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Sample records for allosteric akt inhibitor

  1. Allosteric Small-Molecule Inhibitors of the AKT Kinase

    NASA Astrophysics Data System (ADS)

    Dalafave, D. S.

    This research addresses computational design of small druglike molecules for possible anticancer applications. AKT and SGK are kinases that control important cellular functions. They are highly homologous, having similar activators and targets. Cancers with increased SGK activity may develop resistance to AKT-specific inhibitors. Our goal was to design new molecules that would bind both AKT and SGK, thus preventing the development of drug resistance. Most kinase inhibitors target the kinase ATP-binding site. However, the high similarity in this site among kinases makes it difficult to target specifically. Furthermore, mutations in this site can cause resistance to ATP-competitive kinase inhibitors. We used existing AKT inhibitors as initial templates to design molecules that could potentially bind the allosteric sites of both AKT and SGK. Molecules with no implicit toxicities and optimal drug-like properties were used for docking studies. Binding energies of the stable complexes that the designed molecules formed with AKT and SGK were calculated. Possible applications of the designed putative inhibitors against cancers with overexpressed AKT/SGK is discussed.

  2. Rapid assembly of diverse and potent allosteric Akt inhibitors.

    PubMed

    Wu, Zhicai; Robinson, Ronald G; Fu, Sheng; Barnett, Stanley F; Defeo-Jones, Deborah; Jones, Raymond E; Kral, Astrid M; Huber, Hans E; Kohl, Nancy E; Hartman, George D; Bilodeau, Mark T

    2008-03-15

    This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.

  3. The PI3K/Akt pathway: recent progress in the development of ATP-competitive and allosteric Akt kinase inhibitors.

    PubMed

    Lindsley, Craig W; Barnett, Stanley F; Layton, Mark E; Bilodeau, Mark T

    2008-02-01

    This article describes recent advances in the development and biological evaluation of allosteric and ATP-competitive small molecule inhibitors for the serine/threonine kinase Akt (protein kinase B, PKB). Unregulated activation of the PI3K/Akt/PTEN pathway is a prominent feature of many human cancers and Akt is over-expressed or activated in all major cancers making Akt an exciting new target for cancer therapy. The development of Akt inhibitors has been complicated and hampered by the presence of three Akt isozymes, (Akt1, Akt2 and Akt3) which differ in function and tissue distribution, as well as a lack of Akt specific inhibitors. In the past 18 months, a large number of reports have appeared describing the discovery and development of allosteric Akt kinase inhibitors and classical ATP-competitive Akt kinase inhibitors. This review will discuss the PI3K/Akt/PTEN pathway, allosteric and ATP-competitive Akt kinase inhibitors, their biological evaluation and progress towards target validation.

  4. Targeting AKT1-E17K and the PI3K/AKT Pathway with an Allosteric AKT Inhibitor, ARQ 092

    PubMed Central

    Yu, Yi; Savage, Ronald E.; Eathiraj, Sudharshan; Meade, Justin; Wick, Michael J.; Hall, Terence; Abbadessa, Giovanni; Schwartz, Brian

    2015-01-01

    As a critical component in the PI3K/AKT/mTOR pathway, AKT has become an attractive target for therapeutic intervention. ARQ 092 and a next generation AKT inhibitor, ARQ 751 are selective, allosteric, pan-AKT and AKT1-E17K mutant inhibitors that potently inhibit phosphorylation of AKT. Biochemical and cellular analysis showed that ARQ 092 and ARQ 751 inhibited AKT activation not only by dephosphorylating the membrane-associated active form, but also by preventing the inactive form from localizing into plasma membrane. In endometrial PDX models harboring mutant AKT1-E17K and other tumor models with an activated AKT pathway, both compounds exhibited strong anti-tumor activity. Combination studies conducted in in vivo breast tumor models demonstrated that ARQ 092 enhanced tumor inhibition of a common chemotherapeutic agent (paclitaxel). In a large panel of diverse cancer cell lines, ARQ 092 and ARQ 751 inhibited proliferation across multiple tumor types but were most potent in leukemia, breast, endometrial, and colorectal cancer cell lines. Moreover, inhibition by ARQ 092 and ARQ 751 was more prevalent in cancer cell lines containing PIK3CA/PIK3R1 mutations compared to those with wt-PIK3CA/PIK3R1 or PTEN mutations. For both ARQ 092 and ARQ 751, PIK3CA/PIK3R1 and AKT1-E17K mutations can potentially be used as predictive biomarkers for patient selection in clinical studies. PMID:26469692

  5. Iterative In situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

    PubMed Central

    Millward, Steven W.; Henning, Ryan K.; Kwong, Gabriel A.; Pitram, Suresh; Agnew, Heather D.; Deyle, Kaycie M.; Nag, Arundhati; Hein, Jason; Lee, Su Seong; Lim, Jaehong; Pfeilsticker, Jessica A.; Sharpless, K. Barry; Heath, James R.

    2011-01-01

    We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to pre-inhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties PMID:21962254

  6. Molecular characterization of anastrozole resistance in breast cancer: pivotal role of the Akt/mTOR pathway in the emergence of de novo or acquired resistance and importance of combining the allosteric Akt inhibitor MK-2206 with an aromatase inhibitor.

    PubMed

    Vilquin, Paul; Villedieu, Marie; Grisard, Evelyne; Ben Larbi, Sabrina; Ghayad, Sandra E; Heudel, Pierre-Etienne; Bachelot, Thomas; Corbo, Laura; Treilleux, Isabelle; Vendrell, Julie A; Cohen, Pascale A

    2013-10-01

    Acquisition of resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of estrogen receptor alpha (ERα)-positive breast cancers. The Res-Ana cells, a new model of acquired resistance to anastrozole, were established by long-term exposure of aromatase-overexpressing MCF-7 cells to this drug. These resistant cells developed ER-independent mechanisms of resistance and decreased sensitivity to the AI letrozole or to ERα antagonists. They also displayed a constitutive activation of the PI3K/Akt/mTOR pathway and a deregulated expression of several ErbB receptors. An observed increase in the phospho-Akt/Akt ratio between primary and matched recurrent breast tumors of patients who relapsed under anastrozole adjuvant therapy also argued for a pivotal role of the Akt pathway in acquired resistance to anastrozole. Ectopic overexpression of constitutively active Akt1 in control cells was sufficient to induce de novo resistance to anastrozole. Strikingly, combining anastrozole with the highly selective and allosteric Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings lead to us proposing a model of anastrozole-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to anastrozole and sensitivity to MK-2206. Altogether, our work demonstrated that the Akt/mTOR pathway plays a key role in resistance to anastrozole and that combining anastrozole with Akt/mTOR pathway inhibitors represents a promising strategy in the clinical management of hormone-dependent breast cancer patients.

  7. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

    PubMed

    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

  8. Akt inhibitors reduce glucose uptake independently of their effects on Akt.

    PubMed

    Tan, Shi-Xiong; Ng, Yvonne; James, David E

    2010-11-15

    The protein kinase Akt is involved in various cellular processes, including cell proliferation, growth and metabolism. Hyperactivation of Akt is commonly observed in human tumours and so this pathway has been the focus of targeted drug discovery. However, Akt also plays an essential role in other physiological processes, such as the insulin-regulated transport of glucose into muscle and fat cells. This process, which is essential for whole-body glucose homoeostasis in mammals, is thought to be mediated via Akt-dependent movement of GLUT4 glucose transporters to the plasma membrane. In the present study, we have investigated the metabolic side effects of non-ATP-competitive allosteric Akt inhibitors. In 3T3-L1 adipocytes, these inhibitors caused a decrease in the Akt signalling pathway concomitant with reduced glucose uptake. Surprisingly, a similar reduction in GLUT4 translocation to the plasma membrane was not observed. Further investigation revealed that the inhibitory effects of these compounds on glucose uptake in 3T3-L1 adipocytes were independent of the Akt signalling pathway. The inhibitors also inhibited glucose transport into other cell types, including human erythrocytes and T-47D breast cancer cells, suggesting that these effects are not specific to GLUT4. We conclude that these drugs may, at least in part, inhibit tumorigenesis through inhibition of tumour cell glucose transport.

  9. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities

    PubMed Central

    Hubbard, Paul A.; Moody, Colleen L.; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  10. Allosteric modulation of Ras and the PI3K/AKT/mTOR pathway: emerging therapeutic opportunities.

    PubMed

    Hubbard, Paul A; Moody, Colleen L; Murali, Ramachandran

    2014-01-01

    GTPases and kinases are two predominant signaling modules that regulate cell fate. Dysregulation of Ras, a GTPase, and the three eponymous kinases that form key nodes of the associated phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K)/AKT/mTOR pathway have been implicated in many cancers, including pancreatic cancer, a disease noted for its current lack of effective therapeutics. The K-Ras isoform of Ras is mutated in over 90% of pancreatic ductal adenocarcinomas (PDAC) and there is growing evidence linking aberrant PI3K/AKT/mTOR pathway activity to PDAC. Although these observations suggest that targeting one of these nodes might lead to more effective treatment options for patients with pancreatic and other cancers, the complex regulatory mechanisms and the number of sequence-conserved isoforms of these proteins have been viewed as significant barriers in drug development. Emerging insights into the allosteric regulatory mechanisms of these proteins suggest novel opportunities for development of selective allosteric inhibitors with fragment-based drug discovery (FBDD) helping make significant inroads. The fact that allosteric inhibitors of Ras and AKT are currently in pre-clinical development lends support to this approach. In this article, we will focus on the recent advances and merits of developing allosteric drugs targeting these two inter-related signaling pathways. PMID:25566081

  11. Tumors with AKT1E17K Mutations Are Rational Targets for Single Agent or Combination Therapy with AKT Inhibitors.

    PubMed

    Davies, Barry R; Guan, Nin; Logie, Armelle; Crafter, Claire; Hanson, Lyndsey; Jacobs, Vivien; James, Neil; Dudley, Philippa; Jacques, Kelly; Ladd, Brendon; D'Cruz, Celina M; Zinda, Michael; Lindemann, Justin; Kodaira, Makoto; Tamura, Kenji; Jenkins, Emma L

    2015-11-01

    AKT1(E17K) mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1(E17K) mutation is not known. Expression of exogenous copies of AKT1(E17K) in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1(E17K) mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1(E17K) mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1(E17K) and FGFR3(Y373C) mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1(E17K) mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor.

  12. Aminofurazans as potent inhibitors of AKT kinase

    SciTech Connect

    Rouse, Meagan B.; Seefeld, Mark A.; Leber, Jack D.; McNulty, Kenneth C.; Sun, Lihui; Miller, William H.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Choudhry, Anthony E.; Schaber, Michael D.; Heerding, Dirk A.

    2009-06-24

    AKT inhibitors containing an imidazopyridine aminofurazan scaffold have been optimized. We have previously disclosed identification of the AKT inhibitor GSK690693, which has been evaluated in clinical trials in cancer patients. Herein we describe recent efforts focusing on investigating a distinct region of this scaffold that have afforded compounds (30 and 32) with comparable activity profiles to that of GSK690693.

  13. Context-dependent antagonism between Akt inhibitors and topoisomerase poisons.

    PubMed

    Gálvez-Peralta, Marina; Flatten, Karen S; Loegering, David A; Peterson, Kevin L; Schneider, Paula A; Erlichman, Charles; Kaufmann, Scott H

    2014-05-01

    Signaling through the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, which is aberrantly activated in >50% of carcinomas, inhibits apoptosis and contributes to drug resistance. Accordingly, several Akt inhibitors are currently undergoing preclinical or early clinical testing. To examine the effect of Akt inhibition on the activity of multiple widely used classes of antineoplastic agents, human cancer cell lines were treated with the Akt inhibitor A-443654 [(2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine; ATP-competitive] or MK-2206 (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;dihydrochloride; allosteric inhibitor) or with small interfering RNA (siRNA) targeting phosphoinositide-dependent kinase 1 (PDK1) along with cisplatin, melphalan, camptothecin, or etoposide and assayed for colony formation. Surprisingly different results were observed when Akt inhibitors were combined with different drugs. Synergistic effects were observed in multiple cell lines independent of PI3K pathway status when A-443654 or MK-2206 was combined with the DNA cross-linking agents cisplatin or melphalan. In contrast, effects of the Akt inhibitors in combination with camptothecin or etoposide were more complicated. In HCT116 and DLD1 cells, which harbor activating PI3KCA mutations, A-443654 over a broad concentration range enhanced the effects of camptothecin or etoposide. In contrast, in cell lines lacking activating PI3KCA mutations, partial inhibition of Akt signaling synergized with camptothecin or etoposide, but higher A-443654 or MK-2206 concentrations (>80% inhibition of Akt signaling) or PDK1 siRNA antagonized the topoisomerase poisons by diminishing DNA synthesis, a process that contributes to effective DNA damage and killing by these agents. These results indicate that the effects of combining inhibitors of the PI3K/Akt pathway with certain classes of chemotherapeutic agents might be more

  14. A Novel, Potent, Small Molecule AKT Inhibitor Exhibits Efficacy against Lung Cancer Cells In Vitro

    PubMed Central

    Dinavahi, Saketh S.; Prasanna, Rajagopalan; Dharmarajan, Sriram; Perumal, Yogeeswari; Viswanadha, Srikant

    2015-01-01

    Purpose Anomalies of Akt regulation, including overexpression in lung cancer, impart resistance to conventional chemotherapy and radiation, thereby implicating this kinase as a therapeutic intervention point. A novel scaffold of Akt inhibitors was developed through virtual screening of chemical databases available at Birla Institute of Technology and Science, Pilani, Hyderabad, based on docking studies using Maestro. A benzothienopyrimidine derivative (BIA-6) was identified as a potential lead molecule that inhibited Akt1 enzyme activity with an IC50 of 256 nM. Materials and Methods BIA-6 was tested for in vitro Akt1 inhibition using a fluorescence resonance energy transfer kit. Anti-proliferative activity was tested in NCI-H460, A549, NCI-H1975, and NCI-H2170 cell lines. The effect of the compound on p-Akt (S473) was estimated. Results BIA-6 allosterically caused a dose dependent reduction of growth of cell lines with a half maximal growth inhibition (GI50) range of 0.49 μM to 6.6 μM. Cell cycle analysis indicated that BIA-6 caused a G1 phase arrest at < 100 nM but led to apoptosis at higher doses. BIA-6 also exhibited synergism with standard chemotherapeutic agents. Conclusion BIA-6 is a novel, allosteric Akt inhibitor with potent anti-cancer activity in lung cancer cell lines, that effectively blocks the phosphoinositide-3 kinase/Akt pathway with a high margin selectivity towards normal cells. PMID:25687876

  15. Development of sulfonamide AKT PH domain inhibitors

    PubMed Central

    Ahad, Ali Md.; Zuohe, Song; Du-Cuny, Lei; Moses, Sylvestor A.; Zhou, Li Li; Zhang, Shuxing; Powis, Garth; Meuillet, Emmanuelle J.; Mash, Eugene A.

    2011-01-01

    Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function. PMID:21353784

  16. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review)

    PubMed Central

    NITULESCU, GEORGE MIHAI; MARGINA, DENISA; JUZENAS, PETRAS; PENG, QIAN; OLARU, OCTAVIAN TUDOREL; SALOUSTROS, EMMANOUIL; FENGA, CONCETTINA; SPANDIDOS, DEMETRIOS A.; LIBRA, MASSIMO; TSATSAKIS, ARISTIDIS M.

    2016-01-01

    Targeted cancer therapies are used to inhibit the growth, progression, and metastasis of the tumor by interfering with specific molecular targets and are currently the focus of anticancer drug development. Protein kinase B, also known as Akt, plays a central role in many types of cancer and has been validated as a therapeutic target nearly two decades ago. This review summarizes the intracellular functions of Akt as a pivotal point of converging signaling pathways involved in cell growth, proliferation, apoptotis and neo-angiogenesis, and focuses on the drug design strategies to develop potent anticancer agents targeting Akt. The discovery process of Akt inhibitors has evolved from adenosine triphosphate (ATP)-competitive agents to alternative approaches employing allosteric sites in order to overcome the high degree of structural similarity between Akt isoforms in the catalytic domain, and considerable structural analogy to the AGC kinase family. This process has led to the discovery of inhibitors with greater specificity, reduced side-effects and lower toxicity. A second generation of Akt has inhibitors emerged by incorporating a chemically reactive Michael acceptor template to target the nucleophile cysteines in the catalytic activation loop. The review outlines the development of several promising drug candidates emphasizing the importance of each chemical scaffold. We explore the pipeline of Akt inhibitors and their preclinical and clinical examination status, presenting the potential clinical application of these agents as a monotherapy or in combination with ionizing radiation, other targeted therapies, or chemotherapy. PMID:26698230

  17. Phase 2 Study of MK-2206, an Allosteric Inhibitor of AKT, as Second-Line Therapy for Advanced Gastric and Gastroesophageal Junction Cancer: A SWOG Cooperative Group Trial (S1005)

    PubMed Central

    Ramanathan, Ramesh K.; McDonough, Shannon L.; Kennecke, Hagen F.; Iqbal, Syma; Baranda, Joaquina C.; Seery, Tara E.; Lim, Howard J.; Hezel, Aram F.; Vaccaro, Gina M.; Blanke, Charles D.

    2015-01-01

    BACKGROUND The AKT inhibitor MK-2206 at a dose of 60 mg every other day was evaluated in gastric/gastroesophageal junction cancers. METHODS Patients who had progressed after first-line treatment were eligible. Pertinent eligibility criteria included adequate organ function, a fasting serum glucose level ≤ 150 mg/dL, and less than grade 2 malabsorption or chronic diarrhea. MK-2206 was given orally (60 evaluable patients required). The primary endpoint was overall survival, and a median survival of 6.5 months (power, 89%; significance level, 0.07) was considered encouraging for further investigation. RESULTS Seventy patients were included in the final analyses. The median age was 59.8 years (range, 30.4–86.7 years); 70% were male, 89% were white, and 7% were Asian. There were 2 deaths possibly related to the study drug (cardiac arrest and respiratory failure). Grade 4 adverse events included hyperglycemia, anemia, and lung infection (1 each). Grade 3 adverse events occurred in < 5% of patients except for fatigue (6%). Other adverse events (all grades) included anemia (17%), anorexia (30%), diarrhea (26%), fatigue (50%), hyperglycemia (30%), nausea (40%), vomiting (22%), dry skin (19%), maculopapular rash (30%), and acneiform rash (13%). The response rate was 1%, the median progression-free survival was 1.8 months (95% confidence interval, 1.7–1.8 months), and the median overall survival was 5.1 months (95% confidence interval, 3.7–9.4 months) CONCLUSIONS MK-2206 as second-line therapy was well tolerated by an unselected group of patients with gastric/gastroesophageal junction cancers, but it did not have sufficient activity (response rate, 1%; overall survival, 5.1 months) to warrant further testing in this population. PMID:25827820

  18. An allosteric inhibitor of protein arginine methyltransferase 3.

    PubMed

    Siarheyeva, Alena; Senisterra, Guillermo; Allali-Hassani, Abdellah; Dong, Aiping; Dobrovetsky, Elena; Wasney, Gregory A; Chau, Irene; Marcellus, Richard; Hajian, Taraneh; Liu, Feng; Korboukh, Ilia; Smil, David; Bolshan, Yuri; Min, Jinrong; Wu, Hong; Zeng, Hong; Loppnau, Peter; Poda, Gennadiy; Griffin, Carly; Aman, Ahmed; Brown, Peter J; Jin, Jian; Al-Awar, Rima; Arrowsmith, Cheryl H; Schapira, Matthieu; Vedadi, Masoud

    2012-08-01

    PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

  19. Motexafin gadolinium modulates levels of phosphorylated Akt and synergizes with inhibitors of Akt phosphorylation.

    PubMed

    Ramos, Jason; Sirisawad, Mint; Miller, Richard; Naumovski, Louie

    2006-05-01

    Motexafin gadolinium (MGd, Xcytrin) is a tumor-selective expanded porphyrin that targets oxidative stress-related proteins. MGd treatment of the follicular lymphoma-derived cell line HF-1 resulted in growth suppression and apoptosis whereas MGd treatment of the Burkitt's lymphoma-derived cell line Ramos resulted in growth suppression but not apoptosis. Because phosphorylation status of Akt/protein kinase B is regulated by oxidative stress, we monitored total and phosphorylated Akt (pAkt) in MGd-treated HF-1 and Ramos cells. Levels of pAkt increased within 30 minutes after MGd treatment of HF-1 but after 4 hours began to show a progressive decline to below baseline levels before cells underwent apoptosis. In MGd-treated Ramos cells, pAkt increased approximately 2-fold within 4 hours and remained persistently elevated. Because pAkt activates survival pathways, we determined if MGd-induced cell death could be enhanced by inhibiting phosphorylation of Akt. The addition of specific inhibitors of Akt phosphorylation (Akt inhibitor 1 or SH-5) reduced pAkt levels in MGd-treated HF-1 and Ramos cells and synergistically enhanced MGd-induced cell death. MGd was also evaluated in combination with celecoxib, an inhibitor of Akt phosphorylation, or docetaxel, a microtubule inhibitor that can decrease Akt phosphorylation. The combination of MGd/celecoxib or MGd/docetaxel resulted in decreased Akt phosphorylation and in synergistic cytotoxicity compared with either agent alone. These data point to a potential protective role for pAkt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by combining it with agents that inhibit Akt phosphorylation.

  20. Indole-based allosteric inhibitors of HIV-1 integrase.

    PubMed

    Patel, Pratiq A; Kvaratskhelia, Nina; Mansour, Yara; Antwi, Janet; Feng, Lei; Koneru, Pratibha; Kobe, Mathew J; Jena, Nivedita; Shi, Guqin; Mohamed, Mosaad S; Li, Chenglong; Kessl, Jacques J; Fuchs, James R

    2016-10-01

    Employing a scaffold hopping approach, a series of allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) have been synthesized based on an indole scaffold. These compounds incorporate the key elements utilized in quinoline-based ALLINIs for binding to the IN dimer interface at the principal LEDGF/p75 binding pocket. The most potent of these compounds displayed good activity in the LEDGF/p75 dependent integration assay (IC50=4.5μM) and, as predicted based on the geometry of the five- versus six-membered ring, retained activity against the A128T IN mutant that confers resistance to many quinoline-based ALLINIs. PMID:27568085

  1. A Novel Allosteric Inhibitor of Macrophage Migration Inhibitory Factor (MIF)*

    PubMed Central

    Bai, Fengwei; Asojo, Oluwatoyin A.; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A.; Leng, Lin; Koski, Raymond A.; Powell, Thomas J.; Bucala, Richard; Anthony, Karen G.

