be treated with caution. GSK650394A, on the other hand, selectively inhibits SGK1 and the finding that GSK650394A suppressed insulin-induced Na(+) absorption suggests that this response is dependent upon signalling via PI3K/SGK1.

Stimulation of spinal dorsal horn β2-adrenergic receptor ameliorates neuropathic mechanical hypersensitivity through a reduction of phosphorylation of microglial p38 MAP kinase and astrocytic c-jun N-terminal kinase.

PubMed

Zhang, Fang Fang; Morioka, Norimitsu; Abe, Hiromi; Fujii, Shiori; Miyauchi, Kazuki; Nakamura, Yoki; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

2016-12-01

The noradrenaline-adrenergic system has a crucial role in controlling nociceptive transduction at the spinal level. While α-adrenergic receptors are known to regulate nociceptive neurotransmitter release at the spinal presynaptic level, it is not entirely clear whether β-adrenergic receptors are involved in controlling pain transduction at the spinal level as well. The current study elucidated a role of β-adrenergic receptors in neuropathic pain in mice following a partial sciatic nerve ligation (PSNL). In addition, the cellular and intracellular signaling cascade induced by β-adrenergic receptors in neuropathic mice was elaborated. Intrathecal injection of isoproterenol (1 nmol), a nonselective β-adrenergic receptor agonist, briefly ameliorated hind paw mechanical hypersensitivity of PSNL mice. Isoproterenol's antinociceptive effect was mediated through β2-adrenergic receptors since pretreatment with ICI118551, a selective β2-adrenergic receptor antagonist, but not with CGP20712A, a selective β1-adrenergic receptor antagonist, significantly attenuated isoproterenol's effect. Furthermore, intrathecal treatment with a selective β2-adrenergic receptor agonist, terbutaline, but not a selective β1-adrenergic receptor agonist, dobutamine, also significantly ameliorated neuropathic pain. Fourteen days after PSNL, increased phosphorylation of both p38 Mitogen-activated protein kinase (MAPK) in microglia and c-jun N-terminal kinase (JNK) in astrocytes of ipsilateral spinal dorsal horn were observed. Phosphorylation of both microglial p38 MAPK and astrocytic JNK were downregulated by stimulation of the β2-adrenergic receptor. Together, these results suggest that spinal β2-adrenergic receptor have an inhibitory role in neuropathic nociceptive transduction at the spinal level through a downregulation of glial activity, perhaps through modulation of MAP kinases phosphorylation. Thus, targeting of β2-adrenergic receptors could be an effective therapeutic strategy in treating neuropathic pain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Understanding Mechanisms of Resistance in the Epithelial Growth Factor Receptor in Non‐Small Cell Lung Cancer and the Role of Biopsy at Progression

PubMed Central

Villaruz, Liza C.; Ross, Jeffrey

2016-01-01

Abstract Molecular profiling and the discovery of drugs that target specific activating mutations have allowed the personalization of treatment for non‐small cell lung cancer (NSCLC). The epithelial growth factor receptor (EGFR) is frequently over‐expressed and/or aberrantly activated in different cancers, including NSCLC. The most common activating mutations of EGFR in NSCLC fall within the tyrosine kinase‐binding domain. Three oral EGFR tyrosine kinase inhibitors (TKIs) have been approved by the U.S. Food and Drug Administration (FDA) for first‐line use in patients with EGFR mutation‐positive NSCLC (exon 19 deletions or exon 21 [L858R] substitution mutations), as detected by an FDA‐approved test. However, disease progression is common and is often the result of secondary mutations, of which the EGFR T790M mutation is the most prevalent. Few options were available upon progression until the introduction of osimertinib, a kinase inhibitor that targets the T790M mutation, which was recently approved for use in patients with metastatic EGFR T790M mutation‐positive NSCLC, as detected by an FDA‐approved test, who progressed on or after EGFR TKI therapy. With the introduction of osimertinib, outcomes can now be improved in select patients. Therefore, performing a biopsy at progression to determine the underlying molecular cause of the acquired resistance is important for the enabling of individualized options that may provide the greatest opportunity for improved outcomes. This review discusses the latest updates in molecular testing at progression and outlines treatment options for this difficult‐to‐treat population. Implications for Practice. Although the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs)—gefitinib, erlotinib, and afatinib—have changed the treatment paradigm for non‐small cell lung cancer among those with EGFR mutation positive disease, most patients experience progression after approximately 12 months of treatment. Until recently, options were limited for patients who progressed, but improvements in molecular profiling and the approval of osimertinib, which targets the resistance mutation T790M, afford the opportunity for improved outcomes in many patients with this mutation. This article explains the options available after progression on initial EGFR TKI therapy and the importance of molecular testing at progression in making treatment decisions. PMID:27821794

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

PubMed Central

Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

2015-01-01

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; K i<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug. PMID:26398286

Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility.

PubMed

Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B; Murray, Brion W

2015-01-01

Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2-6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.

Molecular assays in detecting EGFR gene aberrations: an updated HER2-dependent algorithm for interpreting gene signals; a short technical report.

PubMed

Tsiambas, Evangelos; Ragos, Vasileios; Lefas, Alicia Y; Georgiannos, Stavros N; Rigopoulos, Dimitrios N; Georgakopoulos, Georgios; Stamatelopoulos, Athanasios; Grapsa, Dimitra; Syrigos, Konstantinos

2016-01-01

Purpose: Among oncogenes that have already been identified and cloned, Epidermal Growth Factor Receptor (EGFR) remains one of the most significant. Understanding its deregulation mechanisms improves critically patients' selection for personalized therapies based on modern molecular biology and oncology guidelines. Anti-EGFR targeted therapeutic strategies have been developed based on specific genetic profiles and applied in subgroups of patients suffering by solid cancers of different histogenetic origin. Detection of specific EGFR somatic mutations leads to tyrosine kinase inhibitors (TKIs) application in subsets of them. Concerning EGFR gene numerical imbalances, identification of pure gene amplification is critical for targeting the molecule via monoclonal antibodies (mAbs). In the current technical paper we demonstrate the main molecular methods applied in EGFR analyses focused also on new data in interpreting numerical imbalances based on ASCO/ACAP guidelines for HER2 in situ hybridization (ISH) clarifications.