    2012-01-01

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases. PMID:22782901

  2. Biomarkers of Response to Akt Inhibitor MK-2206 in Breast Cancer

    PubMed Central

    Sangai, Takafumi; Akcakanat, Argun; Chen, Huiqin; Tarco, Emily; Wu, Yun; Do, Kim-Anh; Miller, Todd W.; Arteaga, Carlos L.; Mills, Gordon B.; Gonzalez-Angulo, Ana Maria; Meric-Bernstam, Funda

    2013-01-01

    Purpose We tested the hypothesis that allosteric Akt inhibitor MK-2206 inhibits tumor growth, and that PTEN/PIK3CA mutations confer MK-2206 sensitivity. Experimental Design MK-2206 effects on cell signaling were assessed in vitro and in vivo. Its antitumor efficacy was assessed in vitro in a panel of cancer cell lines with differing PIK3CA and PTEN status. Its in vivo efficacy was tested as a single agent and in combination with paclitaxel. Results MK-2206 inhibited Akt signaling and cell-cycle progression, and increased apoptosis in a dose-dependent manner in breast cancer cell lines. Cell lines with PTEN or PIK3CA mutations were significantly more sensitive to MK-2206; however, several lines with PTEN/PIK3CA mutations were MK-2206 resistant. siRNA knockdown of PTEN in breast cancer cells increased Akt phosphorylation concordant with increased MK-2206 sensitivity. Stable transfection of PIK3CA E545K or H1047R mutant plasmids into normal-like MCF10A breast cells enhanced MK-2206 sensitivity. Cell lines that were less sensitive to MK-2206 had lower ratios of Akt1/Akt2 and had less growth inhibition with Akt siRNA knockdown. In PTEN-mutant ZR75-1 breast cancer xenografts, MK-2206 treatment inhibited Akt signaling, cell proliferation, and tumor growth. In vitro, MK-2206 showed a synergistic interaction with paclitaxel in MK-2206–sensitive cell lines, and this combination had significantly greater antitumor efficacy than either agent alone in vivo. Conclusions MK-2206 has antitumor activity alone and in combination with chemotherapy. This activity may be greater in tumors with PTEN loss or PIK3CA mutation, providing a strategy for patient enrichment in clinical trials. PMID:22932669

  3. Allosteric Indole Amide Inhibitors of p97: Identification of a Novel Probe of the Ubiquitin Pathway.

    PubMed

    Alverez, Celeste; Bulfer, Stacie L; Chakrasali, Ramappa; Chimenti, Michael S; Deshaies, Raymond J; Green, Neal; Kelly, Mark; LaPorte, Matthew G; Lewis, Taber S; Liang, Mary; Moore, William J; Neitz, R Jeffrey; Peshkov, Vsevolod A; Walters, Michael A; Zhang, Feng; Arkin, Michelle R; Wipf, Peter; Huryn, Donna M

    2016-02-11

    A high-throughput screen to discover inhibitors of p97 ATPase activity identified an indole amide that bound to an allosteric site of the protein. Medicinal chemistry optimization led to improvements in potency and solubility. Indole amide 3 represents a novel uncompetitive inhibitor with excellent physical and pharmaceutical properties that can be used as a starting point for drug discovery efforts. PMID:26985295

  4. Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2

    PubMed Central

    Rastelli, Giulio; Anighoro, Andrew; Chripkova, Martina; Carrassa, Laura; Broggini, Massimo

    2014-01-01

    Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75–1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells. PMID:24911186

  5. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  6. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  7. Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor.

    PubMed

    Dumble, Melissa; Crouthamel, Ming-Chih; Zhang, Shu-Yun; Schaber, Michael; Levy, Dana; Robell, Kimberly; Liu, Qi; Figueroa, David J; Minthorn, Elisabeth A; Seefeld, Mark A; Rouse, Meagan B; Rabindran, Sridhar K; Heerding, Dirk A; Kumar, Rakesh

    2014-01-01

    Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

  8. Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment.

    PubMed

    García-Echeverría, Carlos

    2010-08-01

    Over the past few years a number of components of the PI3K/mTOR pathway have been the subject of intense drug discovery activities both in pharmaceutical companies and in academia. This review article summarizes progress made in the identification and development of allosteric and ATP-competitive kinase inhibitors of mTOR and their potential therapeutic use in oncology.

  9. Titration of signalling output: insights into clinical combinations of MEK and AKT inhibitors

    PubMed Central

    Stewart, A.; Thavasu, P.; de Bono, J. S.; Banerji, U.

    2015-01-01

    Background We aimed to understand the relative contributions of inhibiting MEK and AKT on cell growth to guide combinations of these agents. Materials and methods A panel of 20 cell lines was exposed to either the MEK inhibitor, PD0325901, or AKT inhibitor, AKT 1/2 inhibitor. p-ERK and p-S6 ELISAs were used to define degrees of MEK and AKT inhibition, respectively. Growth inhibition to different degrees of MEK and AKT inhibition, either singly or in combination using 96-h sulphorhodamine assays was then studied. Results A significantly greater growth inhibition was seen in BRAFM and PIK3CAM cells upon maximal MEK (P = 0.004) and AKT inhibition (P = 0.038), respectively. KRASM and BRAF/PIK3CA/KRASWT cells were not significantly more likely to be sensitive to MEK or AKT inhibition. Significant incremental growth inhibition of the combination of MEK + AKT over either MEK or AKT inhibition alone was seen when MEK + AKT was inhibited maximally and not when sub-maximal inhibition of both MEK + AKT was used (11/20 cell lines versus 1/20 cell lines; P = 0.0012). Conclusions KRASM cells are likely to benefit from combinations of MEK and AKT inhibitors. Sub-maximally inhibiting both MEK and AKT within a combination, in a majority of instances, does not significantly increase growth inhibition compared with maximally inhibiting MEK or AKT alone and alternative phase I trial designs are needed to clinically evaluate such combinations. PMID:25908604

  10. Exploiting an Allosteric Binding Site of PRMT3 Yields Potent and Selective Inhibitors

    PubMed Central

    Liu, Feng; Li, Fengling; Ma, Anqi; Dobrovetsky, Elena; Dong, Aiping; Gao, Cen; Korboukh, Ilia; Liu, Jing; Smil, David; Brown, Peter J.; Frye, Stephen V.; Arrowsmith, Cheryl H.; Schapira, Matthieu; Vedadi, Masoud; Jin, Jian

    2015-01-01

    Protein arginine methyltransferases (PRMTs) play an important role in diverse biological processes. Among the nine known human PRMTs, PRMT3 has been implicated in ribosomal biosynthesis via asymmetric dimethylation of the 40S ribosomal protein S2 and in cancer via interaction with the DAL-1 tumor suppressor protein. However, few selective inhibitors of PRMTs have been discovered. We recently disclosed the first selective PRMT3 inhibitor, which occupies a novel allosteric binding site and is noncompetitive with both the peptide substrate and cofactor. Here we report comprehensive structure–activity relationship studies of this series, which resulted in the discovery of multiple PRMT3 inhibitors with submicromolar potencies. An X-ray crystal structure of compound 14u in complex with PRMT3 confirmed that this inhibitor occupied the same allosteric binding site as our initial lead compound. These studies provide the first experimental evidence that potent and selective inhibitors can be created by exploiting the allosteric binding site of PRMT3. PMID:23445220

  11. Detection of allosteric kinase inhibitors by displacement of active site probes.

    PubMed

    Lebakken, Connie S; Reichling, Laurie J; Ellefson, Jason M; Riddle, Steven M

    2012-07-01

    Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

  12. Drug Resistance Resulting from Kinase Dimerization Is Rationalized by Thermodynamic Factors Describing Allosteric Inhibitor Effects.

    PubMed

    Kholodenko, Boris N

    2015-09-22

    Treatment of cancer patients with ATP-competitive inhibitors of BRAF/CRAF kinases surprisingly increases total kinase activity, especially in wild-type BRAF cells, subverting the desired clinical outcome. Similar inhibition resistance is observed for numerous kinases involving homo/heterodimerization in their activation cycles. Here, I demonstrate that drug resistance resulting from kinase dimerization can be explained using thermodynamic principles. I show that allosteric regulation by inhibitors is described by thermodynamic factors that quantify inhibitor-induced changes in kinase dimerization and the difference in the drug affinity for a free monomer versus a dimer harboring one drug molecule. The analysis extends to kinase homo- and heterodimers, allows for their symmetric and asymmetric conformations, and predicts how thermodynamic factors influence dose-response dependencies. I show how two inhibitors, ineffective on their own, when combined can abolish drug resistance at lower doses than either inhibitor applied alone. Thus, the mechanistic models suggest ways to overcome resistance to kinase inhibitors.

  13. Selective small molecule inhibitor of the Mycobacterium tuberculosis fumarate hydratase reveals an allosteric regulatory site

    PubMed Central

    Kasbekar, Monica; Fischer, Gerhard; Mott, Bryan T.; Yasgar, Adam; Hyvönen, Marko; Boshoff, Helena I. M.; Abell, Chris; Barry, Clifton E.; Thomas, Craig J.

    2016-01-01

    Enzymes in essential metabolic pathways are attractive targets for the treatment of bacterial diseases, but in many cases, the presence of homologous human enzymes makes them impractical candidates for drug development. Fumarate hydratase, an essential enzyme in the tricarboxylic acid (TCA) cycle, has been identified as one such potential therapeutic target in tuberculosis. We report the discovery of the first small molecule inhibitor, to our knowledge, of the Mycobacterium tuberculosis fumarate hydratase. A crystal structure at 2.0-Å resolution of the compound in complex with the protein establishes the existence of a previously unidentified allosteric regulatory site. This allosteric site allows for selective inhibition with respect to the homologous human enzyme. We observe a unique binding mode in which two inhibitor molecules interact within the allosteric site, driving significant conformational changes that preclude simultaneous substrate and inhibitor binding. Our results demonstrate the selective inhibition of a highly conserved metabolic enzyme that contains identical active site residues in both the host and the pathogen. PMID:27325754

  14. Rational design of allosteric regulation of homoserine dehydrogenase by a nonnatural inhibitor L-lysine.

    PubMed

    Chen, Zhen; Rappert, Sugima; Zeng, An-Ping

    2015-02-20

    Allosteric proteins, which can sense different signals, are interesting biological parts for synthetic biology. In particular, the design of an artificial allosteric enzyme to sense an unnatural signal is both challenging and highly desired, for example, for a precise and dynamical control of fluxes of growth-essential but byproduct pathways in metabolic engineering of industrial microorganisms. In this work, we used homoserine dehydrogenase (HSDH) of Corynebacterium glutamicum, which is naturally allosterically regulated by threonine and isoleucine, as an example to demonstrate the feasibility of reengineering an allosteric enzyme to respond to an unnatural inhibitor L-lysine. For this purpose, the natural threonine binding sites of HSD were first predicted and verified by mutagenesis experiments. The threonine binding sites were then engineered to a lysine binding pocket. The reengineered HSD only responds to lysine inhibition but not to threonine. This is a significant step toward the construction of artificial molecular circuits for dynamic control of growth-essential byproduct formation pathway for lysine biosynthesis. PMID:24344690

  15. Is There a Future for AKT Inhibitors in the Treatment of Cancer?

    PubMed

    Jansen, Valerie M; Mayer, Ingrid A; Arteaga, Carlos L

    2016-06-01

    An AKT inhibitor plus an antiestrogen exhibited no significant clinical activity in patients with ER(+)/HER2(-) breast cancer despite laboratory studies supporting an antitumor effect for both drugs combined. These results raise concerns about the development of AKT inhibitors in unselected patients whose tumors have unknown dependence on the PI3K/AKT pathway. Clin Cancer Res; 22(11); 2599-601. ©2016 AACRSee related article by Ma et al., p. 2650.

  16. Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

    PubMed

    Kim, Dong Joon; Roh, Eunmiri; Lee, Mee-Hyun; Oi, Naomi; Lim, Do Young; Kim, Myoung Ok; Cho, Yong-Yeon; Pugliese, Angelo; Shim, Jung-Hyun; Chen, Hanyong; Cho, Eun Jin; Kim, Jong-Eun; Kang, Sun Chul; Paul, Souren; Kang, Hee Eun; Jung, Ji Won; Lee, Sung-Young; Kim, Sung-Hyun; Reddy, Kanamata; Yeom, Young Il; Bode, Ann M; Dong, Zigang

    2016-03-01

    Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials. PMID:26676750

  17. Discovery of allosteric BCR-ABL inhibitors from phenotypic screen to clinical candidate.

    PubMed

    Gray, Nathanael S; Fabbro, Doriano

    2014-01-01

    The development of imatinib, an ATP-competitive inhibitor of the BCR-ABL oncoprotein, has revolutionized the treatment of chronic myelogenous leukemia (CML). Unfortunately, the leukemia eventually becomes resistant imatinib as a result of emergence of cells expressing drug insensitive BCR-ABL mutant proteins. This has motivated the development of several next-generation ATP-competitive drugs. This chapter describes the discovery and development of a complementary strategy involving inhibiting BCR-ABL by targeting an allosteric binding site. Compounds that bind to the myristate-binding pocket of BCR-ABL are able to induce formation of an "inactive" state and are able to overcome resistance mutations located in the ATP-binding pocket including the recalcitrant T315I "gatekeeper" mutation. Myristate-pocket inhibitors are also able to function synergistically with ATP-competitive inhibitors in cellular and murine models of CML and this dual inhibitory strategy is currently being investigated in the clinic.

  18. Identification of an Allosteric Small Molecule Inhibitor Selective for Inducible Form of Heat Shock Protein 70

    PubMed Central

    Howe, Matthew K.; Bodoor, Khaldon; Carlson, David A.; Hughes, Philip F.; Alwarawrah, Yazan; Loiselle, David R.; Jaeger, Alex M.; Darr, David B.; Jordan, Jamie L.; Hunter, Lucas M.; Molzberger, Eileen T.; Gobillot, Theodore A.; Thiele, Dennis J.; Brodsky, Jeffrey L.; Spector, Neil L.; Haystead, Timothy A. J.

    2014-01-01

    Summary Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i. PMID:25500222

  19. Reactivation of AKT signaling following treatment of cancer cells with PI3K inhibitors attenuates their antitumor effects

    SciTech Connect

    Dufour, Marc; Dormond-Meuwly, Anne; Pythoud, Catherine; Demartines, Nicolas; Dormond, Olivier

    2013-08-16

    Highlights: •PI3K inhibitors inhibit AKT only transiently. •Re-activation of AKT limits the anti-cancer effect of PI3K inhibitors. •The results suggest to combine PI3K and AKT inhibitors in cancer therapy. -- Abstract: Targeting the phosphatidylinositol-3-kinase (PI3K) is a promising approach in cancer therapy. In particular, PI3K blockade leads to the inhibition of AKT, a major downstream effector responsible for the oncogenic activity of PI3K. However, we report here that small molecule inhibitors of PI3K only transiently block AKT signaling. Indeed, treatment of cancer cells with PI3K inhibitors results in a rapid inhibition of AKT phosphorylation and signaling which is followed by the reactivation of AKT signaling after 48 h as observed by Western blot. Reactivation of AKT signaling occurs despite effective inhibition of PI3K activity by PI3K inhibitors. In addition, wortmannin, a broad range PI3K inhibitor, did not block AKT reactivation suggesting that AKT signals independently of PI3K. In a therapeutical perspective, combining AKT and PI3K inhibitors exhibit stronger anti-proliferative and pro-apoptotic effects compared to AKT or PI3K inhibitors alone. Similarly, in a tumor xenograft mouse model, concomitant PI3K and AKT blockade results in stronger anti-cancer activity compared with either blockade alone. This study shows that PI3K inhibitors only transiently inhibit AKT which limits their antitumor activities. It also provides the proof of concept to combine PI3K inhibitors with AKT inhibitors in cancer therapy.

  20. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  1. Modulation of lysyl oxidase-like 2 enzymatic activity by an allosteric antibody inhibitor.

    PubMed

    Rodriguez, Hector M; Vaysberg, Maria; Mikels, Amanda; McCauley, Scott; Velayo, Arleene C; Garcia, Carlos; Smith, Victoria

    2010-07-01

    In this report, we assessed the steady-state enzymatic activity of lysyl oxidase-like 2 (LOXL2) against the substrates 1,5-diaminopentane (DAP), spermine, and fibrillar type I collagen. We find that both DAP and spermine are capable of activating LOXL2 to the same extent and have similar Michaelis constants (K(m) approximately 1 mm) and catalytic rates (k(cat) approximately 0.02 s(-1)). We also show that LOXL2 is capable of being inhibited by a known suicide inhibitor of lysyl oxidase (LOX), beta-aminopropionitrile, which we find is a potent inhibitor of LOXL2 activity. The modality of inhibition of beta-aminopropionitrile was also examined and found to be competitive with respect to the substrates DAP and spermine. In addition, we identified an antibody inhibitor (AB0023) of LOXL2 enzymatic function and have found that the inhibition occurs in a non-competitive manner with respect to both spermine and DAP. The binding epitope of AB0023 was mapped to the scavenger receptor cysteine-rich domain four of human LOXL2. AB0023 binds to a region remote from the catalytic domain making AB0023 an allosteric inhibitor of LOXL2. This affords AB0023 several advantages, because it is specific for LOXL2 and inhibits the enzymatic function of LOXL2 in a non-competitive manner thereby allowing inhibition of LOXL2 regardless of substrate concentration. These results suggest that antibody allosteric modulators of enzymatic function represent a novel drug development strategy and, in the context of LOXL2, suggest that inhibitors such as these might be useful therapeutics in oncology, fibrosis, and inflammation.

  2. Identification of allosteric ERK2 inhibitors through in silico biased screening and competitive binding assay.

    PubMed

    Kinoshita, Takayoshi; Sugiyama, Hajime; Mori, Yurika; Takahashi, Naruhide; Tomonaga, Atsushi

    2016-02-01

    Extracellular signal-regulated kinase 2 (ERK2) is a drug target for type 2 diabetes mellitus. A peptide-type ERK2 inhibitor (PEP) was discovered in the previous study through the knowledge-based method and showed physiological effects on the db/db mice model of type 2 diabetes. Here, the crystal structure showed that PEP bound to the allosteric site without the interruption of the ATP competitive inhibitor binding to ERK2. An in silico biased-screening using the focused library rendered three compounds with inhibitory activity of IC50 <100 μM. Among them, two compounds revealed the concentration-dependent competition with PEP and could be lead compounds for antidiabetic medicine.

  3. PARP1 inhibitors attenuate AKT phosphorylation via the upregulation of PHLPP1

    SciTech Connect

    Wang, Shuai; Wang, Huibo; Davis, Ben C.; Liang, Jiyong; Cui, Rutao; Chen, Sai-Juan; Xu, Zhi-Xiang

    2011-08-26

    Highlights: {yields} PARP1 inhibitors cause a cytotoxic effect independent of DNA repair impairment. {yields} PARP1 inhibitors attenuated AKT-FOXO3A signaling by activating PHLPP1. {yields} PHLPP1 regulates the sensitivity of cancer cells to PARP1 inhibitors. -- Abstract: Poly(ADP-ribose) polymerase-1 (PARP1) inhibitors are emerging as an important class of drugs for treating BRCA-deficient cancers. Recent discoveries have shown that PARP1 inhibitors may treat other cancer patients in addition to the relatively small proportion of patients carrying BRCA mutations. However, the additional targets by which PARP1 inhibitor-mediated tumor suppression remain poorly understood. In this study, we show that two PARP1 inhibitors, PJ-34 and 3-AB, attenuate AKT phosphorylation at serine 473 (S473) independent of DNA repair impairment. These inhibitors decrease the AKT-associated phosphorylation of FOXO3A, enhance the nuclear retention of FOXO3A, and activate its transcriptional activity. We further demonstrate that treatment with PJ-34 or 3-AB dramatically increases the level of PHLPP1. Overexpression of PHLPP1 enhances the PARP1 inhibitor-induced downregulation of AKT phosphorylation and increases tumor cell death. In contrast, knockdown of PHLPP1 abrogates the PARP1 inhibitor-mediated AKT inhibition and desensitizes cells to its treatment. Therefore, our findings not only show the robust role of PARP1 inhibitors in AKT inhibition but also develop a novel strategy to increase the effectiveness of cancer treatment via PARP1 inhibitor-induced PHLPP1 upregulation.

  4. Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry

    PubMed Central

    McMillan, Kirk; Adler, Marc; Auld, Douglas S.; Baldwin, John J.; Blasko, Eric; Browne, Leslie J.; Chelsky, Daniel; Davey, David; Dolle, Ronald E.; Eagen, Keith A.; Erickson, Shawn; Feldman, Richard I.; Glaser, Charles B.; Mallari, Cornell; Morrissey, Michael M.; Ohlmeyer, Michael H. J.; Pan, Gonghua; Parkinson, John F.; Phillips, Gary B.; Polokoff, Mark A.; Sigal, Nolan H.; Vergona, Ronald; Whitlow, Marc; Young, Tish A.; Devlin, James J.

    2000-01-01

    Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC 1.14.13.39) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC50 values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (Kd ≈ 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor–heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein–protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies. PMID:10677491

  5. NMR reveals the allosteric opening and closing of Abelson tyrosine kinase by ATP-site and myristoyl pocket inhibitors

    PubMed Central

    Skora, Lukasz; Mestan, Jürgen; Fabbro, Doriano; Jahnke, Wolfgang; Grzesiek, Stephan

    2013-01-01

    Successful treatment of chronic myelogenous leukemia is based on inhibitors binding to the ATP site of the deregulated breakpoint cluster region (Bcr)–Abelson tyrosine kinase (Abl) fusion protein. Recently, a new type of allosteric inhibitors targeting the Abl myristoyl pocket was shown in preclinical studies to overcome ATP-site inhibitor resistance arising in some patients. Using NMR and small-angle X-ray scattering, we have analyzed the solution conformations of apo Abelson tyrosine kinase (c-Abl) and c-Abl complexes with ATP-site and allosteric inhibitors. Binding of the ATP-site inhibitor imatinib leads to an unexpected open conformation of the multidomain SH3-SH2-kinase c-Abl core, whose relevance is confirmed by cellular assays on Bcr-Abl. The combination of imatinib with the allosteric inhibitor GNF-5 restores the closed, inactivated state. Our data provide detailed insights on the poorly understood combined effect of the two inhibitor types, which is able to overcome drug resistance. PMID:24191057

  6. Novel Inhibitors Complexed with Glutamate Dehydrogenase: ALLOSTERIC REGULATION BY CONTROL OF PROTEIN DYNAMICS

    SciTech Connect

    Li, Ming; Smith, Christopher J.; Walker, Matthew T.; Smith, Thomas J.

    2009-12-01

    Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate using NAD(P){sup +} as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

  7. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

    NASA Astrophysics Data System (ADS)

    Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

    2013-11-01

    Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

  8. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

  9. Investigating the allosteric reverse signalling of PARP inhibitors with microsecond molecular dynamic simulations and fluorescence anisotropy.

    PubMed

    Marchand, Jean-Rémy; Carotti, Andrea; Passeri, Daniela; Filipponi, Paolo; Liscio, Paride; Camaioni, Emidio; Pellicciari, Roberto; Gioiello, Antimo; Macchiarulo, Antonio

    2014-10-01

    The inhibition of the poly(ADP-ribose) polymerase (PARP) family members is a strategy pursued for the development of novel therapeutic agents in a range of diseases, including stroke, cardiac ischemia, cancer, inflammation and diabetes. Even though some PARP-1 inhibitors have advanced to clinical setting for cancer therapy, a great deal of attention is being devoted to understand the polypharmacology of current PARP inhibitors. Besides blocking the catalytic activity, recent works have shown that some PARP inhibitors exhibit a poisoning activity, by trapping the enzyme at damaged sites of DNA and forming cytotoxic complexes. In this study we have used microsecond molecular dynamics to study the allosteric reverse signalling that is at the basis of such an effect. We show that Olaparib, but not Veliparib and HYDAMTIQ, is able to induce a specific conformational drift of the WGR domain of PARP-1, which stabilizes PARP-1/DNA complex through the locking of several salt bridge interactions. Fluorescence anisotropy assays support such a mechanism, providing the first experimental evidence that HYDAMTIQ, a potent PARP inhibitor with neuroprotective properties, is less potent than Olaparib to trap PARP-1/DNA complex.

  10. Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase

    PubMed Central

    Danley, Dennis E; Baima, Eric T; Mansour, Mahmoud; Fennell, Kimberly F; Chrunyk, Boris A; Mueller, John P; Liu, Shenping; Qiu, Xiayang

    2015-01-01

    Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL−1). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae. PMID:25287857

  11. Discovery and structural characterization of an allosteric inhibitor of bacterial cis-prenyltransferase.

    PubMed

    Danley, Dennis E; Baima, Eric T; Mansour, Mahmoud; Fennell, Kimberly F; Chrunyk, Boris A; Mueller, John P; Liu, Shenping; Qiu, Xiayang

    2015-01-01

    Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL(-1) ). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae. PMID:25287857

  12. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

    PubMed Central

    Chugh, Pauline; Bradel-Tretheway, Birgit; Monteiro-Filho, Carlos MR; Planelles, Vicente; Maggirwar, Sanjay B; Dewhurst, Stephen; Kim, Baek

    2008-01-01

    Background Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs. Results Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production. Conclusion Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs. PMID:18237430

  13. Optimization of allosteric MEK inhibitors. Part 2: Taming the sulfamide group balances compound distribution properties.

    PubMed

    Hartung, Ingo V; Hammer, Stefanie; Hitchcock, Marion; Neuhaus, Roland; Scholz, Arne; Siemeister, Gerhard; Bohlmann, Rolf; Hillig, Roman C; Pühler, Florian

    2016-01-01

    Recently, we had identified an unexplored pocket adjacent to the known binding site of allosteric MEK inhibitors which allowed us to design highly potent and in vivo efficacious novel inhibitors. We now report that our initial preclinical candidate, featuring a phenoxy side chain with a sulfamide capping group, displayed human carbonic anhydrase off-target activity and species-dependent blood cell accumulation, which prevented us from advancing this candidate further. Since this sulfamide MEK inhibitor displayed an exceptionally favorable PK profile with low brain penetration potential despite being highly oral bioavailable, we elected to keep the sulfamide capping group intact while taming its unwanted off-target activity by optimizing the structural surroundings. Introduction of a neighboring fluorine atom or installation of a methylene linker reduced hCA potency sufficiently, at the cost of MEK target potency. Switching to a higher fluorinated central core reinstated high MEK potency, leading to two new preclinical candidates with long half-lives, high bioavailabilities, low brain penetration potential and convincing efficacy in a K-Ras-mutated A549 xenograft model. PMID:26611920

  14. Optimization of allosteric MEK inhibitors. Part 2: Taming the sulfamide group balances compound distribution properties.

    PubMed

    Hartung, Ingo V; Hammer, Stefanie; Hitchcock, Marion; Neuhaus, Roland; Scholz, Arne; Siemeister, Gerhard; Bohlmann, Rolf; Hillig, Roman C; Pühler, Florian

    2016-01-01

    Recently, we had identified an unexplored pocket adjacent to the known binding site of allosteric MEK inhibitors which allowed us to design highly potent and in vivo efficacious novel inhibitors. We now report that our initial preclinical candidate, featuring a phenoxy side chain with a sulfamide capping group, displayed human carbonic anhydrase off-target activity and species-dependent blood cell accumulation, which prevented us from advancing this candidate further. Since this sulfamide MEK inhibitor displayed an exceptionally favorable PK profile with low brain penetration potential despite being highly oral bioavailable, we elected to keep the sulfamide capping group intact while taming its unwanted off-target activity by optimizing the structural surroundings. Introduction of a neighboring fluorine atom or installation of a methylene linker reduced hCA potency sufficiently, at the cost of MEK target potency. Switching to a higher fluorinated central core reinstated high MEK potency, leading to two new preclinical candidates with long half-lives, high bioavailabilities, low brain penetration potential and convincing efficacy in a K-Ras-mutated A549 xenograft model.

  15. Repression of AKT signaling by ARQ 092 in cells and tissues from patients with Proteus syndrome

    PubMed Central

    Lindhurst, Marjorie J.; Yourick, Miranda R.; Yu, Yi; Savage, Ronald E.; Ferrari, Dora; Biesecker, Leslie G.

    2015-01-01

    A somatic activating mutation in AKT1, c.49G>A, pGlu17Lys, that results in elevated AKT signaling in mutation-positive cells, is responsible for the mosaic overgrowth condition, Proteus syndrome. ARQ 092 is an allosteric pan-AKT inhibitor under development for treatment in cancer. We tested the efficacy of this drug for suppressing AKT signaling in cells and tissues from patients with Proteus syndrome. ARQ 092 reduced phosphorylation of AKT and downstream targets of AKT in a concentration-dependent manner in as little as two hours. While AKT signaling was suppressed with ARQ 092 treatment, cells retained their ability to respond to growth factor stimulation by increasing pAKT levels proportionally to untreated cells. At concentrations sufficient to decrease AKT signaling, little reduction in cell viability was seen. These results indicate that ARQ 092 can suppress AKT signaling and warrants further development as a therapeutic option for patients with Proteus syndrome. PMID:26657992

  16. Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase.

    PubMed

    Tso, Shih-Chia; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Qi, Xiangbing; Skvora, Kristen J; Dork, Kenneth; Wallace, Amy L; Morlock, Lorraine K; Lee, Brendan H; Hutson, Susan M; Strom, Stephen C; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

    2014-07-25

    The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 μM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1⁄2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations. PMID:24895126

  17. Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase

    PubMed Central

    Liu, Mengping; Gentles, Robert G.; Ding, Min; Voss, Stacey; Pelosi, Lenore A.; Wang, Ying-Kai; Rigat, Karen L.; Mosure, Kathleen W.; Bender, John A.; Knipe, Jay O.; Colonno, Richard; Meanwell, Nicholas A.; Kadow, John F.; Santone, Kenneth S.; Roberts, Susan B.; Gao, Min

    2014-01-01

    BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing. PMID:24733465

  18. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization.

    PubMed

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation.

  19. Novel Inhibitors of AKT: Assessment of a Different Approach Targeting the Pleckstrin Homology Domain

    PubMed Central

    Meuillet, E.J.

    2014-01-01

    Protein kinase B/AKT plays a central role in cancer. The serine/threonine kinase is overexpressed or constitutively active in many cancers and has been validated as a therapeutic target for cancer treatment. However, targeting the kinase activity has revealed itself to be a challenge due to non-selectivity of the compounds towards other kinases. This review summarizes other approaches scientists have developed to inhibit the activity and function of AKT. They consist of targeting the pleckstrin homology (PH) domain of AKT. Indeed, upon the generation of 3-phosphorylated phosphatidylinositol phosphates (PI3Ps) by PI3-kinase (PI3K), AKT translocates from the cytosol to the plasma membrane and binds to the PI3Ps via its PH domain. Thus, several analogs of PI3Ps (PI Analogs or PIAs), alkylphospholipids (APLs), such as edelfosine or inositol phosphates (IPs) have been described to inhibit the binding of the PH domain to PI3Ps. Recent allostetic inhibitors and small molecules that do not bind the kinase domain but affect the kinase activity of AKT, presumably by interacting with the PH domain, have been also identified. Finally, several drug screening studies spawned novel chemical scaffolds that bind the PH domain of AKT. Together, these approaches have been more or less sucessful in vitro and to some extent translated in preclinical studies. Several of these new AKT PH domain inhibitors exhibit promising anti-tumor activity in mouse models and some of them show synergy with ionizing radiation and chemotherapy. Early clinical trials have started and results will attest to the validity and efficacy of such approaches in the near future. PMID:21649580

  20. Structure of a small-molecule inhibitor complexed with GlmU from Haemophilus influenzae reveals an allosteric binding site

    SciTech Connect

    Mochalkin, Igor; Lightle, Sandra; Narasimhan, Lakshmi; Bornemeier, Dirk; Melnick, Michael; VanderRoest, Steven; McDowell, Laura

    2008-04-02

    N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU) is an essential enzyme in aminosugars metabolism and an attractive target for antibiotic drug discovery. GlmU catalyzes the formation of uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc), an important precursor in the peptidoglycan and lipopolisaccharide biosynthesis in both Gram-negative and Gram-positive bacteria. Here we disclose a 1.9 {angstrom} resolution crystal structure of a synthetic small-molecule inhibitor of GlmU from Haemophilus influenzae (hiGlmU). The compound was identified through a high-throughput screening (HTS) configured to detect inhibitors that target the uridyltransferase active site of hiGlmU. The original HTS hit exhibited a modest micromolar potency (IC{sub 50} - 18 {mu}M in a racemic mixture) against hiGlmU and no activity against Staphylococcus aureus GlmU (saGlmU). The determined crystal structure indicated that the inhibitor occupies an allosteric site adjacent to the GlcNAc-1-P substrate-binding region. Analysis of the mechanistic model of the uridyltransferase reaction suggests that the binding of this allosteric inhibitor prevents structural rearrangements that are required for the enzymatic reaction, thus providing a basis for structure-guided design of a new class of mechanism-based inhibitors of GlmU.

  1. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling

    PubMed Central

    Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Yokokawa, Fumiaki

    2016-01-01

    Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a “de novo” initiation mechanism. Crystal structures of the flavivirus RdRp revealed a “closed” conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the “GDD” active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed “N pocket”). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1–2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses. PMID:27500641

  2. Potent Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and Resistance Profiling.

    PubMed

    Lim, Siew Pheng; Noble, Christian Guy; Seh, Cheah Chen; Soh, Tingjin Sherryl; El Sahili, Abbas; Chan, Grace Kar Yarn; Lescar, Julien; Arora, Rishi; Benson, Timothy; Nilar, Shahul; Manjunatha, Ujjini; Wan, Kah Fei; Dong, Hongping; Xie, Xuping; Shi, Pei-Yong; Yokokawa, Fumiaki

    2016-08-01

    Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses. PMID:27500641

  3. Exploitation of the ability of γ-tocopherol to facilitate membrane co-localization of Akt and PHLPP1 to develop PHLPP1-targeted Akt inhibitors.

    PubMed

    Yan, Ribai; Chuang, Hsiao-Ching; Kapuriya, Naval; Chou, Chih-Chien; Lai, Po-Ting; Chang, Hsin-Wen; Yang, Chia-Ning; Kulp, Samuel K; Chen, Ching-Shih

    2015-03-12

    Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.

  4. Insight into the structural mechanism for PKBα allosteric inhibition by molecular dynamics simulations and free energy calculations.

    PubMed

    Chen, Shi-Feng; Cao, Yang; Han, Shuang; Chen, Jian-Zhong

    2014-03-01

    Protein kinase B (PKB/Akt) is an attractive target for the treatment of tumor. Unlike PKB's ATP-competitive inhibitors, its allosteric inhibitors can maintain PKB's inactive state via its binding in a pocket between PH domain and kinase domain, which specifically inhibit PKB by preventing the phosphorylations of Thr308 and Ser473. In the present studies, MD simulations were performed on three allosteric inhibitors with different inhibitory potencies (IC50) to investigate the interaction modes between the inhibitors and PKBα. MM/GB(PB)SA were further applied to calculate the binding free energies of these inhibitors binding to PKBα. The computed binding free energies were consistent with the ranking of their experimental bioactivities. The key residues of PKBα interacting with the allosteric inhibitor were further discussed by analyzing the different interaction modes of these three inhibitors binding to PKBα and by calculating binding free energy contributions of corresponding residues around the binding pocket. The structural requirements were then summarized for the allosteric inhibitor binding to PKBα. A possible structural mechanism of PKBα inhibition induced by the binding of allosteric inhibitor was formulated. The current studies indicate that there should be an optimum balance between the van der Waals and total electrostatic interactions for further designing of PKBα allosteric inhibitors. PMID:24374242

  5. Synthesis and SAR studies of dual AKT/NF-κB inhibitors against melanoma.

    PubMed

    Barile, Elisa; De, Surya K; Feng, Yongmei; Chen, Vida; Yang, Li; Ronai, Ze'ev; Pellecchia, Maurizio

    2013-11-01

    The protein Kinase B alpha (AKT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways are central regulators of cellular signaling events at the basis of tumor development and progression. Both pathways are often up-regulated in different tumor types including melanoma. We recently reported the identification of compound 1 (BI-69A11) as inhibitor of the AKT and the NF-κB pathways. Here, we describe SAR studies that led to novel fluorinated derivatives with increased cellular potency, reflected in efficient inhibition of AKT and IKKs. Selected compounds demonstrated effective toxicity on melanoma, breast, and prostate cell lines. Finally, a representative derivative showed promising efficacy in an in vivo melanoma xenograft model. PMID:23790042

  6. Akt inhibitor MK2206 prevents influenza pH1N1 virus infection in vitro.

    PubMed

    Denisova, Oxana V; Söderholm, Sandra; Virtanen, Salla; Von Schantz, Carina; Bychkov, Dmitrii; Vashchinkina, Elena; Desloovere, Jens; Tynell, Janne; Ikonen, Niina; Theisen, Linda L; Nyman, Tuula A; Matikainen, Sampsa; Kallioniemi, Olli; Julkunen, Ilkka; Muller, Claude P; Saelens, Xavier; Verkhusha, Vladislav V; Kainov, Denis E

    2014-07-01

    The influenza pH1N1 virus caused a global flu pandemic in 2009 and continues manifestation as a seasonal virus. Better understanding of the virus-host cell interaction could result in development of better prevention and treatment options. Here we show that the Akt inhibitor MK2206 blocks influenza pH1N1 virus infection in vitro. In particular, at noncytotoxic concentrations, MK2206 alters Akt signaling and inhibits endocytic uptake of the virus. Interestingly, MK2206 is unable to inhibit H3N2, H7N9, and H5N1 viruses, indicating that pH1N1 evolved specific requirements for efficient infection. Thus, Akt signaling could be exploited further for development of better therapeutics against pH1N1 virus. PMID:24752266

  7. Allosteric Inhibitors at the Heterodimer Interface of Imidazole Glycerol Phosphate Synthase

    NASA Astrophysics Data System (ADS)

    Snoeberger, Ning-Shiuan Nicole

    Imidazole glycerol phosphate synthase (IGPS) from Thermotoga maritima is a heterodimeric enzyme composed of the HisH and HisF proteins. It is attractive as a pathological target since it is absent in mammals but found in plant and opportunistic human pathogens. IGPS was experimentally determined to be a V-type allosteric enzyme that is involved in an essential biosynthetic pathway of microorganisms. The enzyme catalyzes the hydrolysis of glutamine to form NH3 in the HisH protein, followed by cyclization of NH3 with N'-[(5'-phosphoribulosyl)imino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) in the HisF subunit, forming imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamide ribotide (AICAR) that enter the histidine and purine biosynthetic pathways. Allosteric motions induced upon the binding of the effector PRFAR to HisF propagate through the non-covalent HisH/HisF interface and synchronize catalytic activity at the two distant active sites. However, the nature of the allosteric pathway and the feasibility of manipulating signal transduction by using allosteric drug-like molecules remain to be established. Molecular docking studies of commercial drugs at the HisH/HisF interface were used to identify stable candidates with a potential allosteric effect on the reaction mechanism. Molecular dynamic simulations and calculations of NMR chemical shifts were combined to elucidate the allosteric pathway of IGPS.

  8. A novel PKB/Akt inhibitor, MK-2206, effectively inhibits insulin-stimulated glucose metabolism and protein synthesis in isolated rat skeletal muscle.

    PubMed

    Lai, Yu-Chiang; Liu, Yang; Jacobs, Roxane; Rider, Mark H

    2012-10-01

    PKB (protein kinase B), also known as Akt, is a key component of insulin signalling. Defects in PKB activation lead to insulin resistance and metabolic disorders, whereas PKB overactivation has been linked to tumour growth. Small-molecule PKB inhibitors have thus been developed for cancer treatment, but also represent useful tools to probe the roles of PKB in insulin action. In the present study, we examined the acute effects of two allosteric PKB inhibitors, MK-2206 and Akti 1/2 (Akti) on PKB signalling in incubated rat soleus muscles. We also assessed the effects of the compounds on insulin-stimulated glucose uptake, glycogen and protein synthesis. MK-2206 dose-dependently inhibited insulin-stimulated PKB phosphorylation, PKBβ activity and phosphorylation of PKB downstream targets (including glycogen synthase kinase-3α/β, proline-rich Akt substrate of 40 kDa and Akt substrate of 160 kDa). Insulin-stimulated glucose uptake, glycogen synthesis and glycogen synthase activity were also decreased by MK-2206 in a dose-dependent manner. Incubation with high doses of MK-2206 (10 μM) inhibited insulin-induced p70 ribosomal protein S6 kinase and 4E-BP1 (eukaryotic initiation factor 4E-binding protein-1) phosphorylation associated with increased eEF2 (eukaryotic elongation factor 2) phosphorylation. In contrast, Akti only modestly inhibited insulin-induced PKB and mTOR (mammalian target of rapamycin) signalling, with little or no effect on glucose uptake and protein synthesis. MK-2206, rather than Akti, would thus be the tool of choice for studying the role of PKB in insulin action in skeletal muscle. The results point to a key role for PKB in mediating insulin-stimulated glucose uptake, glycogen synthesis and protein synthesis in skeletal muscle.

  9. Novel Kinase Inhibitors Targeting the PH Domain of AKT for Preventing and Treating Cancer | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute's Medical Oncology Branch is seeking statements of capability or interest from parties interested in licensing and co-development collaborative research to further develop, evaluate, or commercialize novel kinase inhibitors targeting the PH domain of AKT.

  10. Identification of an allosteric small-molecule inhibitor selective for the inducible form of heat shock protein 70.

    PubMed

    Howe, Matthew K; Bodoor, Khaldon; Carlson, David A; Hughes, Philip F; Alwarawrah, Yazan; Loiselle, David R; Jaeger, Alex M; Darr, David B; Jordan, Jamie L; Hunter, Lucas M; Molzberger, Eileen T; Gobillot, Theodore A; Thiele, Dennis J; Brodsky, Jeffrey L; Spector, Neil L; Haystead, Timothy A J

    2014-12-18

    Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i.

  11. Novel Cancer Chemotherapy Hits by Molecular Topology: Dual Akt and Beta-Catenin Inhibitors

    PubMed Central

    Morell, Cecilia; Rodríguez-Henche, Nieves; Recio-Iglesias, Maria Carmen; Garcia-Domenech, Ramon

    2015-01-01

    Background and Purpose Colorectal and prostate cancers are two of the most common types and cause of a high rate of deaths worldwide. Therefore, any strategy to stop or at least slacken the development and progression of malignant cells is an important therapeutic choice. The aim of the present work is the identification of novel cancer chemotherapy agents. Nowadays, many different drug discovery approaches are available, but this paper focuses on Molecular Topology, which has already demonstrated its extraordinary efficacy in this field, particularly in the identification of new hit and lead compounds against cancer. This methodology uses the graph theoretical formalism to numerically characterize molecular structures through the so called topological indices. Once obtained a specific framework, it allows the construction of complex mathematical models that can be used to predict physical, chemical or biological properties of compounds. In addition, Molecular Topology is highly efficient in selecting and designing new hit and lead drugs. According to the aforementioned, Molecular Topology has been applied here for the construction of specific Akt/mTOR and β-catenin inhibition mathematical models in order to identify and select novel antitumor agents. Experimental Approach Based on the results obtained by the selected mathematical models, six novel potential inhibitors of the Akt/mTOR and β-catenin pathways were identified. These compounds were then tested in vitro to confirm their biological activity. Conclusion and Implications Five of the selected compounds, CAS n° 256378-54-8 (Inhibitor n°1), 663203-38-1 (Inhibitor n°2), 247079-73-8 (Inhibitor n°3), 689769-86-6 (Inhibitor n°4) and 431925-096 (Inhibitor n°6) gave positive responses and resulted to be active for Akt/mTOR and/or β-catenin inhibition. This study confirms once again the Molecular Topology’s reliability and efficacy to find out novel drugs in the field of cancer. PMID:25910265

  12. Effects of AKT inhibitor therapy in response and resistance to BRAF inhibition in melanoma

    PubMed Central

    2014-01-01

    Background The clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro. Methods Anti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures. Results More than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 < 1.5 μM). The combination of dabrafenib and AKTi synergistically potentiated growth inhibition in the majority of cell lines with IC50 > 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line. Conclusions AKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations. PMID:24735930

  13. Preclinical pharmacology, antitumor activity and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930

    PubMed Central

    Yap, Timothy A.; Walton, Mike I.; Hunter, Lisa-Jane K.; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D.; Ruddle, Ruth; Heaton, Simon P.; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J.; Thompson, Neil T.; Aherne, Wynne; Raynaud, Florence I.; Eccles, Suzanne A.; Workman, Paul; Collins, Ian; Garrett, Michelle D.

    2016-01-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G1 arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective and potent AKT inhibitor, which blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being employed in clinical trials. PMID:21191045

  14. Preclinical pharmacology, antitumor activity, and development of pharmacodynamic markers for the novel, potent AKT inhibitor CCT128930.

    PubMed

    Yap, Timothy A; Walton, Mike I; Hunter, Lisa-Jane K; Valenti, Melanie; de Haven Brandon, Alexis; Eve, Paul D; Ruddle, Ruth; Heaton, Simon P; Henley, Alan; Pickard, Lisa; Vijayaraghavan, Gowri; Caldwell, John J; Thompson, Neil T; Aherne, Wynne; Raynaud, Florence I; Eccles, Suzanne A; Workman, Paul; Collins, Ian; Garrett, Michelle D

    2011-02-01

    AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials. PMID:21191045

  15. Recent Syntheses of PI3K/Akt/mTOR Signaling Pathway Inhibitors

    PubMed Central

    2013-01-01

    This review focuses on the syntheses of PI3K/Akt/mTOR inhibitors that have been reported outside of the patent literature in the last 5 years but is largely centered on synthetic work reported in 2011 and 2012. While focused on syntheses of inhibitors, some information on in vitro and in vivo testing of compounds is also included. Many of these reported compounds are reversible, competitive adenosine triphosphate (ATP) binding inhibitors, so given the structural similarities of many of these compounds to the adenine core, this review presents recent work on inhibitors based on where the synthetic chemistry was started, i.e. inhibitor syntheses which started with purines/pyrimidines are followed by inhibitor syntheses which began with pyridines, pyrazines, azoles, and triazines then moves to inhibitors which bear no structural resemblance to adenine: liphagal, wortmannin and quercetin analogs. The review then finishes with a short section on recent syntheses of phosphotidyl inositol (PI) analogs since competitive PI binding inhibitors represent an alternative to the competitive ATP binding inhibitors which have received the most attention. PMID:23735831

  16. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

    PubMed Central

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin A.; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

  17. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    PubMed

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  18. Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket.

    PubMed

    Ganesan, Ramya; Mahankali, Madhu; Alter, Gerald; Gomez-Cambronero, Julian

    2015-03-01

    Phospholipase D (PLD) has been implicated in many physiological functions, such as chemotaxis and phagocytosis, as well as pathological functions, such as cancer cell invasion and metastasis. New inhibitors have been described that hamper the role of PLD in those pathologies but their site of action is not known. We have characterized the biochemical and biological behavior of the PLD1/2 dual inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), and the specific PLD2 inhibitor, N-[2-[1-(3-Fluorophenyl)-4-oxo-1,3,-8-triazaspiro[4.5]dec-8-yl]ethyl]-2-naphthalenecarboxamide (NFOT), and found that both FIPI and NFOT are mixed-kinetics inhibitors. Mutagenesis studies indicate that FIPI binds at S757 of PLD2, which is within the HKD2 catalytic site of the enzyme, whereas NFOT binds to PLD2 at two different sites, one being at S757/S648 and another to an allosteric site that is a natural site occupied by PIP2 (R210/R212). This latter site, along with F244/L245/L246, forms a hydrophobic pocket in the PH domain. The mechanism of action of FIPI is a direct effect on the catalytic site (and as such inhibits both PLD1 and PLD2 isoforms), whereas PLD2 affects both the catalytic site (orthosteric) and blocks PIP2 binding to PLD2 (allosteric), which negates the natural enhancing role of PIP2. Moreover, NFOT prevents cell invasion of cancer cells, which does not occur in cells overexpressing PLD2-F244A/L245A/L246A, or PLD2-R210A/R212A, or PLD2-S757/S648 mutants. This study provides new specific knowledge of enzyme regulation and mechanisms of activation and inhibition of PLD2 that are necessary to understand its role in cell signaling and to develop new inhibitors for cancer cell invasion and metastasis.

  19. BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model.

    PubMed

    Perna, Daniele; Karreth, Florian A; Rust, Alistair G; Perez-Mancera, Pedro A; Rashid, Mamunur; Iorio, Francesco; Alifrangis, Constantine; Arends, Mark J; Bosenberg, Marcus W; Bollag, Gideon; Tuveson, David A; Adams, David J

    2015-02-10

    BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

  20. BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model.

    PubMed

    Perna, Daniele; Karreth, Florian A; Rust, Alistair G; Perez-Mancera, Pedro A; Rashid, Mamunur; Iorio, Francesco; Alifrangis, Constantine; Arends, Mark J; Bosenberg, Marcus W; Bollag, Gideon; Tuveson, David A; Adams, David J

    2015-02-10

    BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes. PMID:25624498

  1. Lovastatin Inhibits VEGFR and AKT Activation: Synergistic Cytotoxicity in Combination with VEGFR Inhibitors

    PubMed Central

    Addison, Christina L.; Dimitroulakos, Jim

    2010-01-01

    Background In a recent study, we demonstrated the ability of lovastatin, a potent inhibitor of mevalonate synthesis, to inhibit the function of the epidermal growth factor receptor (EGFR). Lovastatin attenuated ligand-induced receptor activation and downstream signaling through the PI3K/AKT pathway. Combining lovastatin with gefitinib, a potent EGFR inhibitor, induced synergistic cytotoxicity in a variety of tumor derived cell lines. The vascular endothelial growth factor receptor (VEGFR) and EGFR share similar activation, internalization and downstream signaling characteristics. Methodology/Principal Findings The VEGFRs, particularly VEGFR-2 (KDR, Flt-1), play important roles in regulating tumor angiogenesis by promoting endothelial cell proliferation, survival and migration. Certain tumors, such as malignant mesothelioma (MM), also express both the VEGF ligand and VEGFRs that act in an autocrine loop to directly stimulate tumor cell growth and survival. In this study, we have shown that lovastatin inhibits ligand-induced VEGFR-2 activation through inhibition of receptor internalization and also inhibits VEGF activation of AKT in human umbilical vein endothelial cells (HUVEC) and H28 MM cells employing immunofluorescence and Western blotting. Combinations of lovastatin and a VEGFR-2 inhibitor showed more robust AKT inhibition than either agent alone in the H28 MM cell line. Furthermore, combining 5 µM lovastatin treatment, a therapeutically relevant dose, with two different VEGFR-2 inhibitors in HUVEC and the H28 and H2052 mesothelioma derived cell lines demonstrated synergistic cytotoxicity as demonstrated by MTT cell viability and flow cytometric analyses. Conclusions/Significance These results highlight a novel mechanism by which lovastatin can regulate VEGFR-2 function and a potential therapeutic approach for MM through combining statins with VEGFR-2 inhibitors. PMID:20838437

  2. Histone deacetylase inhibitor AR42 regulates telomerase activity in human glioma cells via an Akt-dependent mechanism.

    PubMed

    Yang, Ya-Luen; Huang, Po-Hsien; Chiu, Hao-Chieh; Kulp, Samuel K; Chen, Ching-Shih; Kuo, Cheng-Ju; Chen, Huan-Da; Chen, Chang-Shi

    2013-05-24

    Epigenetic regulation via abnormal activation of histone deacetylases (HDACs) is a mechanism that leads to cancer initiation and promotion. Activation of HDACs results in transcriptional upregulation of human telomerase reverse transcriptase (hTERT) and increases telomerase activity during cellular immortalization and tumorigenesis. However, the effects of HDAC inhibitors on the transcription of hTERT vary in different cancer cells. Here, we studied the effects of a novel HDAC inhibitor, AR42, on telomerase activity in a PTEN-null U87MG glioma cell line. AR42 increased hTERT mRNA in U87MG glioma cells, but suppressed total telomerase activity in a dose-dependent manner. Further analyses suggested that AR42 decreases the phosphorylation of hTERT via an Akt-dependent mechanism. Suppression of Akt phosphorylation and telomerase activity was also observed with PI3K inhibitor LY294002 further supporting the hypothesis that Akt signaling is involved in suppression of AR42-induced inhibition of telomerase activity. Finally, ectopic expression of a constitutive active form of Akt restored telomerase activity in AR42-treated cells. Taken together, our results demonstrate that the novel HDAC inhibitor AR42 can suppress telomerase activity by inhibiting Akt-mediated hTERT phosphorylation, indicating that the PI3K/Akt pathway plays an important role in the regulation of telomerase activity in response to this HDAC inhibitor.

  3. Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes.

    PubMed

    Watts, Ciorsdaidh A; Richards, Frances M; Bender, Andreas; Bond, Peter J; Korb, Oliver; Kern, Oliver; Riddick, Michelle; Owen, Paul; Myers, Rebecca M; Raff, Jordan; Gergely, Fanni; Jodrell, Duncan I; Ley, Steven V

    2013-11-21

    Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics.

  4. Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

    PubMed

    Court, John J; Poisson, Carl; Ardzinski, Andrzej; Bilimoria, Darius; Chan, Laval; Chandupatla, Kishan; Chauret, Nathalie; Collier, Philip N; Das, Sanjoy Kumar; Denis, Francois; Dorsch, Warren; Iyer, Ganesh; Lauffer, David; L'Heureux, Lucille; Li, Pan; Luisi, Brian S; Mani, Nagraj; Nanthakumar, Suganthi; Nicolas, Olivier; Rao, B Govinda; Ronkin, Steven; Selliah, Subajini; Shawgo, Rebecca S; Tang, Qing; Waal, Nathan D; Yannopoulos, Constantin G; Green, Jeremy

    2016-07-14

    The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

  5. Hydrogen/Deuterium Exchange Kinetics Demonstrate Long Range Allosteric Effects of Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase.

    PubMed

    Deredge, Daniel; Li, Jiawen; Johnson, Kenneth A; Wintrode, Patrick L

    2016-05-01

    New nonnucleoside analogs are being developed as part of a multi-drug regimen to treat hepatitis C viral infections. Particularly promising are inhibitors that bind to the surface of the thumb domain of the viral RNA-dependent RNA polymerase (NS5B). Numerous crystal structures have been solved showing small molecule non-nucleoside inhibitors bound to the hepatitis C viral polymerase, but these structures alone do not define the mechanism of inhibition. Our prior kinetic analysis showed that nonnucleoside inhibitors binding to thumb site-2 (NNI2) do not block initiation or elongation of RNA synthesis; rather, they block the transition from the initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex. Here we have mapped the effect of three NNI2 inhibitors on the conformational dynamics of the enzyme using hydrogen/deuterium exchange kinetics. All three inhibitors rigidify an extensive allosteric network extending >40 Å from the binding site, thus providing a structural rationale for the observed disruption of the transition from distributive initiation to processive elongation. The two more potent inhibitors also suppress slow cooperative unfolding in the fingers extension-thumb interface and primer grip, which may contribute their stronger inhibition. These results establish that NNI2 inhibitors act through long range allosteric effects, reveal important conformational changes underlying normal polymerase function, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:27006396

  6. Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage

    PubMed Central

    2013-01-01

    Background LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction. Results We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket. Conclusion Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral

  7. Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth.

    PubMed

    Lai, Wei-Ting; Cheng, Kai-Lin; Baruchello, Riccardo; Rondanin, Riccardo; Marchetti, Paolo; Simoni, Daniele; Lee, Ray M; Guh, Jih-Hwa; Hsu, Lih-Ching

    2016-08-01

    We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion.

  8. Hemiasterlin derivative (R)(S)(S)-BF65 and Akt inhibitor MK-2206 synergistically inhibit SKOV3 ovarian cancer cell growth.

    PubMed

    Lai, Wei-Ting; Cheng, Kai-Lin; Baruchello, Riccardo; Rondanin, Riccardo; Marchetti, Paolo; Simoni, Daniele; Lee, Ray M; Guh, Jih-Hwa; Hsu, Lih-Ching

    2016-08-01

    We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion. PMID:27328368

  9. Conversion of UMP, an allosteric inhibitor of carbamyl phosphate synthetase, to an activator by modification of the UMP ribose moiety.

    PubMed

    Boettcher, B; Meister, A

    1981-06-25

    UMP is known to be an allosteric inhibitor of carbamyl phosphate synthetase, whereas IMP activates the enzyme. Surprisingly, dialdehyde UMP (prepared by periodate oxidation of UMP) was found to be a potent activator of the enzyme. Dialdehyde IMP, like IMP, produced activation. The corresponding dialcohol analogs of UMP and IMP (prepared by borohydride reduction of the dialdehyde analogs) had no effect on activity. These nucleotide interactions were further characterized by sedimentation velocity studies and by examination of the effects of inorganic phosphate on enzymatic activity. Although UMP promotes formation of an enzyme dimer, and IMP promotes formation of a tetramer (Powers, S. G., Meister, A., and Haschemeyer, R. H. (1980) J. Biol. Chem. 255, 1554-1558), the dialdehyde analogs of UMP and IMP both promote formation of mixed species. Low levels (less than 10 mM) of inorganic phosphate decrease the extent of activation by IMP, dialdehyde IMP, and dialdehyde UMP, but increase the extent of inhibition by UMP. The marked activation observed with dialdehyde UMP, and other considerations, suggest that the binding sites on the enzyme for IMP and UMP may overlap substantially. The findings also suggest that physiological levels of inorganic phosphate function in the modulation of the allosteric regulation of this enzyme by nucleotides. PMID:7240186

  10. Discovery of an Allosteric Inhibitor Binding Site in 3-Oxo-acyl-ACP Reductase from Pseudomonas aeruginosa

    PubMed Central

    2013-01-01

    3-Oxo-acyl-acyl carrier protein (ACP) reductase (FabG) plays a key role in the bacterial fatty acid synthesis II system in pathogenic microorganisms, which has been recognized as a potential drug target. FabG catalyzes reduction of a 3-oxo-acyl-ACP intermediate during the elongation cycle of fatty acid biosynthesis. Here, we report gene deletion experiments that support the essentiality of this gene in P. aeruginosa and the identification of a number of small molecule FabG inhibitors with IC50 values in the nanomolar to low micromolar range and good physicochemical properties. Structural characterization of 16 FabG-inhibitor complexes by X-ray crystallography revealed that the compounds bind at a novel allosteric site located at the FabG subunit–subunit interface. Inhibitor binding relies primarily on hydrophobic interactions, but specific hydrogen bonds are also observed. Importantly, the binding cavity is formed upon complex formation and therefore would not be recognized by virtual screening approaches. The structure analysis further reveals that the inhibitors act by inducing conformational changes that propagate to the active site, resulting in a displacement of the catalytic triad and the inability to bind NADPH. PMID:24015914

  11. Analogues of the Allosteric Heat Shock Protein 70 (Hsp70) Inhibitor, MKT-077, As Anti-Cancer Agents

    PubMed Central

    2013-01-01

    The rhodacyanine, MKT-077, has antiproliferative activity against cancer cell lines through its ability to inhibit members of the heat shock protein 70 (Hsp70) family of molecular chaperones. However, MKT-077 is rapidly metabolized, which limits its use as either a chemical probe or potential therapeutic. We report the synthesis and characterization of MKT-077 analogues designed for greater stability. The most potent molecules, such as 30 (JG-98), were at least 3-fold more active than MKT-077 against the breast cancer cell lines MDA-MB-231 and MCF-7 (EC50 values of 0.4 ± 0.03 and 0.7 ± 0.2 μM, respectively). The analogues modestly destabilized the chaperone clients, Akt1 and Raf1, and induced apoptosis in these cells. Further, the microsomal half-life of JG-98 was improved at least 7-fold (t1/2 = 37 min) compared to MKT-077 (t1/2 < 5 min). Finally, NMR titration experiments suggested that these analogues bind an allosteric site that is known to accommodate MKT-077. These studies advance MKT-077 analogues as chemical probes for studying Hsp70s roles in cancer. PMID:24312699

  12. Feedback loops blockade potentiates apoptosis induction and antitumor activity of a novel AKT inhibitor DC120 in human liver cancer.

    PubMed

    Yang, F; Deng, R; Qian, X-J; Chang, S-H; Wu, X-Q; Qin, J; Feng, G-K; Ding, K; Zhu, X-F

    2014-01-01

    The serine/threonine kinase AKT is generally accepted as a promising anticancer therapeutic target. However, the relief of feedback inhibition and enhancement of other survival pathways often attenuate the anticancer effects of AKT inhibitors. These compensatory mechanisms are very complicated and remain poorly understood. In the present study, we found a novel 2-pyrimidyl-5-amidothiazole compound, DC120, as an ATP competitive AKT kinase inhibitor that suppressed proliferation and induced apoptosis in liver cancer cells both in vitro and in vivo. DC120 blocked the phosphorylation of downstream molecules in the AKT signal pathway in dose- and time-dependent manners both in vitro and in vivo. However, unexpectedly, DC120 activated mammalian target of rapamycin complex 1 (mTORC1) pathway that was suggested by increased phosphorylation of 70KD ribosomal protein S6 kinase (P70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The activated mTORC1 signal was because of increase of intracellular Ca(2+) via Ca(2+)/calmodulin (CaM)/ signaling to human vacuolar protein sorting 34 (hVps34) upon AKT inhibition. Meanwhile, DC120 attenuated the inhibitory effect of AKT on CRAF by decreasing phosphorylation of CRAF at Ser259 and thus activated the mitogen-activated protein kinase (MAPK) pathway. The activation of the mTORC1 and MAPK pathways by DC120 was not mutually dependent, and the combination of DC120 with mTORC1 inhibitor and/or MEK inhibitor induced significant apoptosis and growth inhibition both in vitro and in vivo. Taken together, the combination of AKT, mTORC1 and/or MEK inhibitors would be a promising therapeutic strategy for liver cancer treatment. PMID:24625973

  13. The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer

    PubMed Central

    Diaz, R; Nguewa, P A; Diaz-Gonzalez, J A; Hamel, E; Gonzalez-Moreno, O; Catena, R; Serrano, D; Redrado, M; Sherris, D; Calvo, A

    2009-01-01

    Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 μM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg−1 P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer. PMID:19240717

  14. Modulation of curcumin-induced Akt phosphorylation and apoptosis by PI3K inhibitor in MCF-7 cells

    SciTech Connect

    Kizhakkayil, Jaleel; Thayyullathil, Faisal; Chathoth, Shahanas; Hago, Abdulkader; Patel, Mahendra; Galadari, Sehamuddin

    2010-04-09

    Curcumin has been shown to induce apoptosis in various malignant cancer cell lines. One mechanism of curcumin-induced apoptosis is through the PI3K/Akt signaling pathway. Akt, also known as protein kinase B (PKB), is a member of the family of phosphatidylinositol 3-OH-kinase regulated Ser/Thr kinases. The active Akt regulates cell survival and proliferation; and inhibits apoptosis. In this study we found that curcumin induces apoptotic cell death in MCF-7 cells, as assessed by MTT assay, DNA ladder formation, PARP cleavage, p53 and Bax induction. At apoptotic inducing concentration, curcumin induces a dramatic Akt phosphorylation, accompanied by an increased phosphorylation of glycogen synthase kinase 3{beta} (GSK3{beta}), which has been considered to be a pro-growth signaling molecule. Combining curcumin with PI3K inhibitor, LY290042, synergizes the apoptotic effect of curcumin. The inhibitor LY290042 was capable of attenuating curcumin-induced Akt phosphorylation and activation of GSK3{beta}. All together, our data suggest that blocking the PI3K/Akt survival pathway sensitizes the curcumin-induced apoptosis in MCF-7 cells.

  15. The Akt inhibitor MK-2206 enhances the cytotoxicity of paclitaxel (Taxol) and cisplatin in ovarian cancer cells.

    PubMed

    Lin, Ying-Hsi; Chen, Bert Yu-Hung; Lai, Wei-Ting; Wu, Shao-Fu; Guh, Jih-Hwa; Cheng, Ann-Lii; Hsu, Lih-Ching

    2015-01-01

    Abnormalities in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway are commonly observed in human cancers and contribute to chemotherapy resistance. Combination therapy, involving the use of molecular targeted agents and traditional cytotoxic drugs, may represent a promising strategy to lower resistance and enhance cytotoxicity. Here, we demonstrate the efficacy of an Akt inhibitor, MK-2206, in increasing the cytotoxic effect of either paclitaxel (Taxol) or cisplatin against the ovarian cancer cell lines SKOV3 (with constitutively active Akt) and ES2 (with inactive Akt). Sequential treatment of Taxol or cisplatin, followed by MK-2206, induced a synergistic inhibition of cell proliferation and effectively promoted cell death, either by inhibiting the phosphorylation of Akt and its downstream effectors 4E-BP1 and p70S6K in SKOV3 cells or by restoring p53 levels, which were downregulated after Taxol or cisplatin treatment, in ES2 cells. Combination treatment also downregulated the pro-survival protein Bcl-2 in both SKOV3 and ES2 cells, which may have contributed to cell death. In addition, we discovered that Taxol/MK-2206 or cisplatin/MK-2206 combination treatment resulted in significant enhancement of intracellular reactive oxygen species (ROS) induced by MK-2206, in both SKOV3 and ES2 cells; however, MK-2206-induced growth inhibition was reversed by a ROS scavenger only in ES2 cells. MK-2206 also suppressed DNA repair, particularly in SKOV3 cells. Taken together, our results demonstrate that the Akt inhibitor MK-2206 enhances the efficacy of cytotoxic agents in both Akt-active and Akt-inactive ovarian cancer cells but through different mechanisms.

  16. Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme Oxygenase (HemO) of Pseudomonas aeruginosa.

    PubMed

    Heinzl, Geoffrey A; Huang, Weiliang; Yu, Wenbo; Giardina, Bennett J; Zhou, Yue; MacKerell, Alexander D; Wilks, Angela; Xue, Fengtian

    2016-07-28

    New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of Pseudomonas aeruginosa, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound 23 showed a binding affinity of 5.7 μM and an MIC50 of 52.3 μg/mL against P. aeruginosa PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound 23 showed an EC50 of 11.3 μM. The compounds showed increased activity against clinical isolates of P. aeruginosa, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS). PMID:27353344

  17. Additive effect by combination of Akt inhibitor, MK-2206, and PDGFR inhibitor, tyrphostin AG 1296, in suppressing anaplastic thyroid carcinoma cell viability and motility

    PubMed Central

    Che, Huan-yong; Guo, Hang-yuan; Si, Xu-wei; You, Qiao-ying; Lou, Wei-ying

    2014-01-01

    The phosphatidylinositol-3-kinase/Akt pathway and receptor tyrosine kinases regulate many tumorigenesis related cellular processes including cell metabolism, cell survival, cell motility, and angiogenesis. Anaplastic thyroid carcinoma (ATC) is a rare type of thyroid cancer with no effective systemic therapy. It has been shown that Akt activation is associated with tumor progression in ATC. Here we observed the additive effect between an Akt inhibitor (MK-2206) and a novel platelet-derived growth factor receptor inhibitor (tyrphostin AG 1296) in ATC therapy. We found an additive effect between MK-2206 and tyrphostin AG 1296 in suppressing ATC cell viability. The combination of MK-2206 and tyrphostin AG 1296 induces additive apoptosis, additive suppression of the Akt signaling pathway, as well as additive inhibition of cell migration and invasion of ATC cells. Furthermore, the combination of MK-2206 and tyrphostin AG 1296 induced additive suppression of ATC tumor growth in vivo. In summary, our studies suggest that the combination of Akt and receptor tyrosine kinase inhibitors may be an efficient therapeutic strategy for ATC treatment, which might shed new light on ATC therapy. PMID:24665203

  18. Higher risk of infections with PI3K-AKT-mTOR pathway inhibitors in patients with advanced solid tumors on Phase I clinical trials

    PubMed Central

    Rafii, Saeed; Roda, Desamparados; Geuna, Elena; Jimenez, Begona; Rihawi, Karim; Capelan, Marta; Yap, Timothy A; Molife, L Rhoda; Kaye, Stanley B; de Bono, Johann S; Banerji, Udai

    2015-01-01

    Novel antitumor therapies against the PI3K-AKT-mTOR pathway are increasingly used to treat cancer, either as single agents or in combination with chemotherapy or other targeted therapies. Although these agents are not known to be myelosuppressive, an increased risk of infection has been reported with rapamycin analogs. However, the risk of infection with new inhibitors of this pathway such as PI3K, AKT, mTORC 1/2 or multi-kinase inhibitors is unknown. Methods In this retrospective case-control study, we determined the incidence of infection in a group of 432 patients who were treated on 15 phase I clinical trials involving PI3K-AKT-mTOR pathway inhibitors (cases) vs a group of 100 patients on 10 phase I clinical trials of single agent non-PI3K-AKT-mTOR pathway inhibitors (controls) which did not involve conventional cytotoxic agents. We also collected data from 42 patients who were treated with phase I trials of combinations of PI3K-AKT-mTOR inhibitors and MEK inhibitors and 24 patients with combinations of PI3K-AKT-mTOR inhibitors and cytotoxic chemotherapies. Results The incidence of all grade infection was significantly higher with all single agent PI3K-AKT-mTOR inhibitors compared to the control group (27% vs 8% respectively, OR: 4.26, 95% CI: 1.9-9.1, p=0.0001). The incidence of grade 3 and 4 infection was also significantly higher with PI3K-AKT-mTOR inhibitors compared to the control group (10.3% vs 3%, OR: 3.74, 95% CI: 1.1-12.4, p=0.02). Also the combination of PI3K-AKT-mTOR inhibitors and chemotherapy was associated with a significantly higher incidence of all grade (OR: 4.79, 95% CI: 2.0-11.2, p=0.0001) and high grade (OR: 2.87, 95% CI: 1.0-7.6, p=0.03) infection when compared with single agent PI3K-AKT-mTOR inhibitors. Conclusion Inhibitors of the PI3K-AKT-mTOR pathway can be associated with a higher risk of infection. Combinations of PI3K-AKT-mTOR inhibitors and cytotoxic chemotherapy significantly increase the risk of infection. This should be taken

  19. Synthesis of new pyrrolo[1,2-a]quinoxaline derivatives as potential inhibitors of Akt kinase.

    PubMed

    Desplat, Vanessa; Geneste, Ambre; Begorre, Marc-Antoine; Fabre, Solene Belisle; Brajot, Stephane; Massip, Stephane; Thiolat, Denis; Mossalayi, Djavad; Jarry, Christian; Guillon, Jean

    2008-10-01

    Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC(50) of 4.5 microM, and 1h that inhibited U937 and MCF7 cell lines with IC(50) of 5 and 8 microM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.

  20. Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

    SciTech Connect

    Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram; Chen, Albert H.; Dolinski, Brian; Xu, Youyuan; Keilhack, Heike; Nguyen, Thi; Wiznerowicz, Maciej; Li, Lixia; Lutterbach, Bart A.; Chi, An; Paweletz, Cloud; Allison, Timothy; Yan, Youwei; Munshi, Sanjeev K.; Klippel, Anke; Kraus, Manfred; Bobkova, Ekaterina V.; Deshmukh, Sujal; Xu, Zangwei; Mueller, Uwe; Szewczak, Alexander A.; Pan, Bo-Sheng; Richon, Victoria; Pollock, Roy; Blume-Jensen, Peter; Northrup, Alan; Andersen, Jannik N.

    2013-11-20

    Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.

  1. Active Glutaminase C Self-assembles into a Supratetrameric Oligomer That Can Be Disrupted by an Allosteric Inhibitor*

    PubMed Central

    Ferreira, Amanda Petrina Scotá; Cassago, Alexandre; Gonçalves, Kaliandra de Almeida; Dias, Marília Meira; Adamoski, Douglas; Ascenção, Carolline Fernanda Rodrigues; Honorato, Rodrigo Vargas; de Oliveira, Juliana Ferreira; Ferreira, Igor Monteze; Fornezari, Camila; Bettini, Jefferson; Oliveira, Paulo Sérgio Lopes; Paes Leme, Adriana Franco; Portugal, Rodrigo Villares; Ambrosio, Andre Luis Berteli; Dias, Sandra Martha Gomes

    2013-01-01

    The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop 321LRFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism. PMID:23935106

  2. Allosteric competitive inhibitors of the glucose-1-phosphate thymidylyltransferase (RmlA) from Pseudomonas aeruginosa.

    PubMed

    Alphey, Magnus S; Pirrie, Lisa; Torrie, Leah S; Boulkeroua, Wassila Abdelli; Gardiner, Mary; Sarkar, Aurijit; Maringer, Marko; Oehlmann, Wulf; Brenk, Ruth; Scherman, Michael S; McNeil, Michael; Rejzek, Martin; Field, Robert A; Singh, Mahavir; Gray, David; Westwood, Nicholas J; Naismith, James H

    2013-02-15

    Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

  3. Substituted tetrahydroquinolines as potent allosteric inhibitors of reverse transcriptase and its key mutants

    SciTech Connect

    Su, Dai-Shi; Lim, John J.; Tinney, Elizabeth; Wan, Bang-Lin; Young, Mary Beth; Anderson, Kenneth D.; Rudd, Deanne; Munshi, Vandna; Bahnck, Carolyn; Felock, Peter J.; Lu, Meiqing; Lai, Ming-Tain; Touch, Sinoeun; Moyer, Gregory; DiStefano, Daniel J.; Flynn, Jessica A.; Liang, Yuexia; Sanchez, Rosa; Prasad, Sridhar; Yan, Youwei; Perlow-Poehnelt, Rebecca; Torrent, Maricel; Miller, Mike; Vacca, Joe P.; Williams, Theresa M.; Anthony, Neville J.; Merck

    2010-09-27

    Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are key elements of multidrug regimens, called HAART (Highly Active Antiretroviral Therapy), that are used to treat HIV-1 infections. Elucidation of the structure-activity relationships of the thiocarbamate moiety of the previous published lead compound 2 provided a series of novel tetrahydroquinoline derivatives as potent inhibitors of HIV-1 RT with nanomolar intrinsic activity on the WT and key mutant enzymes and potent antiviral activity in infected cells. The SAR optimization, mutation profiles, preparation of compounds, and pharmacokinetic profile of compounds are described.

  4. Galantamine, an acetylcholinesterase inhibitor and positive allosteric modulator of nicotinic acetylcholine receptors, attenuates nicotine taking and seeking in rats.

    PubMed

    Hopkins, Thomas J; Rupprecht, Laura E; Hayes, Matthew R; Blendy, Julie A; Schmidt, Heath D

    2012-09-01

    Current smoking cessation pharmacotherapies have limited efficacy in preventing relapse and maintaining abstinence during withdrawal. Galantamine is an acetylcholinesterase inhibitor that also acts as a positive allosteric modulator of nicotinic acetylcholine receptors. Galantamine has recently been shown to reverse nicotine withdrawal-induced cognitive impairments in mice, which suggests that galantamine may function to prevent relapse in human smokers. However, there are no studies examining whether galantamine administration modulates nicotine self-administration and/or reinstatement of nicotine seeking in rodents. The present experiments were designed to determine the effects of galantamine administration on nicotine taking and reinstatement of nicotine-seeking behavior, an animal model of relapse. Moreover, the effects of galantamine on sucrose-maintained responding and sucrose seeking were also examined to determine whether galantamine's effects generalized to other reinforced behaviors. An inverted U-shaped dose-response curve was obtained when animals self-administered different unit doses of nicotine with the highest responding for 0.03 mg/kg per infusion of nicotine. Acute galantamine administration (5.0 mg/kg, i.p.) attenuated nicotine self-administration when animals were maintained on either a fixed-ratio 5 (FR5) or progressive ratio (PR) schedule of reinforcement. Galantamine administration also attenuated the reinstatement of nicotine-seeking behavior. No significant effects of galantamine on sucrose self-administration or sucrose reinstatement were noted. Acetylcholinesterase inhibitors have also been shown to produce nausea and vomiting in humans. However, at doses required to attenuate nicotine self-administration, no effects of galantamine on nausea/malaise as measured by pica were noted. These results indicate that increased extracellular acetylcholine levels and/or nicotinic acetylcholine receptor stimulation is sufficient to attenuate

  5. In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

    PubMed Central

    Moses, Sylvestor A.; Ali, M. Ahad; Zuohe, Song; Du-Cuny, Lei; Zhou, Li Li; Lemos, Robert; Ihle, Nathan; Skillman, A. Geoffrey; Zhang, Shuxing; Mash, Eugene A.; Powis, Garth; Meuillet, Emmanuelle J.

    2010-01-01

    The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl) benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties. PMID:19491272

  6. Wortmannin, PI3K/Akt signaling pathway inhibitor, attenuates thyroid injury associated with severe acute pancreatitis in rats.

    PubMed

    Abliz, Ablikim; Deng, Wenhong; Sun, Rongze; Guo, Wenyi; Zhao, Liang; Wang, Weixing

    2015-01-01

    Increasing evidences suggest that PI3K/AKT pathway plays an important role in the pathogenesis of inflammatory diseases such as acute pancreatitis. However, the exact effect of PI3K/AKT on thyroid injury associated with acute pancreatitis has not been investigated. This study aimed to investigate the protective effects of wortmannin, PI3K/AKT inhibitor, on thyroid injury in a rat model of severe acute pancreatitis (SAP). Sixty male SD rats were randomly divided into four groups: sham operating group (SO), SAP group, wortmannin treatment (WOR) group and drug control (WOR-CON) group. Serum amylase (AMY), lipase (LIP) and thyroid hormone levels were evaluated. The morphological change of thyroid tissue was analyzed under the light and transmission electron microscopy. AKT, P38MAPK and NF-κB expression in the thyroid tissue was evaluated by immunohistochemical staining. Oxidative stress and inflammatory cytokines were detected. Results showed that wortmannin attenuated the following: (1) serum AMY, LIP and thyroid hormone (2) pancreatic and thyroid pathological injuries (3) thyroid MDA, (4) thyroid ultrastructural change, (5) serum TNF-α, IL-6 and IL-1β (6) AKT, MAPKP38 and NF-κB expression in thyroid tissues. These results suggested that wortmannin attenuates thyroid injury in SAP rats, presumably because of its role on prevent ROS generation and inhibits the activation of P38MAPK, NF-κB pathway. Our findings provide new therapeutic targets for thyroid injury associated with SAP. PMID:26823696

  7. Wortmannin, PI3K/Akt signaling pathway inhibitor, attenuates thyroid injury associated with severe acute pancreatitis in rats.

    PubMed

    Abliz, Ablikim; Deng, Wenhong; Sun, Rongze; Guo, Wenyi; Zhao, Liang; Wang, Weixing

    2015-01-01

    Increasing evidences suggest that PI3K/AKT pathway plays an important role in the pathogenesis of inflammatory diseases such as acute pancreatitis. However, the exact effect of PI3K/AKT on thyroid injury associated with acute pancreatitis has not been investigated. This study aimed to investigate the protective effects of wortmannin, PI3K/AKT inhibitor, on thyroid injury in a rat model of severe acute pancreatitis (SAP). Sixty male SD rats were randomly divided into four groups: sham operating group (SO), SAP group, wortmannin treatment (WOR) group and drug control (WOR-CON) group. Serum amylase (AMY), lipase (LIP) and thyroid hormone levels were evaluated. The morphological change of thyroid tissue was analyzed under the light and transmission electron microscopy. AKT, P38MAPK and NF-κB expression in the thyroid tissue was evaluated by immunohistochemical staining. Oxidative stress and inflammatory cytokines were detected. Results showed that wortmannin attenuated the following: (1) serum AMY, LIP and thyroid hormone (2) pancreatic and thyroid pathological injuries (3) thyroid MDA, (4) thyroid ultrastructural change, (5) serum TNF-α, IL-6 and IL-1β (6) AKT, MAPKP38 and NF-κB expression in thyroid tissues. These results suggested that wortmannin attenuates thyroid injury in SAP rats, presumably because of its role on prevent ROS generation and inhibits the activation of P38MAPK, NF-κB pathway. Our findings provide new therapeutic targets for thyroid injury associated with SAP.

  8. Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake.

    PubMed

    Zhang, Peng; Li, Shufen; Gao, Yang; Lu, Wenbo; Huang, Ke; Ye, Deyong; Li, Xi; Chu, Yong

    2014-12-15

    Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC50=8 μM) and BTO-5s (IC50=10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.

  9. Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

    PubMed Central

    Fritsche-Guenther, Raphaela; Witzel, Franziska; Kempa, Stefan; Brummer, Tilman; Sers, Christine; Blüthgen, Nils

    2016-01-01

    Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell. PMID:26799289

  10. Molecular pharmacology and antitumor activity of PHT-427 a novel AKT/PDPK1 pleckstrin homology domain inhibitor

    PubMed Central

    Meuillet, Emmanuelle J.; Zuohe, Song; Lemos, Robert; Ihle, Nathan; Kingston, John; Watkins, Ryan; Moses, Sylvestor A.; Zhang, Shuxing; Cuny, Lei Du; Herbst, Roy; Jacoby, Jörg J.; Zhou, Li Li; Ahad, Ali M.; Mash, Eugene A; Kirkpatrick, D Lynn; Powis, Garth

    2010-01-01

    Phosphatidylinositol 3-kinase (PIK3)/ PtdIns dependent protein kinase-1(PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427 a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogs with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDKP1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice with up to 80% inhibition in the most sensitive tumors and showed greater activity than analogs with C4, C6 or C8 alkyl chains. Inhibition of PDKP1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive and K-Ras mutant tumors the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer, and with erlotinib in NSC lung cancer. When given over 5 days PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. PMID:20197390

  11. Molecular Recognition of the Catalytic Zinc(II) Ion in MMP-13: Structure-Based Evolution of an Allosteric Inhibitor to Dual Binding Mode Inhibitors with Improved Lipophilic Ligand Efficiencies

    PubMed Central

    Fischer, Thomas; Riedl, Rainer

    2016-01-01

    Matrix metalloproteinases (MMPs) are a class of zinc dependent endopeptidases which play a crucial role in a multitude of severe diseases such as cancer and osteoarthritis. We employed MMP-13 as the target enzyme for the structure-based design and synthesis of inhibitors able to recognize the catalytic zinc ion in addition to an allosteric binding site in order to increase the affinity of the ligand. Guided by molecular modeling, we optimized an initial allosteric inhibitor by addition of linker fragments and weak zinc binders for recognition of the catalytic center. Furthermore we improved the lipophilic ligand efficiency (LLE) of the initial inhibitor by adding appropriate zinc binding fragments to lower the clogP values of the inhibitors, while maintaining their potency. All synthesized inhibitors showed elevated affinity compared to the initial hit, also most of the novel inhibitors displayed better LLE. Derivatives with carboxylic acids as the zinc binding fragments turned out to be the most potent inhibitors (compound 3 (ZHAWOC5077): IC50 = 134 nM) whereas acyl sulfonamides showed the best lipophilic ligand efficiencies (compound 18 (ZHAWOC5135): LLE = 2.91). PMID:26938528

  12. Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

    PubMed Central

    Moriconi, Alessio; Cunha, Thiago M.; Souza, Guilherme R.; Lopes, Alexandre H.; Cunha, Fernando Q.; Carneiro, Victor L.; Pinto, Larissa G.; Brandolini, Laura; Aramini, Andrea; Bizzarri, Cinzia; Bianchini, Gianluca; Beccari, Andrea R.; Fanton, Marco; Bruno, Agostino; Costantino, Gabriele; Bertini, Riccardo; Galliera, Emanuela; Locati, Massimo; Ferreira, Sérgio H.; Teixeira, Mauro M.; Allegretti, Marcello

    2014-01-01

    Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR−/− mice compared with WT mice. Furthermore, treatment of C5aR−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain. PMID:25385614

  13. Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound.

    PubMed

    Zhao, Gengxiang; Haskins, Nantaporn; Jin, Zhongmin; M Allewell, Norma; Tuchman, Mendel; Shi, Dashuang

    2013-08-01

    Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in L-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by L-arginine, although L-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by L-arginine, we have determined the structure of the mmNAGS/K complexed with L-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when L-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by L-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism. PMID:23850694

  14. Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line

    PubMed Central

    Kumar, Amit; Abbas, Wasim; Colin, Laurence; Khan, Kashif Aziz; Bouchat, Sophie; Varin, Audrey; Larbi, Anis; Gatot, Jean-Stéphane; Kabeya, Kabamba; Vanhulle, Caroline; Delacourt, Nadège; Pasquereau, Sébastien; Coquard, Laurie; Borch, Alexandra; König, Renate; Clumeck, Nathan; De Wit, Stephane; Rohr, Olivier; Rouzioux, Christine; Fulop, Tamas; Van Lint, Carine; Herbein, Georges

    2016-01-01

    Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine473 and threonine308. In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients. PMID:27076174

  15. DC120, a novel AKT inhibitor, preferentially suppresses nasopharyngeal carcinoma cancer stem-like cells by downregulating Sox2

    PubMed Central

    Tang, Jun; Yang, Fen; Feng, Gong-Kan; Chen, Wen-Dan; Wu, Xiao-Qi; Qian, Xiao-Jun; Ding, Ke; Zhu, Xiao-Feng

    2015-01-01

    Side population (SP) contains cancer stem-like cells (CSLCs). In this study, we characterized SP cells from nasopharyngeal carcinoma (NPC) cell lines and found that SP cells had a higher self-renewal ability in vitro and greater tumorigenicity in vivo. The AKT pathway was activated in NPC SP cells. DC120, a 2-pyrimidyl-5-amidothiazole inhibitor of the ATP binding site of AKT, inhibited phosphorylation of FKHRL1 and GSK-3β. DC120 inhibited SP fraction, the sphere-forming ability in vitro and growth of primary xenografts as well as secondary xenografts’ tumor recurrence. This inhibition was accompanied by reduced expression of stem-related gene Sox2 due to induction of p27 and miR-30a. A combination of DC120 and CDDP more effectively inhibited NPC cells compared with monotherapy in vitro and in vivo. Clinical evaluation of DC120 is warranted. PMID:25749514

  16. Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation.

    PubMed

    Li, Jiawen; Johnson, Kenneth A

    2016-05-01

    Replication of the hepatitis C viral genome is catalyzed by the NS5B (nonstructural protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information. PMID:26851276

  17. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

    PubMed Central

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J.; Stockwell, Simon R.; Stokes, Jamie E.; Tan, Yaw S.; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J.; Hyvönen, Marko; Spring, David R.; McKenzie, Grahame J.; Venkitaraman, Ashok R.

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  18. Allosteric modulation of caspases.

    PubMed

    Häcker, Hans-Georg; Sisay, Mihiret Tekeste; Gütschow, Michael

    2011-11-01

    Caspases are proteolytic enzymes mainly involved in the induction and execution phases of apoptosis. This type of programmed cell death is an essential regulatory process required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis is attributed a key role in many human diseases, including neurodegenerative disorders, ischemic damage, autoimmune diseases and cancer. Allosteric modulation of the function of a protein occurs when the regulatory trigger, such as the binding of a small effector or inhibitor molecule, takes place some distance from the protein's active site. In recent years, several caspases have been identified that possess allosteric sites and binding of small molecule to these sites resulted in the modulation of enzyme activities. Regulation of caspase activity by small molecule allosteric modulators is believed to be of great therapeutic importance. In this review we give brief highlights on recent developments in identifying and characterizing natural and synthetic allosteric inhibitors as well as activators of caspases and discuss their potential in drug discovery and protein engineering. PMID:21807025

  19. Compensatory activation of Akt in response to mTOR and Raf inhibitors - a rationale for dual-targeted therapy approaches in neuroendocrine tumor disease.

    PubMed

    Zitzmann, Kathrin; Rüden, Janina von; Brand, Stephan; Göke, Burkhard; Lichtl, Jennifer; Spöttl, Gerald; Auernhammer, Christoph J

    2010-09-01

    Several studies have established a link between aberrant PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K-Akt-mTOR- and Ras-Raf-MEK-Erk1/2 signaling in NET disease.

  20. Structure-based design, synthesis and biological evaluation of diphenylmethylamine derivatives as novel Akt1 inhibitors.

    PubMed

    Liu, Tao; Zhan, Wenhu; Wang, Yanming; Zhang, Liangren; Yang, Bo; Dong, Xiaowu; Hu, Yongzhou

    2014-02-12

    A series of diphenylmethylamine derivatives were rationally designed, synthesized and biologically evaluated. Most of them exhibited moderate to good Akt1 inhibitory activities, as well as promising anti-proliferative efficacy against cancer cell lines. Besides, molecular docking studies were carried out to probe their binding modes with Akt1. Further kinase selectivity studies of compound 22c were performed, indicating its excellent selectivity against Aurora A, Drak, IKKβ, GSK3β, SYK and JAK2, and moderate selectivity against PKC and BRAF. Finally, a refined pharmacophore model was generated using the most active compounds 2, 12c and 22c via application of HipHop program. PMID:24389511

  1. Structure-based design, synthesis and biological evaluation of diphenylmethylamine derivatives as novel Akt1 inhibitors.

    PubMed

    Liu, Tao; Zhan, Wenhu; Wang, Yanming; Zhang, Liangren; Yang, Bo; Dong, Xiaowu; Hu, Yongzhou

    2014-02-12

    A series of diphenylmethylamine derivatives were rationally designed, synthesized and biologically evaluated. Most of them exhibited moderate to good Akt1 inhibitory activities, as well as promising anti-proliferative efficacy against cancer cell lines. Besides, molecular docking studies were carried out to probe their binding modes with Akt1. Further kinase selectivity studies of compound 22c were performed, indicating its excellent selectivity against Aurora A, Drak, IKKβ, GSK3β, SYK and JAK2, and moderate selectivity against PKC and BRAF. Finally, a refined pharmacophore model was generated using the most active compounds 2, 12c and 22c via application of HipHop program.

  2. The A128T resistance mutation reveals aberrant protein multimerization as the primary mechanism of action of allosteric HIV-1 integrase inhibitors.

    PubMed

    Feng, Lei; Sharma, Amit; Slaughter, Alison; Jena, Nivedita; Koh, Yasuhiro; Shkriabai, Nikolozi; Larue, Ross C; Patel, Pratiq A; Mitsuya, Hiroaki; Kessl, Jacques J; Engelman, Alan; Fuchs, James R; Kvaratskhelia, Mamuka

    2013-05-31

    Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance. PMID:23615903

  3. TALEN Knockout of the PSIP1 Gene in Human Cells: Analyses of HIV-1 Replication and Allosteric Integrase Inhibitor Mechanism

    PubMed Central

    Morrison, James H.; Saenz, Dyana T.; Fuchs, James R.; Kvaratskhelia, Mamuka; Ekker, Stephen C.

    2014-01-01

    ABSTRACT HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T and Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole-gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited, and spreading viral replication was severely impaired in PSIP1−/− Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested that LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1−/− cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions noninfectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75 independent. The block to HIV-1 propagation in PSIP1−/− human CD4+ T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure. IMPORTANCE LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus. Once inside the nucleus, lentiviral preintegration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering

  4. Molecular Motions as a Drug Target: Mechanistic Simulations of Anthrax Toxin Edema Factor Function Led to the Discovery of Novel Allosteric Inhibitors

    PubMed Central

    Laine, Élodie; Martínez, Leandro; Ladant, Daniel; Malliavin, Thérèse; Blondel, Arnaud

    2012-01-01

    Edema Factor (EF) is a component of Bacillus anthracis toxin essential for virulence. Its adenylyl cyclase activity is induced by complexation with the ubiquitous eukaryotic cellular protein, calmodulin (CaM). EF and its complexes with CaM, nucleotides and/or ions, have been extensively characterized by X-ray crystallography. Those structural data allowed molecular simulations analysis of various aspects of EF action mechanism, including the delineation of EF and CaM domains through their association energetics, the impact of calcium binding on CaM, and the role of catalytic site ions. Furthermore, a transition path connecting the free inactive form to the CaM-complexed active form of EF was built to model the activation mechanism in an attempt to define an inhibition strategy. The cavities at the surface of EF were determined for each path intermediate to identify potential sites where the binding of a ligand could block activation. A non-catalytic cavity (allosteric) was found to shrink rapidly at early stages of the path and was chosen to perform virtual screening. Amongst 18 compounds selected in silico and tested in an enzymatic assay, 6 thiophen ureidoacid derivatives formed a new family of EF allosteric inhibitors with IC50 as low as 2 micromolars. PMID:23012649

  5. Activated AKT pathway promotes establishment of endometriosis.

    PubMed

    Kim, Tae Hoon; Yu, Yanni; Luo, Lily; Lydon, John P; Jeong, Jae-Wook; Kim, J Julie

    2014-05-01

    The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice

  6. Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors

    PubMed Central

    Wheler, Jennifer J.; Atkins, Johnique T.; Janku, Filip; Moulder, Stacy L.; Stephens, Philip J.; Yelensky, Roman; Valero, Vicente; Miller, Vincent; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-01-01

    There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway. PMID:27489863

  7. Presence of both alterations in FGFR/FGF and PI3K/AKT/mTOR confer improved outcomes for patients with metastatic breast cancer treated with PI3K/AKT/mTOR inhibitors.

    PubMed

    Wheler, Jennifer J; Atkins, Johnique T; Janku, Filip; Moulder, Stacy L; Stephens, Philip J; Yelensky, Roman; Valero, Vicente; Miller, Vincent; Kurzrock, Razelle; Meric-Bernstam, Funda

    2016-01-01

    There is limited data on co-expression of FGFR/FGR amplifications and PI3K/ AKT/mTOR alterations in breast cancer. Tumors from patients with metastatic breast cancer referred to our Phase I Program were analyzed by next generation sequencing (NGS). Genomic libraries were selected for all exons of 236 (or 182) cancer-related genes sequenced to average depth of >500× in a CLIA laboratory (Foundation Medicine, Cambridge, MA, USA) and analyzed for all classes of genomic alterations. We report genomic profiles of 112 patients with metastatic breast cancer, median age 55 years (range, 27-78). Twenty-four patients (21%) had at least one amplified FGFR or FGF. Fifteen of the 24 patients (63%) also had an alteration in the PI3K/ AKT/mTOR pathway. There was no association between alterations in FGFR/FGF and PI3K/AKT/mTOR (P=0.49). Patients with simultaneous amplification in FGFR/FGF signaling and the PI3K/AKT/mTOR pathway had a higher rate of SD≥6 months/PR/ CR when treated with therapies targeting the PI3K/AKT/mTOR pathway than patients with only alterations in the PI3K/AKT/mTOR pathway (73% vs. 34%; P=0.0376) and remained on treatment longer (6.8 vs. 3.7 months; P=0.053). Higher response rates were seen in patients with simultaneous amplification in FGFR/FGF signaling and alterations in the PI3K/AKT/mTOR pathway who were treated with inhibitors of that pathway. PMID:27489863

  8. mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms.

    PubMed

    Wilson-Edell, Kathleen A; Yevtushenko, Mariya A; Rothschild, Daniel E; Rogers, Aric N; Benz, Christopher C

    2014-04-01

    Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR-/+, HER2-/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.

  9. Matrine induces the apoptosis of lung cancer cells through downregulation of inhibitor of apoptosis proteins and the Akt signaling pathway.

    PubMed

    Niu, Huiyan; Zhang, Yifei; Wu, Baogang; Zhang, Yi; Jiang, Hongfang; He, Ping

    2014-09-01

    Lung cancer is the leading cause of cancer‑related mortality in humans. The prognosis for advanced lung cancer patients is extremely poor. Current standard care is rather ineffective for prolonging patient life while preserving satisfactory quality of life due to adverse side-effects. Matrine extracted from the traditional Chinese herbal plant Sophora flavescens was shown to induce cancer cell death in vitro. The aim of this study was to investigate the effect of matrine on the proliferation and apoptosis of lung cancer cells and the molecular basis of matrine-induced apoptosis. The results showed that matrine inhibited cell proliferation and induced apoptosis in lung cancer A549 and 95D cells in a dose- and time-dependent manner. The apoptotic effects of matrine on lung cancer cells appeared to act via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway and downregulation of the expression of the inhibitor of apoptosis protein (IAP) family proteins. Matrine exerts its cancer-killing effect via promoting apoptosis in lung cancer cells and may be a useful adjuvant therapeutic scheme for treating advanced lung cancer patients.

  10. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

    PubMed Central

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F. J.; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT. PMID:26800261

  11. Combination of PI3K/Akt/mTOR inhibitors and PDT in endothelial and tumor cells

    NASA Astrophysics Data System (ADS)

    Fateye, Babasola; Chen, Bin

    2011-02-01

    The PI3/Akt/mTOR kinase signaling pathway is a major signaling pathway in eukaryotic cells, and dysregulation of this signaling pathway has been implicated in tumorigenesis and malignancy in several cancers including prostate cancer. We assessed the effects of combination PI3K pathway inhibition on the efficacy of PDT in human prostate tumor cell line (PC3) and SV40-transformed mouse endothelial cell line (SVEC-40). Combination of PDT and BEZ 235 (BEZ), a pan-PI3/ mTOR kinase inhibitor additively enhanced efficacy of sub-lethal PDT in both cell lines. The combination of the pan-PI3/ mTOR kinase inhibitor LY294002 (LY) with PDT also enhanced efficacy of PDT in PC3 in an additive manner but synergistically in SVEC. In order to determine the mechanism of enhancement of efficacy, we assessed apoptosis and autophagy following PDT. PDT-mediated apoptosis was enhanced in endothelial cells, by both BEZ and LY rapidly after treatment. Compared to SVEC, PC3 cells are apoptosis-deficient and apoptosis was not significantly enhanced by either LY or BEZ. However, lethal PDT of PC3 cells induced a delayed autophagic response which may be enhanced by combination, depending on PI3K inhibitor and dose.

  12. Chloroquine or Chloroquine-PI3K/Akt Pathway Inhibitor Combinations Strongly Promote γ-Irradiation-Induced Cell Death in Primary Stem-Like Glioma Cells

    PubMed Central

    Firat, Elke; Weyerbrock, Astrid; Gaedicke, Simone; Grosu, Anca-Ligia; Niedermann, Gabriele

    2012-01-01

    We asked whether inhibitors of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is highly active in cancer stem cells (CSCs) and upregulated in response to genotoxic treatments, promote γ-irradiationγIR)-induced cell death in highly radioresistant, patient-derived stem-like glioma cells (SLGCs). Surprisingly, in most cases the inhibitors did not promote γIR-induced cell death. In contrast, the strongly cytostatic Ly294002 and PI-103 even tended to reduce it. Since autophagy was induced we examined whether addition of the clinically applicable autophagy inhibitor chloroquine (CQ) would trigger cell death in SLGCs. Triple therapy with CQ at doses as low as 5 to 10 µM indeed caused strong apoptosis. At slightly higher doses, CQ alone strongly promoted γIR-induced apoptosis in all SLGC lines examined. The strong apoptosis in combinations with CQ was invariably associated with strong accumulation of the autophagosomal marker LC3-II, indicating inhibition of late autophagy. Thus, autophagy-promoting effects of PI3K/Akt pathway inhibitors apparently hinder cell death induction in γ-irradiated SLGCs. However, as we show here for the first time, the late autophagy inhibitor CQ strongly promotes γIR-induced cell death in highly radioresistant CSCs, and triple combinations of CQ, γIR and a PI3K/Akt pathway inhibitor permit reduction of the CQ dose required to trigger cell death. PMID:23091617

  13. Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2.

    PubMed

    Marchbank, Tania; Mahmood, Asif; Playford, Raymond J

    2013-08-01

    Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

  14. Novel agents and associated toxicities of inhibitors of the pi3k/Akt/mtor pathway for the treatment of breast cancer

    PubMed Central

    Chia, S.; Gandhi, S.; Joy, A.A.; Edwards, S.; Gorr, M.; Hopkins, S.; Kondejewski, J.; Ayoub, J.P.; Califaretti, N.; Rayson, D.; Dent, S.F.

    2015-01-01

    The pi3k/Akt/mtor (phosphatidylinositol 3 kinase/ Akt/mammalian target of rapamycin) signalling pathway is an established driver of oncogenic activity in human malignancies. Therapeutic targeting of this pathway holds significant promise as a treatment strategy. Everolimus, an mtor inhibitor, is the first of this class of agents approved for the treatment of hormone receptor–positive, human epidermal growth factor receptor 2–negative advanced breast cancer. Everolimus has been associated with significant improvements in progression-free survival; however, it is also associated with increased toxicity related to its specific mechanism of action. Methods A comprehensive review of the literature conducted using a focused medline search was combined with a search of current trials at http://ClinicalTrials.gov/. Summary tables of the toxicities of the various classes of pi3k/Akt/mtor inhibitors were created. A broad group of Canadian health care professionals was assembled to review the data and to produce expert opinion and summary recommendations for possible best practices in managing the adverse events associated with these pathway inhibitors. Results Differing toxicities are associated with the various classes of pi3k/Akt/mtor pathway inhibitors. The most common unique adverse events observed in everolimus clinical trials in breast cancer include stomatitis (all grades: approximately 60%), noninfectious pneumonitis (15%), rash (40%), hyperglycemia (15%), and immunosuppression (40%). To minimize grades 3 and 4 toxicities and to attempt to attain optimal outcomes, effective management of those adverse events is critical. Management should be interdisciplinary and should use approaches that include education, early recognition, active intervention, and potentially prophylactic strategies. Discussion Everolimus likely represents the first of many complex oral targeted therapies for the treatment of breast cancer. Using this agent as a template, it is essential to

  15. Discovery of a selective irreversible BMX inhibitor for prostate cancer.

    PubMed

    Liu, Feiyang; Zhang, Xin; Weisberg, Ellen; Chen, Sen; Hur, Wooyoung; Wu, Hong; Zhao, Zheng; Wang, Wenchao; Mao, Mao; Cai, Changmeng; Simon, Nicholas I; Sanda, Takaomi; Wang, Jinhua; Look, A Thomas; Griffin, James D; Balk, Steven P; Liu, Qingsong; Gray, Nathanael S

    2013-07-19

    BMX is a member of the TEC family of nonreceptor tyrosine kinases. We have used structure-based drug design in conjunction with kinome profiling to develop a potent, selective, and irreversible BMX kinase inhibitor, BMX-IN-1, which covalently modifies Cys496. BMX-IN-1 inhibits the proliferation of Tel-BMX-transformed Ba/F3 cells at two digit nanomolar concentrations but requires single digit micromolar concentrations to inhibit the proliferation of prostate cancer cell lines. Using a combinatorial kinase inhibitor screening strategy, we discovered that the allosteric Akt inhibitor, MK2206, is able to potentiate BMX inhibitor's antiproliferation efficacy against prostate cancer cells. PMID:23594111

  16. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

    SciTech Connect

    Rubio, V.; Cervera, J.; Bendala, E. ); Lusty, C.J. ); Britton, H.G. )

    1991-01-29

    The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH{sub 3} and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of ({sup 14}C)UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with ({sup 14}C)UMP. The ({sup 14}C)UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH{sub 2} half, which is involved in the binding of the molecule of ATP that yields P{sub i}, close to the regulatory domain.

  17. The novel AKT inhibitor afuresertib shows favorable safety, pharmacokinetics, and clinical activity in multiple myeloma

    PubMed Central

    Spencer, Andrew; Yoon, Sung-Soo; Harrison, Simon J.; Morris, Shannon R.; Smith, Deborah A.; Brigandi, Richard A.; Gauvin, Jennifer; Kumar, Rakesh; Opalinska, Joanna B.

    2014-01-01

    The PI3K/AKT pathway is constitutively active in hematologic malignancies, providing proliferative and antiapoptotic signals and possibly contributing to drug resistance. We conducted an open-label phase 1 study to evaluate the maximum tolerated dose (MTD), safety, pharmacokinetics, and clinical activity of afuresertib—an oral AKT inhibitor—in patients with advanced hematologic malignancies. Seventy-three patients were treated at doses ranging from 25 to 150 mg per day. The MTD was established at 125 mg per day because of 2 dose-limiting toxicities in the 150-mg cohort (liver function test abnormalities). The most frequent adverse events were nausea (35.6%), diarrhea (32.9%), and dyspepsia (24.7%). Maximum plasma concentrations and area under the plasma concentration-time curves from time 0 to 24 hours were generally dose proportional at >75-mg doses; the median time to peak plasma concentrations was 1.5 to 2.5 hours post dose, with a half-life of approximately 1.7 days. Three multiple myeloma patients attained partial responses; an additional 3 attained minimal responses. Clinical activity was also observed in non-Hodgkin lymphoma, Langerhan's cell histiocytosis, and Hodgkin disease. Single-agent afuresertib showed a favorable safety profile and demonstrated clinical activity against hematologic malignancies, including multiple myeloma. This trial was registered at www.clinicaltrials.gov as #NCT00881946. PMID:25075128

  18. The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation

    PubMed Central

    Mercado-Pimentel, ME; Igarashi, S; Dunn, AM; Behbahani, M; Miller, C; Read, CM; Jacob, A

    2016-01-01

    Activation of PKB/AKT signaling, which requires PDK1 and PDK2 function, drives Vestibular Schwannoma (VS) and meningioma growth. PDK2 function is defined as a molecule that phosphorylates AKT-Ser473. Integrin-Linked Kinase (ILK) functions as PDK2 in PKB/AKT activation in many cancers; therefore, we hypothesized that OSU-T315, a small molecule ILK inhibitor, will inhibit the ILK-PDK2 function in PKB/AKT signaling activation in VS and meningioma cell growth. OSU-T315 decreased cell viability at IC50 < 2μM in VS (HEI193) and meningioma (Ben-Men-1) cell lines, in primary cells at < 3.5μM, while in normal primary Schwann cells at 7.1μM. OSU-T315 inhibits AKT signaling by decreasing phosphorylation at AKT-Ser473, AKT-Thr308, ILK-Ser246 and ILK-Thr173. In addition, OSU-T315 affected the phosphorylation or expression levels of AKT downstream proliferation effectors as well as autophagy markers. Flow cytometry shows that OSU-T315 increased the percentage of cells arrested at G2/M for both, HEI193 (39.99%) and Ben-Men-1 (26.96%) cells, compared to controls (21.54%, 8.47%). Two hours of OSU-T315 treatment increased cell death in both cell lines (34.3%, 9.1%) versus untreated (12.1%, 8.1%). Though longer exposure increased cell death in Ben-Men-1, TUNEL assays showed that OSU-T315 does not induce apoptosis. OSU-T315 was primarily cytotoxic for HEI193 and Ben-Men-1 inducing a dysregulated autophagy. Our studies suggest that OSU-T315 has translational potential as a chemotherapeutic agent against VS and meningioma.

  19. Histone deacetylase inhibitors reduce WB-F344 oval cell viability and migration capability by suppressing AKT/mTOR signaling in vitro.

    PubMed

    Zhang, Peng; Zhu, Xiaofeng; Wu, Ying; Hu, Ronglin; Li, Dongming; Du, Jun; Jiao, Xingyuan; He, Xiaoshun

    2016-01-15

    Histone deacetylase (HDAC) can blockDNA replication and transcription and altered HDAC expression was associated with tumorigenesis. This study investigated the effects of HDAC inhibitors on hepatic oval cells and aimed to delineate the underlying molecular events. Hepatic oval cells were treated with two different HDAC inhibitors, suberoylanilidehydroxamic acid (SAHA) and trichostatin-A (TSA). Cells were subjected to cell morphology, cell viability, cell cycle, and wound healing assays. The expression of proteins related to both apoptosis and the cell cycle, and proteins of the AKT/mammalian target of rapamycin (mTOR) signaling pathway were analyzed by Western blot. The data showed that HDAC inhibitors reduced oval cell viability and migration capability, and arrested oval cells at the G0/G1 and S phases of the cell cycle, in a dose- and time-dependent manner. HDAC inhibitors altered cell morphology and reduced oval cell viability, and downregulated the expression of PCNA, cyclinD1, c-Myc and Bmi1 proteins, while also suppressing AKT/mTOR and its downstream target activity. In conclusion, this study demonstrates that HDAC inhibitors affect oval cells by suppressing AKT/mTOR signaling. PMID:26558695

  20. Molecular dynamics simulations and modelling of the residue interaction networks in the BRAF kinase complexes with small molecule inhibitors: probing the allosteric effects of ligand-induced kinase dimerization and paradoxical activation.

    PubMed

    Verkhivker, G M

    2016-10-20

    Protein kinases are central to proper functioning of cellular networks and are an integral part of many signal transduction pathways. The family of protein kinases represents by far the largest and most important class of therapeutic targets in oncology. Dimerization-induced activation has emerged as a common mechanism of allosteric regulation in BRAF kinases, which play an important role in growth factor signalling and human diseases. Recent studies have revealed that most of the BRAF inhibitors can induce dimerization and paradoxically stimulate enzyme transactivation by conferring an active conformation in the second monomer of the kinase dimer. The emerging connections between inhibitor binding and BRAF kinase domain dimerization have suggested a molecular basis of the activation mechanism in which BRAF inhibitors may allosterically modulate the stability of the dimerization interface and affect the organization of residue interaction networks in BRAF kinase dimers. In this work, we integrated structural bioinformatics analysis, molecular dynamics and binding free energy simulations with the protein structure network analysis of the BRAF crystal structures to determine dynamic signatures of BRAF conformations in complexes with different types of inhibitors and probe the mechanisms of the inhibitor-induced dimerization and paradoxical activation. The results of this study highlight previously unexplored relationships between types of BRAF inhibitors, inhibitor-induced changes in the residue interaction networks and allosteric modulation of the kinase activity. This study suggests a mechanism by which BRAF inhibitors could promote or interfere with the paradoxical activation of BRAF kinases, which may be useful in informing discovery efforts to minimize the unanticipated adverse biological consequences of these therapeutic agents.

  1. RES-529: a PI3K/AKT/mTOR pathway inhibitor that dissociates the mTORC1 and mTORC2 complexes

    PubMed Central

    2016-01-01

    RES-529 (previously named Palomid 529, P529) is a phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway inhibitor that interferes with the pathway through both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) dissociation. This compound is currently being developed in oncology and ophthalmology. The oncology focus is for the treatment of glioblastoma, where it has received orphan designation by the US Food and Drug Administration, and prostate cancer. We present a review of the PI3K/AKT/mTOR pathway, its role in tumorigenesis, and the potential of RES-529 in cancer treatment. RES-529 inhibits mTORC1/mTORC2 activity in various cancer cell lines, as noted by decreased phosphorylation of substrates including ribosomal protein S6, 4E-BP1, and AKT, leading to cell growth inhibition and death, with activity generally in the range of 5–15 μmol/l. In animal tumor models where the PI3K/AKT/mTOR pathway is abnormally activated (i.e. glioblastoma, prostate cancer, and breast cancer), RES-529 reduces tumor growth by as much as 78%. RES-529 treatment is synergistic with radiation therapy, chemotherapy, and hormonal therapy in reducing tumor growth, potentially by preventing PI3K/AKT/mTOR pathway activation associated with these treatments. Furthermore, this compound has shown antiangiogenic activity in several animal models. mTORC1 and mTORC2 have redundant and distinct activities that contribute toward oncogenesis. Current inhibitors of this pathway have primarily targeted mTORC1, but have shown limited clinical efficacy. Inhibitors of mTORC1 and mTORC2 such as RES-529 may therefore have the potential to overcome the deficiencies found in targeting only mTORC1. PMID:26918392

  2. PIM and AKT kinase inhibitors show synergistic cytotoxicity in acute myeloid leukaemia that is associated with convergence on mTOR and MCL1 pathways.

    PubMed

    Meja, Koremu; Stengel, Chloe; Sellar, Rob; Huszar, Dennis; Davies, Barry R; Gale, Rosemary E; Linch, David C; Khwaja, Asim

    2014-10-01

    PIM kinases (PIM1, 2 and 3) are involved in cell proliferation and survival signalling and are emerging targets for the therapy of various malignancies. We found that a significant proportion of primary acute myeloid leukaemia (AML) samples showed PIM1 and PIM2 expression by quantitative reverse transcription polymerase chain reaction. Therefore, we investigated the effects of a novel ATP-competitive pan-PIM inhibitor, AZD1897, on AML cell growth and survival. PIM inhibition showed limited single agent activity in AML cell lines and primary AML cells, including those with or without FLT3-internal tandem duplication (ITD) mutation. However, significant synergy was seen when AZD1897 was combined with the Akt inhibitor AZD5363, a compound that is in early-phase clinical trials. AML cells from putative leukaemia stem cell subsets, including CD34+38- and CD34+38+ fractions, were equivalently affected by dual PIM/Akt inhibition when compared with bulk tumour cells. Analysis of downstream signalling pathways showed that combined PIM/Akt inhibition downregulated mTOR outputs (phosphorylation of 4EBP1 and S6) and markedly reduced levels of the anti-apoptotic protein MCL1. The combination of PIM and Akt inhibition holds promise for the treatment of AML. PMID:24975213

  3. Status of PI3K/Akt/mTOR pathway inhibitors in lymphoma.

    PubMed

    Westin, Jason R

    2014-10-01

    The phosphatidylinositol-3-kinase (PI3K) pathway is well known to regulate a wide variety of essential cellular functions, including glucose metabolism, translational regulation of protein synthesis, cell proliferation, apoptosis, and survival. Aberrations in the PI3K pathway are among the most frequently observed in cancer, and include amplifications, rearrangements, mutations, and loss of regulators. As a net result of these anomalies, the PI3K pathway is activated in many malignancies, including in Hodgkin and non-Hodgkin lymphomas, and yields a competitive growth and survival advantage, increased metastatic ability, and resistance to conventional therapy. Numerous inhibitors targeting various nodes in the PI3K pathway are undergoing clinical development, and their current status in lymphoma will be the focus of this review.

  4. Compound library screening identified Akt/PKB kinase pathway inhibitors as potential key molecules for the development of new chemotherapeutics against schistosomiasis

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Lescuyer, Arlette; Lancelot, Julien; Dissous, Colette

    2014-01-01

    Protein kinases (PKs) are one of the largest protein families in most eukaryotic organisms. These enzymes are involved in the control of cell proliferation, differentiation and metabolism and a large number of the anticancer drugs currently used are directed against PKs. The structure and function of PKs are well conserved throughout evolution. In schistosome parasites, PKs were shown to be involved in essential functions at every stage of the parasite life cycle, making these enzymes promising anti-parasite drug targets. In this study, we tested a panel of commercial inhibitors for various PKs and analyzed their effects on pairing and egg production by schistosomes as well as their toxicity towards schistosomula larvae. Results obtained confirmed the deleterious effect of PK targeting on Schistosoma mansoni physiology and the important function of different tyrosine and serine/threonine kinases in the biology and reproduction of this parasite. They also indicated for the first time that the Protein kinase B (also called Akt) which is a major downstream target of many receptor tyrosine kinases and a central player at the crossroads of signal transduction pathways activated in response to growth factors and insulin, can constitute a novel target for anti-schistosome chemotherapy. Structural and functional studies have shown that SmAkt is a conserved kinase and that its activity can be inhibited by commercially available Akt inhibitors. In treated adult worms, Akt/PKB kinase pathway inhibitors induced profound alterations in pairing and egg laying and they also greatly affected the viability of schistosomula larvae. PMID:25516836

  5. Novel Cancer Therapeutics with Allosteric Modulation of the Mitochondrial C-Raf-DAPK Complex by Raf Inhibitor Combination Therapy.

    PubMed

    Tsai, Yi-Ta; Chuang, Mei-Jen; Tang, Shou-Hung; Wu, Sheng-Tang; Chen, Yu-Chi; Sun, Guang-Huan; Hsiao, Pei-Wen; Huang, Shih-Ming; Lee, Hwei-Jen; Yu, Cheng-Ping; Ho, Jar-Yi; Lin, Hui-Kuan; Chen, Ming-Rong; Lin, Chung-Chih; Chang, Sun-Yran; Lin, Victor C; Yu, Dah-Shyong; Cha, Tai-Lung

    2015-09-01

    Mitochondria are the powerhouses of cells. Mitochondrial C-Raf is a potential cancer therapeutic target, as it regulates mitochondrial function and is localized to the mitochondria by its N-terminal domain. However, Raf inhibitor monotherapy can induce S338 phosphorylation of C-Raf (pC-Raf(S338)) and impede therapy. This study identified the interaction of C-Raf with S308 phosphorylated DAPK (pDAPK(S308)), which together became colocalized in the mitochondria to facilitate mitochondrial remodeling. Combined use of the Raf inhibitors sorafenib and GW5074 had synergistic anticancer effects in vitro and in vivo, but targeted mitochondrial function, rather than the canonical Raf signaling pathway. C-Raf depletion in knockout MEF(C-Raf-/-) or siRNA knockdown ACHN renal cancer cells abrogated the cytotoxicity of combination therapy. Crystal structure simulation showed that GW5074 bound to C-Raf and induced a C-Raf conformational change that enhanced sorafenib-binding affinity. In the presence of pDAPK(S308), this drug-target interaction compromised the mitochondrial targeting effect of the N-terminal domain of C-Raf, which induced two-hit damages to cancer cells. First, combination therapy facilitated pC-Raf(S338) and pDAPK(S308) translocation from mitochondria to cytoplasm, leading to mitochondrial dysfunction and reactive oxygen species (ROS) generation. Second, ROS facilitated PP2A-mediated dephosphorylation of pDAPK(S308) to DAPK. PP2A then dissociated from the C-Raf-DAPK complex and induced profound cancer cell death. Increased pDAPK(S308) modification was also observed in renal cancer tissues, which correlated with poor disease-free survival and poor overall survival in renal cancer patients. Besides mediating the anticancer effect, pDAPK(S308) may serve as a predictive biomarker for Raf inhibitors combination therapy, suggesting an ideal preclinical model that is worthy of clinical translation.

  6. Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents.

    PubMed

    Sun, Jing; Niu, Yan; Wang, Chao; Zhang, Hao; Xie, Bingyu; Xu, Fengrong; Jin, Hongwei; Peng, Yihong; Liang, Lei; Xu, Ping

    2016-08-15

    Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55nM and 60nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells. PMID:27288186

  7. Heat Shock Protein 70 Inhibitors. 2. 2,5′-Thiodipyrimidines, 5-(Phenylthio)pyrimidines, 2-(Pyridin-3-ylthio)pyrimidines, and 3-(Phenylthio)pyridines as Reversible Binders to an Allosteric Site on Heat Shock Protein 70

    PubMed Central

    2015-01-01

    The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue (10.1021/jm401551n), we have described structure–activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands. PMID:24548239

  8. Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70

    PubMed Central

    2015-01-01

    Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure–activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics. PMID:24548207

  9. Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents.

    PubMed

    Sun, Jing; Niu, Yan; Wang, Chao; Zhang, Hao; Xie, Bingyu; Xu, Fengrong; Jin, Hongwei; Peng, Yihong; Liang, Lei; Xu, Ping

    2016-08-15

    Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55nM and 60nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.

  10. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain.

    PubMed

    Rubio, V; Cervera, J; Lusty, C J; Bendala, E; Britton, H G

    1991-01-29

    The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1989678

  11. Lactoferrin attenuates fatty acid-induced lipotoxicity via Akt signaling in hepatocarcinoma cells.

    PubMed

    Morishita, Satoru; Tomita, Keiko; Ono, Tomoji; Murakoshi, Michiaki; Saito, Kenji; Sugiyama, Keikichi; Nishino, Hoyoku; Kato, Hisanori

    2015-12-01

    Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). The excess influx of fatty acids (FAs) into the liver is recognized as a main cause of simple steatosis formation and progression to NASH. Recently, administration of lactoferrin (LF), a glycoprotein present in milk, was suggested to prevent NAFLD development. However, the effect of LF on the contribution of FA to NAFLD development remains unclear. In this study, the effects of LF on FA mixture (FAm)-induced lipotoxicity using human hepatocarcinoma G2 cells were assessed. FAm significantly decreased cell viability and increased intracellular lipid accumulation, whereas LF significantly recovered cell viability without affecting lipid accumulation. FAm-induced lactic dehydrogenase (LDH) and caspase-3/7 activities were significantly decreased by LF and SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. We also found that LF added to FAm-treated cells induced Akt phosphorylation, which contributed to inhibition of JNK signaling pathway-dependent apoptosis. Akt inhibitor VIII, an allosteric Akt inhibitor, significantly attenuated the effect of LF on LDH activity and abrogated the ones on cell viability and caspase-3/7 activity. In summary, the present study has revealed that LF has a protective effect on FAm-induced lipotoxicity in a HepG2 model of NAFLD and identified the activation of the Akt signaling pathway as a possibly major mechanism.

  12. The juxtamembrane sequence of the Hepatitis C virus polymerase can affect RNA synthesis and inhibition by allosteric polymerase inhibitors.

    PubMed

    Wen, Y; Lin, X; Fan, B; Ranjith-Kumar, C T; Kao, C C

    2015-08-01

    The Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), nonstructural protein 5B (NS5B), is anchored in the membrane through a C-terminal helix. A sequence of ca. 12 residues that connects the catalytically competent portion of the RdRp and the C-terminal helix, the juxtamembrane sequence (JMS), has a poorly defined role in RdRp function in a large part since it is translated from a cis-acting RNA element (CRE) that is essential for HCV replication. Using a HCV replicon that transposed a second copy of CRE to the 3' UTR of the HCV replicon, we demonstrate that amino acid substitutions in the JMS were detrimental for HCV replicon replication. Substitutions in the JMS also resulted in a defect in de novo-initiated RNAs synthesis in vitro and in a cell-based reporter assay. A nonnucleoside inhibitor of the NS5B that binds to the catalytic pocket was less potent in inhibiting NS5B in the presence of JMS mutations. The JMS mutants exhibit reduced stability in thermodenaturation assays, suggesting that the JMS helps confer a more stable conformation to NS5B that could impact RNA synthesis. PMID:25895103

  13. Cyclic AMP Analog Blocks Kinase Activation by Stabilizing Inactive Conformation: Conformational Selection Highlights a New Concept in Allosteric Inhibitor Design*

    PubMed Central

    Badireddy, Suguna; Yunfeng, Gao; Ritchie, Mark; Akamine, Pearl; Wu, Jian; Kim, Choel W.; Taylor, Susan S.; Qingsong, Lin; Swaminathan, Kunchithapadam; Anand, Ganesh S.

    2011-01-01

    The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound “B” and C-subunit-bound “H”-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through “induced fit” alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially “select” inactive conformations of target proteins by satisfying all “binding” constraints alone without inducing conformational changes necessary for activation. PMID:21081668

  14. Synthesis and evaluation of the antiproliferative activity of novel pyrrolo[1,2-a]quinoxaline derivatives, potential inhibitors of Akt kinase. Part II.

    PubMed

    Desplat, Vanessa; Moreau, Stephane; Gay, Aurore; Fabre, Solene Belisle; Thiolat, Denis; Massip, Stephane; Macky, Gregory; Godde, Frederic; Mossalayi, Djavad; Jarry, Christian; Guillon, Jean

    2010-04-01

    Attenuation of protein kinases by selective inhibitors is an extremely active field of activity in anticancer drug development. Therefore, Akt, a serine/threonine protein kinase, also known as protein kinase B (PKB), represents an attractive potential target for therapeutic intervention. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel inhibitors with various heterocycle scaffolds. Based on previous results obtained on the antiproliferative activities of new pyrrolo[1,2-a]quinoxalines, a novel series was designed and synthesized from various substituted phenyl-1H-pyrrole-2-carboxylic acid alkyl esters via a multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937, and HL60, and the breast cancer cell line MCF7. The first biological evaluation of our new substituted pyrrolo[1,2-a]quinoxalines showed antiproliferative activity against the tested cell lines. From a general SAR point of view, these preliminary biological results highlight the importance of substitution at the C-4 position of the pyrroloquinoxaline scaffold by a benzylpiperidinyl fluorobenzimidazole group, and also the need for a functionalization on the pyrrole ring.

  15. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer

    PubMed Central

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R.; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E.; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L.

    2014-01-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  16. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

    PubMed

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy.

  17. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

    PubMed

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  18. Abrogation of Mitogen-Activated Protein Kinase and Akt Signaling by Vandetanib Synergistically Potentiates Histone Deacetylase Inhibitor-Induced Apoptosis in Human Glioma Cells

    PubMed Central

    Jane, Esther P.; Premkumar, Daniel R.; Addo-Yobo, Steven O.

    2009-01-01

    Vandetanib is a multitargeted tyrosine kinase inhibitor. Our initial studies demonstrated that this agent blocks vascular endothelial growth factor receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor phosphorylation and mitogen-activated protein kinase (MAPK)-mediated signaling in glioma cell lines in a dose-dependent manner. Despite these effects, we observed that vandetanib had little effect on apoptosis induction at clinically achievable concentrations. Because histone deacetylase inhibitors (HDACIs) have been suggested to regulate signaling protein transcription and downstream interactions via modulation of protein chaperone function through the 90-kDa heat shock protein, we investigated whether combining vandetanib with an HDACI could synergistically potentiate signaling pathway inhibition and apoptosis induction in a panel of malignant human glioma cell lines. Proliferation assays, apoptosis induction studies, and Western immunoblot analysis were conducted in cells treated with vandetanib and HDACIs as single agents or in combination. Vandetanib and suberoylanalide hydroxamic acid reduced proliferation in all cell lines when used as single agents, and the combination produced marked potentiation of growth inhibition as assessed by combinatorial methods. These effects were paralleled by potentiation of Akt signaling inhibition and apoptosis induction. Our results indicate that inhibition of histone deacetylation enhances the antiproliferative effect of vandetanib in malignant human glioma cell lines by enhancing inhibition of MAPK, Akt, and other downstream effectors that may have application in combinatorial therapeutics for these tumors. PMID:19622715

  19. Anti-tumour activity in RAS-driven tumours by blocking AKT and MEK

    PubMed Central

    Tolcher, Anthony W.; Khan, Khurum; Ong, Michael; Banerji, Udai; Papadimitrakopoulou, Vassiliki; Gandara, David R.; Patnaik, Amita; Baird, Richard D.; Olmos, David; Garrett, Christopher R.; Skolnik, Jeffrey M.; Rubin, Eric H.; Smith, Paul D.; Huang, Pearl; Learoyd, Maria; Shannon, Keith A.; Morosky, Anne; Tetteh, Ernestina; Jou, Ying-Ming; Papadopoulos, Kyriakos P.; Moreno, Victor; Kaiser, Brianne; Yap, Timothy A.; Yan, Li; de Bono, Johann S.

    2014-01-01

    Purpose KRAS is the most commonly mutated oncogene in human tumours. KRAS-mutant cells may exhibit resistance to the allosteric MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) and allosteric AKT inhibitors (such as MK-2206), the combination of which may overcome resistance to both monotherapies. Experimental Design We conducted a dose/schedule-finding study evaluating MK-2206 and selumetinib in patients with advanced treatment-refractory solid tumours. Recommended dosing schedules were defined as MK-2206 135 mg weekly and selumetinib 100 mg once-daily. Results Grade 3 rash was the most common dose-limiting toxicity (DLT); other DLTs included grade 4 lipase increase, grade 3 stomatitis, diarrhoea, and fatigue, and grade 3 and grade 2 retinal pigment epithelium detachment. There were no meaningful pharmacokinetic drug-drug interactions. Clinical anti-tumour activity included RECIST 1.0-confirmed partial responses in non-small cell lung cancer and low-grade ovarian carcinoma. Conclusion Responses in KRAS-mutant cancers were generally durable. Clinical co-targeting of MEK and AKT signalling may be an important therapeutic strategy in KRAS-driven human malignancies (Trial NCT number NCT01021748). PMID:25516890

  20. Bortezomib induces apoptosis and growth suppression in human medulloblastoma cells, associated with inhibition of AKT and NF-ĸB signaling, and synergizes with an ERK inhibitor.

    PubMed

    Yang, Fan; Jove, Veronica; Chang, Shirley; Hedvat, Michael; Liu, Lucy; Buettner, Ralf; Tian, Yan; Scuto, Anna; Wen, Wei; Yip, M L Richard; Van Meter, Timothy; Yen, Yun; Jove, Richard

    2012-04-01

    Medulloblastoma is the most common brain tumor in children. Here, we report that bortezomib, a proteasome inhibitor, induced apoptosis and inhibited cell proliferation in two established cell lines and a primary culture of human medulloblastomas. Bortezomib increased the release of cytochrome c to cytosol and activated caspase-9 and caspase-3, resulting in cleavage of PARP. Caspase inhibitor (Z-VAD-FMK) could rescue medulloblastoma cells from the cytotoxicity of bortezomib. Phosphorylation of AKT and its upstream regulator mTOR were reduced by bortezomib treatment in medulloblastoma cells. Bortezomib increased the expression of Bad and Bak, pro-apoptotic proteins, and p21Cip1 and p27Kip1, negative regulators of cell cycle progression, which are associated with the growth suppression and induction of apoptosis in these tumor cells. Bortezomib also increased the accumulation of phosphorylated IĸBα, and decreased nuclear translocation of NF-ĸB. Thus, NF-ĸB signaling and activation of its downstream targets are suppressed. Moreover, ERK inhibitors or downregulating ERK with ERK siRNA synergized with bortezomib on anticancer effects in medulloblastoma cells. Bortezomib also inhibited the growth of human medulloblastoma cells in a mouse xenograft model. These findings suggest that proteasome inhibitors are potentially promising drugs for treatment of pediatric medulloblastomas.

  1. 50th anniversary of the word "allosteric".

    PubMed

    Changeux, Jean-Pierre

    2011-07-01

    A brief historical account on the origin and meaning of the word "allosteric" is presented. The word was coined in an attempt to qualify the chemical mechanism of the feedback inhibition of bacterial enzymes by regulatory ligands. The data lead to the proposal that, at variance with the classical mechanism of mutual exclusion by steric hindrance, the inhibition takes place through an "allosteric" interaction between "no overlapping", stereospecifically distinct, sites for substrate and feedback inhibitor, mediated by a discrete reversible alteration of the molecular structure of the protein. PMID:21574197

  2. PI3K/PTEN/Akt pathway status affects the sensitivity of high-grade glioma cell cultures to the insulin-like growth factor-1 receptor inhibitor NVP-AEW541.

    PubMed

    Hägerstrand, Daniel; Lindh, Maja Bradic; Peña, Cristina; Garcia-Echeverria, Carlos; Nistér, Monica; Hofmann, Francesco; Ostman, Arne

    2010-09-01

    IGF-1 receptor signaling contributes to the growth of many solid tumors, including glioblastoma. This study analyzed the sensitivity of 8 glioblastoma cultures to the IGF-1 receptor inhibitor NVP-AEW541. Growth reduction, caused by a combination of antiproliferative and proapoptotic effects, varied between 20% and 100%. Growth-inhibitory effects of IGF-1 receptor siRNA were also demonstrated in 2 of the cultures. Activating mutations in PIK3CA were found in 2 cultures, and 2 other cultures displayed ligand-independent Akt phosphorylation. Growth inhibition was significantly reduced in cultures with PIK3CA mutations or ligand-independent Akt phosphorylation. PTEN siRNA experiments supported the notion that the status of the PI3K/PTEN/Akt pathway is involved in determining NVP-AEW541 sensitivity. Combination treatments with either PI3 kinase or mTOR inhibitors together with NVP-AEW541 were performed. These experiments demonstrated the effects of NVP-AEW541 in cells not responding to mono-treatment with the IGF-1 receptor inhibitor, when used together with either of the 2 other inhibitors. Together, the studies support continued clinical development of IGF-1 receptor antagonists for glioblastomas and identify links between PI3K/PTEN/Akt status and sensitivity to mono-treatment with NVP-AEW541. Furthermore, the studies suggest that NVP-AEW541 is also active together with PI3 kinase and mTOR inhibitors in cultures with a dysregulated PI3K/PTEN/Akt pathway. These studies should assist in future clinical development of IGF-1 receptor antagonists for glioblastoma and other tumors.

  3. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation.

    PubMed

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-09-18

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  4. Agonistic aptamer to the insulin receptor leads to biased signaling and functional selectivity through allosteric modulation

    PubMed Central

    Yunn, Na-Oh; Koh, Ara; Han, Seungmin; Lim, Jong Hun; Park, Sehoon; Lee, Jiyoun; Kim, Eui; Jang, Sung Key; Berggren, Per-Olof; Ryu, Sung Ho

    2015-01-01

    Due to their high affinity and specificity, aptamers have been widely used as effective inhibitors in clinical applications. However, the ability to activate protein function through aptamer-protein interaction has not been well-elucidated. To investigate their potential as target-specific agonists, we used SELEX to generate aptamers to the insulin receptor (IR) and identified an agonistic aptamer named IR-A48 that specifically binds to IR, but not to IGF-1 receptor. Despite its capacity to stimulate IR autophosphorylation, similar to insulin, we found that IR-A48 not only binds to an allosteric site distinct from the insulin binding site, but also preferentially induces Y1150 phosphorylation in the IR kinase domain. Moreover, Y1150-biased phosphorylation induced by IR-A48 selectively activates specific signaling pathways downstream of IR. In contrast to insulin-mediated activation of IR, IR-A48 binding has little effect on the MAPK pathway and proliferation of cancer cells. Instead, AKT S473 phosphorylation is highly stimulated by IR-A48, resulting in increased glucose uptake both in vitro and in vivo. Here, we present IR-A48 as a biased agonist able to selectively induce the metabolic activity of IR through allosteric binding. Furthermore, our study also suggests that aptamers can be a promising tool for developing artificial biased agonists to targeted receptors. PMID:26245346

  5. PARP-inhibitor treatment prevents hypertension induced cardiac remodeling by favorable modulation of heat shock proteins, Akt-1/GSK-3β and several PKC isoforms.

    PubMed

    Deres, Laszlo; Bartha, Eva; Palfi, Anita; Eros, Krisztian; Riba, Adam; Lantos, Janos; Kalai, Tamas; Hideg, Kalman; Sumegi, Balazs; Gallyas, Ferenc; Toth, Kalman; Halmosi, Robert

    2014-01-01

    Spontaneously hypertensive rat (SHR) is a suitable model for studies of the complications of hypertension. It is known that activation of poly(ADP-ribose) polymerase enzyme (PARP) plays an important role in the development of postinfarction as well as long-term hypertension induced heart failure. In this study, we examined whether PARP-inhibitor (L-2286) treatment could prevent the development of hypertensive cardiopathy in SHRs. 6-week-old SHR animals were treated with L-2286 (SHR-L group) or placebo (SHR-C group) for 24 weeks. Wistar-Kyoto rats were used as aged-matched, normotensive controls (WKY group). Echocardiography was performed, brain-derived natriuretic peptide (BNP) activity and blood pressure were determined at the end of the study. We detected the extent of fibrotic areas. The amount of heat-shock proteins (Hsps) and the phosphorylation state of Akt-1(Ser473), glycogen synthase kinase (GSK)-3β(Ser9), forkhead transcription factor (FKHR)(Ser256), mitogen activated protein kinases (MAPKs), and protein kinase C (PKC) isoenzymes were monitored. The elevated blood pressure in SHRs was not influenced by PARP-inhibitor treatment. Systolic left ventricular function and BNP activity did not differ among the three groups. L-2286 treatment decreased the marked left ventricular (LV) hypertrophy which was developed in SHRs. Interstitial collagen deposition was also decreased by L-2286 treatment. The phosphorylation of extracellular signal-regulated kinase (ERK)1/2(Thr183-Tyr185), Akt-1(Ser473), GSK-3β(Ser9), FKHR(Ser256), and PKC ε(Ser729) and the level of Hsp90 were increased, while the activity of PKC α/βII(Thr638/641), ζ/λ(410/403) were mitigated by L-2286 administration. We could detect signs of LV hypertrophy without congestive heart failure in SHR groups. This alteration was prevented by PARP inhibition. Our results suggest that PARP-inhibitor treatment has protective effect already in the early stage of hypertensive myocardial remodeling. PMID

  6. The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere

    PubMed Central

    2013-01-01

    Introduction Activation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo. Case preparation To further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere. Conclusion Our results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling. PMID:24088382

  7. Polypharmacology within CXCR4: Multiple binding sites and allosteric behavior

    NASA Astrophysics Data System (ADS)

    Planesas, Jesús M.; Pérez-Nueno, Violeta I.; Borrell, José I.; Teixidó, Jordi

    2014-10-01

    CXCR4 is a promiscuous receptor, which binds multiple diverse ligands. As usual in promiscuous proteins, CXCR4 has a large binding site, with multiple subsites, and high flexibility. Hence, it is not surprising that it is involved in the phenomenon of allosteric modulation. However, incomplete knowledge of allosteric ligand-binding sites has hampered an in-depth molecular understanding of how these inhibitors work. For example, it is known that lipidated fragments of intracellular GPCR loops, so called pepducins, such as pepducin ATI-2341, modulate CXCR4 activity using an agonist allosteric mechanism. Nevertheless, there are also examples of small organic molecules, such as AMD11070 and GSK812397, which may act as antagonist allosteric modulators. Here, we give new insights into this issue by proposing the binding interactions between the CXCR4 receptor and the above-mentioned allosteric modulators. We propose that CXCR4 has minimum two topographically different allosteric binding sites. One allosteric site would be in the intracellular loop 1 (ICL1) where pepducin ATI-2341 would bind to CXCR4, and the second one, in the extracellular side of CXCR4 in a subsite into the main orthosteric binding pocket, delimited by extracellular loops n° 1, 2, and the N-terminal end, where antagonists AMD11070 and GSK812397 would bind. Prediction of allosteric interactions between CXCR4 and pepducin ATI-2341 were studied first by rotational blind docking to determine the main binding region and a subsequent refinement of the best pose was performed using flexible docking methods and molecular dynamics. For the antagonists AMD11070 and GSK812397, the entire CXCR4 protein surface was explored by blind docking to define the binding region. A second docking analysis by subsites of the identified binding region was performed to refine the allosteric interactions. Finally, we identified the binding residues that appear to be essential for CXCR4 (agonists and antagonists) allosteric

  8. Targeting the PI3K/Akt pathway in murine MDS/MPN driven by hyperactive Ras

    PubMed Central

    Akutagawa, Jon; Huang, Tannie Q.; Epstein, Inbal; Chang, Tiffany; Quirindongo-Crespo, Maricel; Cottonham, Charisa L.; Dail, Monique; Slusher, Barbara S.; Friedman, Lori S.; Sampath, Deepak; Braun, Benjamin S.

    2016-01-01

    Chronic and juvenile myelomonocytic leukemias (CMML and JMML) are myelodysplastic/myeloproliferative neoplasia (MDS/MPN) overlap syndromes that respond poorly to conventional treatments. Aberrant Ras activation due to NRAS, KRAS, PTPN11, CBL, and NF1 mutations is common in CMML and JMML. However, no mechanism-based treatments currently exist for cancers with any of these mutations. An alternative therapeutic strategy involves targeting Ras-regulated effector pathways that are aberrantly activated in CMML and JMML, which include the Raf/MEK/ERK and phosphoinositide-3´-OH kinase (PI3K)/Akt cascades. Mx1-Cre, KrasD12 and Mx1-Cre, Nf1flox/− mice accurately model many aspects of CMML and JMML. Treating Mx1-Cre, KrasD12 mice with GDC-0941 (also referred to as pictilisib), an orally bioavailable inhibitor of class I PI3K isoforms, reduced leukocytosis, anemia, and splenomegaly while extending survival. However, GDC-0941 treatment attenuated activation of both PI3K/Akt and Raf/MEK/ERK pathways in primary hematopoietic cells, suggesting it could be acting through suppression of Raf/MEK/ERK signals. To interrogate the importance of the PI3K/Akt pathway specifically, we treated mice with the allosteric Akt inhibitor MK-2206. This compound had no effect on Raf/MEK/ERK signaling, yet it also induced robust hematologic responses in Kras and Nf1 mice with MPN. These data support investigating PI3K/Akt pathway inhibitors as a therapeutic strategy in JMML and CMML patients. PMID:26965285

  9. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  10. Allosteric modulation of an excitatory amino acid transporter: the subtype-selective inhibitor UCPH-101 exerts sustained inhibition of EAAT1 through an intramonomeric site in the trimerization domain.

    PubMed

    Abrahamsen, Bjarke; Schneider, Nicole; Erichsen, Mette N; Huynh, Tri H V; Fahlke, Christoph; Bunch, Lennart; Jensen, Anders A

    2013-01-16

    In the present study, the mechanism of action and molecular basis for the activity of the first class of selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rodent ortholog GLAST are elucidated. The previously reported specificity of UCPH-101 and UCPH-102 for EAAT1 over EAAT2 and EAAT3 is demonstrated to extend to the EAAT4 and EAAT5 subtypes as well. Interestingly, brief exposure to UCPH-101 induces a long-lasting inactive state of EAAT1, whereas the inhibition exerted by closely related analogs is substantially more reversible in nature. In agreement with this, the kinetic properties of UCPH-101 unblocking of the transporter are considerably slower than those of UCPH-102. UCPH-101 exhibits noncompetitive inhibition of EAAT1, and its binding site in GLAST has been delineated in an elaborate mutagenesis study. Substitutions of several residues in TM3, TM4c, and TM7a of GLAST have detrimental effects on the inhibitory potency and/or efficacy of UCPH-101 while not affecting the pharmacological properties of (S)-glutamate or the competitive EAAT inhibitor TBOA significantly. Hence, UCPH-101 is proposed to target a predominantly hydrophobic crevice in the "trimerization domain" of the GLAST monomer, and the inhibitor is demonstrated to inhibit the uptake through the monomer that it binds to exclusively and not to affect substrate translocation through the other monomers in the GLAST trimer. The allosteric mode of UCPH-101 inhibition underlines the functional importance of the trimerization domain of the EAAT and demonstrates the feasibility of modulating transporter function through ligand binding to regions distant from its "transport domain." PMID:23325245

  11. Crosstalk between the IGF-1R/AKT/mTORC1 pathway and the tumor suppressors p53 and p27 determines cisplatin sensitivity and limits the effectiveness of an IGF-1R pathway inhibitor

    PubMed Central

    Davaadelger, Batzaya; Duan, Lei; Perez, Ricardo E.; Gitelis, Steven; Maki, Carl G.

    2016-01-01

    The insulin-like growth factor-1 receptor (IGF-1R) signaling pathway is aberrantly activated in multiple cancers and can promote proliferation and chemotherapy resistance. Multiple IGF-1R inhibitors have been developed as potential therapeutics. However, these inhibitors have failed to increase patient survival when given alone or in combination with chemotherapy agents. The reason(s) for the disappointing clinical effect of these inhibitors is not fully understood. Cisplatin (CP) activated the IGF-1R/AKT/mTORC1 pathway and stabilized p53 in osteosarcoma (OS) cells. p53 knockdown reduced IGF-1R/AKT/mTORC1 activation by CP, and IGF-1R inhibition reduced the accumulation of p53. These data demonstrate positive crosstalk between p53 and the IGF-1R/AKT/mTORC1 pathway in response to CP. Further studies showed the effect of IGF-1R inhibition on CP response is dependent on p53 status. In p53 wild-type cells treated with CP, IGF-1R inhibition increased p53s apoptotic function but reduced p53-dependent senescence, and had no effect on long term survival. In contrast, in p53-null/knockdown cells, IGF-1R inhibition reduced apoptosis in response to CP and increased long term survival. These effects were due to p27 since IGF-1R inhibition stabilized p27 in CP-treated cells, and p27 depletion restored apoptosis and reduced long term survival. Together, the results demonstrate 1) p53 expression determines the effect of IGF-1R inhibition on cancer cell CP response, and 2) crosstalk between the IGF-1R/AKT/mTORC1 pathway and p53 and p27 can reduce cancer cell responsiveness to chemotherapy and may ultimately limit the effectiveness of IGF-1R pathway inhibitors in the clinic. PMID:27050276

  12. New paradigm for allosteric regulation of Escherichia coli aspartate transcarbamoylase.

    PubMed

    Cockrell, Gregory M; Zheng, Yunan; Guo, Wenyue; Peterson, Alexis W; Truong, Jennifer K; Kantrowitz, Evan R

    2013-11-12

    For nearly 60 years, the ATP activation and the CTP inhibition of Escherichia coli aspartate transcarbamoylase (ATCase) has been the textbook example of allosteric regulation. We present kinetic data and five X-ray structures determined in the absence and presence of a Mg(2+) concentration within the physiological range. In the presence of 2 mM divalent cations (Mg(2+), Ca(2+), Zn(2+)), CTP does not significantly inhibit the enzyme, while the allosteric activation by ATP is enhanced. The data suggest that the actual allosteric inhibitor of ATCase in vivo is the combination of CTP, UTP, and a divalent cation, and the actual allosteric activator is a divalent cation with ATP or ATP and GTP. The structural data reveals that two NTPs can bind to each allosteric site with a divalent cation acting as a bridge between the triphosphates. Thus, the regulation of ATCase is far more complex than previously believed and calls many previous studies into question. The X-ray structures reveal that the catalytic chains undergo essentially no alternations; however, several regions of the regulatory chains undergo significant structural changes. Most significant is that the N-terminal region of the regulatory chains exists in different conformations in the allosterically activated and inhibited forms of the enzyme. Here, a new model of allosteric regulation is proposed.

  13. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    PubMed

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  14. Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

    PubMed Central

    Henry, Brian L.; Desai, Umesh R.

    2014-01-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  15. Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

    PubMed

    Henry, Brian L; Desai, Umesh R

    2014-11-01

    Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

  16. Structure-based design of a benzodiazepine scaffold yields a potent allosteric inhibitor of hepatitis C NS5B RNA polymerase.

    PubMed

    Vandyck, Koen; Cummings, Maxwell D; Nyanguile, Origène; Boutton, Carlo W; Vendeville, Sandrine; McGowan, David; Devogelaere, Benoit; Amssoms, Katie; Last, Stefaan; Rombauts, Klara; Tahri, Abdellah; Lory, Pedro; Hu, Lili; Beauchamp, Derek A; Simmen, Kenny; Raboisson, Pierre

    2009-07-23

    HCV NS5B polymerase, an essential and virus-specific enzyme, is an important target for drug discovery. Using structure-based design, we optimized a 1,5-benzodiazepine NS5B polymerase inhibitor chemotype into a new sulfone-containing scaffold. The design yielded potent inhibitor (S)-4c (K(D) = 0.79 nM), which has approximately 20-fold greater affinity for NS5B than its carbonyl analogue (R)-2c.

  17. Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 may protect against cognitive impairment in rats of chronic cerebral hypoperfusion via PI3K/AKT signaling.

    PubMed

    Su, Shao-Hua; Wang, Yue-Qing; Wu, Yi-Fang; Wang, Da-Peng; Lin, Qi; Hai, Jian

    2016-10-15

    The present study further investigated the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase (FAAH) inhibitor URB597 (URB) on chronic cerebral hypoperfusion (CCH)-induced cognitive impairment in rats. Spatial learning and memory were assessed with the Morris water maze and by measuring Long-term potentiation. The expression of microtubule-associated protein-2 (MAP)-2, growth-associated protein-43 (GAP)-43, synaptophysin, cannabinoid receptor 1 (CB1), brain-derived neurotrophic factor (BDNF), FAAH, N-acylphosphatidylethanolamine phospholipase D(NAPE-PLD) and monoacyl glycerol lipase (MGL) as well as phosphoinositide 3-kinase (PI3K)/AKT signaling pathway molecules and downstream targets including AKT, phosphorylated (p-)AKT, cyclic AMP response element- binding protein (CREB), p-CREB, Bcl-2-associated death protein (BAD), p-BAD, glycogen synthase kinase (GSK)-3β, p-GSK-3β, forkhead box protein (FOXO) 3A and p-FOXO3A was determined by western blotting. WIN and URB treatment improved learning and memory performance, effects that were abolished by co-administration of the PI3K/AKT inhibitor LY294002. Moreover, WIN and URB reversed the decreases in MAP-2 and synaptophysin expression resulting from CCH, and stimulated BDNF and CB1 expression as well as CREB, FOXO3A, GSK-3β, and BAD phosphorylation, confirming that WIN and URB mediate neuroprotection by preventing neuronal apoptosis and improving cognition via PI3K/AKT signaling. These findings suggest that WIN and URB are promising agents for therapeutic management of CCH. PMID:27424778

  18. M2698 is a potent dual-inhibitor of p70S6K and Akt that affects tumor growth in mouse models of cancer and crosses the blood-brain barrier

    PubMed Central

    Machl, Andreas; Wilker, Erik W; Tian, Hui; Liu, Xiaohong; Schroeder, Patricia; Clark, Anderson; Huck, Bayard R

    2016-01-01

    Dysregulated PI3K/Akt/mTOR (PAM) pathway signaling occurs in ~30% of human cancers, making it a rational target for new therapies; however, the effectiveness of some PAM pathway inhibitors, such as mTORC rapalogs, may be compromised by a compensatory feedback loop leading to Akt activation. In this study, the p70S6K/Akt dual inhibitor, M2698 (previously MSC2363318A), was characterized as a potential anti-cancer agent through examination of its pharmacokinetic, pharmacodynamic and metabolic properties, and anti-tumor activity. M2698 was highly potent in vitro (IC50 1 nM for p70S6K, Akt1 and Akt3 inhibition; IC50 17 nM for pGSK3β indirect inhibition) and in vivo (IC50 15 nM for pS6 indirect inhibition), and relatively selective (only 6/264 kinases had an IC50 within 10-fold of p70S6K). Orally administered M2698 crossed the blood-brain barrier in rats and mice, with brain tumor exposure 4-fold higher than non-disease brain. Dose-dependent inhibition of target substrate phosphorylation was observed in vitro and in vivo, indicating that M2698 blocked p70S6K to provide potent PAM pathway inhibition while simultaneously targeting Akt to overcome the compensatory feedback loop. M2698 demonstrated dose-dependent tumor growth inhibition in mouse xenograft models derived from PAM pathway-dysregulated human triple-negative (MDA-MB-468) and Her2-expressing breast cancer cell lines (MDA-MB-453 and JIMT-1), and reduced brain tumor burden and prolonged survival in mice with orthotopically implanted U251 glioblastoma. These findings highlight M2698 as a promising PAM pathway inhibitor whose unique mechanism of action and capacity to pass the blood-brain barrier warrant clinical investigation in cancers with PAM pathway dysregulation, and those with central nervous system involvement. PMID:27186432

  19. Identification of 4-(2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a Novel Inhibitor of AKT Kinase

    SciTech Connect

    Heerding, Dirk A.; Rhodes, Nelson; Leber, Jack D.; Clark, Tammy J.; Keenan, Richard M.; Lafrance, Louis V.; Li, Mei; Safonov, Igor G.; Takata, Dennis T.; Venslavsky, Joseph W.; Yamashita, Dennis S.; Choudhry, Anthony E.; Copeland, Robert A.; Lai, Zhihong; Schaber, Michael D.; Tummino, Peter J.; Strum, Susan L.; Wood, Edgar R.; Duckett, Derek R.; Eberwein, Derek; Knick, Victoria B.; Lansing, Timothy J.; McConnell, Randy T.; Zhang, ShuYun; Minthorn, Elisabeth A.; Concha, Nestor O.; Warren, Gregory L.; Kumar, Rakesh

    2009-07-22

    Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC{sub 50} values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3{beta}. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3{beta} phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.

  20. A novel AKT inhibitor, AZD5363, inhibits phosphorylation of AKT downstream molecules, and activates phosphorylation of mTOR and SMG-1 dependent on the liver cancer cell type

    PubMed Central

    ZHANG, YUNCHENG; ZHENG, YUANWEN; FAHEEM, ALI; SUN, TIANTONG; LI, CHUNYOU; LI, ZHE; ZHAO, DIANTANG; WU, CHAO; LIU, JUN

    2016-01-01

    Due to frequent phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway dysregulation, AKT is typically accepted as a promising anticancer therapeutic target. mTOR, in particular, represents a suitable therapeutic target for hepatocellular carcinoma, whilst suppressor with morphogenetic effect on genitalia family member-1 (SMG-1) is believed to serve a potential tumor suppressor role in human cancer. Despite SMG-1 and mTOR belonging to the same PI3K-related kinase family, the interactions between them are not yet fully understood. In the present study, a novel pyrrolopyrimidine-derived compound, AZD5363, was observed to suppress proliferation in liver cancer Hep-G2 and Huh-7 cells by inhibiting the phosphorylation of downstream molecules in the AKT signal pathway, in a dose- and time-dependent manner. AZD5363 activated the phosphorylation of mTOR, dependent on the liver cancer cell type, as it may have differing effects in various liver cancer cell lines. Additionally, AZD5363 also activated SMG-1 within the same liver cancer cells types, which subsequently activated the phosphorylation of mTOR. In conclusion, the present study indicates that AZD5363 inhibited phosphorylation of AKT downstream molecules, and activated phosphorylation of mTOR and SMG-1, dependent on the liver cancer type. PMID:26998062

  1. Decreased expression of B7-H3 reduces the glycolytic capacity and sensitizes breast cancer cells to AKT/mTOR inhibitors

    PubMed Central

    Nunes-Xavier, Caroline E.; Karlsen, Karine Flem; Tekle, Christina; Pedersen, Cathrine; Øyjord, Tove; Hongisto, Vesa; Nesland, Jahn M.; Tan, Ming; Sahlberg, Kristine Kleivi; Fodstad, Øystein

    2016-01-01

    B7 family proteins are important immune response regulators, and can mediate oncogenic signaling and cancer development. We have used human triple-negative breast cancer cell lines with different expression levels of B7-H3 to evaluate its effects on the sensitivity to 22 different anticancer compounds in a drug screen. API-2 (triciribidine) and everolimus (RAD-001), two inhibitors that target the PI3K/AKT/mTOR pathway, showed enhanced inhibition of cell viability and proliferation in B7-H3 knockdown tumor cells compared to their B7-H3 expressing counterparts. Similar inhibition was seen in control cells treated with an anti-B7-H3 monoclonal antibody. In B7-H3 overexpressing cells, the effects of the two drugs were reduced, supported also by in vivo experiments in which B7-H3 overexpressing xenografts were less sensitive to everolimus than control tumors. In API-2 and everolimus-treated B7-H3 overexpressing cells, phospho-mTOR levels were decreased. However, phosphorylation of p70S6K was differentially regulated in B7-H3 cells treated with API-2 or everolimus, suggesting a different B7-H3-mediated mechanism downstream of mTOR. Both API-2 and everolimus decreased the glycolysis of the cells, whereas knockdown of B7-H3 decreased and B7-H3 overexpression increased the glycolytic capacity. In conclusion, we have unveiled a previously unknown relationship between B7-H3 expression and glycolytic capacity in tumor cells, and found that B7-H3 confers resistance to API-2 and everolimus. The results provide novel insights into the function of B7-H3 in cancer, and suggest that targeting of B7-H3 may be a novel alternative to improve current anticancer therapies. PMID:26771843

  2. Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

    PubMed

    Pierra Rouvière, Claire; Amador, Agnès; Badaroux, Eric; Convard, Thierry; Da Costa, Daniel; Dukhan, David; Griffe, Ludovic; Griffon, Jean-François; LaColla, Massimiliano; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna Giulia; McCarville, Joe; Mascia, Valeria; Milhau, Julien; Onidi, Loredana; Paparin, Jean-Laurent; Rahali, Rachid; Sais, Efisio; Seifer, Maria; Surleraux, Dominique; Standring, David; Dousson, Cyril

    2016-09-15

    The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study. PMID:27520942

  3. Synthesis of potent and broad genotypically active NS5B HCV non-nucleoside inhibitors binding to the thumb domain allosteric site 2 of the viral polymerase.

    PubMed

    Pierra Rouvière, Claire; Amador, Agnès; Badaroux, Eric; Convard, Thierry; Da Costa, Daniel; Dukhan, David; Griffe, Ludovic; Griffon, Jean-François; LaColla, Massimiliano; Leroy, Frédéric; Liuzzi, Michel; Loi, Anna Giulia; McCarville, Joe; Mascia, Valeria; Milhau, Julien; Onidi, Loredana; Paparin, Jean-Laurent; Rahali, Rachid; Sais, Efisio; Seifer, Maria; Surleraux, Dominique; Standring, David; Dousson, Cyril

    2016-09-15

    The hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase (RdRp) plays a central role in virus replication. NS5B has no functional equivalent in mammalian cells and, as a consequence, is an attractive target for selective inhibition. This Letter describes the discovery of a new family of HCV NS5B non-nucleoside inhibitors, based on the bioisosterism between amide and phosphonamidate functions. As part of this program, SAR in this new series led to the identification of IDX17119, a potent non-nucleoside inhibitor, active on the genotypes 1b, 2a, 3a and 4a. The structure and binding domain of IDX17119 were confirmed by X-ray co-crystallization study.

  4. Allosteric modulators of the hERG K(+) channel: radioligand binding assays reveal allosteric characteristics of dofetilide analogs.

    PubMed

    Yu, Zhiyi; Klaasse, Elisabeth; Heitman, Laura H; Ijzerman, Adriaan P

    2014-01-01

    Drugs that block the cardiac K(+) channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K(+) channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [(3)H]astemizole and [(3)H]dofetilide to the hERG K(+) channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC50 values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC50 values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K(+) channel, which is discussed in the light of findings on other ion channels. PMID:24200993

  5. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  6. Enhancing allosteric inhibition in Thermus thermophilus Phosphofructokinase.

    PubMed

    McGresham, Maria S; Reinhart, Gregory D

    2015-01-27

    The coupling between the binding of the substrate Fru-6-P and the inhibitor phospho(enol)pyruvate (PEP) in phosphofructokinase (PFK) from the extreme thermophile Thermus thermophilus is much weaker than that seen in a PFK from Bacillus stearothermophilus. From the crystal structures of Bacillus stearothermophilus PFK (BsPFK) the residues at positions 59, 158, and 215 in BsPFK are located on the path leading from the allosteric site to the nearest active site and are part of the intricate hydrogen-bonding network connecting the two sites. Substituting the corresponding residues in Thermus thermophilus PFK (TtPFK) with the amino acids found at these positions in BsPFK allowed us to enhance the allosteric inhibition by PEP by nearly 3 kcal mol(-1) (50-fold) to a value greater than or equal to the coupling observed in BsPFK. Interestingly, each single variant N59D, A158T, and S215H produced a roughly 1 kcal mol(-1) increase in coupling free energy of inhibition. The effects of these variants were essentially additive in the three combinations of double variants N59D/A158T, N59D/S215H, and A158T/S215H as well as in the triple variant N59D/A158T/S215H. Consequently, while the hydrogen-bonding network identified is likely involved in the inhibitory allosteric communication, a model requiring a linked chain of interactions connecting the sites is not supported by these data. Despite the fact that the allosteric activator of the bacterial PFK, MgADP, binds at the same allosteric site, the substitutions at positions 59, 158, and 215 do not have an equally dramatic effect on the binding affinity and the allosteric activation by MgADP. The effect of the S215H and N59D/A158T/S215H substitutions on the activation by MgADP could not be determined because of a dramatic drop in MgADP binding affinity that resulted from the S215H substitution. The single variants N59D and A158T supported binding but showed little change in the free energy of activation by MgADP compared to the wild

  7. Molecular and Functional Characterization of Three Different Postzygotic Mutations in PIK3CA-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

    PubMed Central

    Forte, Giovanna; Bagnulo, Rosanna; Stella, Alessandro; Lastella, Patrizia; Cutrone, Mario; Benedicenti, Francesco; Susca, Francesco C.; Patruno, Margherita; Varvara, Dora; Germani, Aldo; Chessa, Luciana; Laforgia, Nicola; Tenconi, Romano; Simone, Cristiano; Resta, Nicoletta

    2015-01-01

    Background PIK3CA-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic PIK3CA mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed. Methods We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 in vitro. Results and Conclusion Our data indicate that patients’ cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients. PMID:25915946

  8. Antitumor effect of a selective COX-2 inhibitor, celecoxib, may be attributed to angiogenesis inhibition through modulating the PTEN/PI3K/Akt/HIF-1 pathway in an H₂₂ murine hepatocarcinoma model.

    PubMed

    Sui, Wenwen; Zhang, Yueying; Wang, Zhaopeng; Wang, Zhaoxia; Jia, Qing; Wu, Licun; Zhang, Weidong

    2014-05-01

    Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has recently been shown to affect the development of different types of cancer. The present study utilized a murine H22 hepatocarcinoma model to investigate the molecular mechanisms involved in celecoxib-induced inhibition of tumor angiogenesis. Tumor-bearing mice were randomly divided into five groups: i) control; ii) low-dose celecoxib (50 mg/kg); iii) high-dose celecoxib (200 mg/kg); iv) 5-fluorouracil (5-FU), (20 mg/kg) and v) combination of 5-FU and celecoxib (50 mg/kg). The antitumor effect of celecoxib was determined by measuring tumor volume. Tumor angiogenesis was evaluated by microvessel density (MVD). Tumor histology and immunostaining for CD34 in endothelial cells were performed to detect MVD. The expression levels of phosphatase and tensin homologue deleted from chromosome 10 (PTEN), phosphatidylinositol 3-kinase (PI3K), phospho‑Akt (P-Akt), COX-2, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor-A (VEGF-A) were detected by ELISA, immunohistochemistry and western blotting, respectively. We discovered substantial growth delay in murine H22 hepatoma as a result of celecoxib treatment. The inhibition rate of tumor growth induced by high-dose and low-dose celecoxib was 49.3 and 37.0%, respectively (P<0.05). The expression of PI3K, P-Akt, COX-2, HIF-1α, VEGF-A and PTEN in tumor tissues treated with celecoxib was demonstrated by immunohistochemistry, and the MVD was decreased in a dose-dependent manner (P<0.05). Reduced PI3K and P-Akt was particularly apparent in the high-dose celecoxib group (P<0.05). ELISA and western blotting data showed that the expression of PI3K, P-Akt, COX-2, HIF-1α and VEGF-A were reduced and PTEN was increased after treatment with celecoxib. In conclusion, the impact of celecoxib-induced tumor growth delay of murine H22 hepatocarcinoma may correlate with the inhibition of angiogenesis by reducing PI3K, P-Akt, COX-2, HIF-1α and VEGF

  9. A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction

    PubMed Central

    Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

    2015-01-01

    The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the

  10. A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

    PubMed

    Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O

    2015-06-19

    The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the

  11. The phosphatidylinositol-3-kinase inhibitor NVP-BKM120 overcomes resistance signals derived from microenvironment by regulating the Akt/FoxO3a/Bim axis in chronic lymphocytic leukemia cells

    PubMed Central

    Rosich, Laia; Saborit-Villarroya, Ifigènia; López-Guerra, Mónica; Xargay-Torrent, Sílvia; Montraveta, Arnau; Aymerich, Marta; Villamor, Neus; Campo, Elias; Pérez-Galán, Patricia; Roué, Gaël; Colomer, Dolors

    2013-01-01

    Phosphatidylinositol-3-kinase pathway is constitutively activated in chronic lymphocytic leukemia mainly due to microenvironment signals, including stromal cell interaction and CXCR4 and B-cell receptor activation. Because of the importance of phosphatidylinositol-3-kinase signaling in chronic lymphocytic leukemia, we investigated the activity of the NVP-BKM120, an orally available pan class I phosphatidylinositol-3-kinase inhibitor. Sensitivity to NVP-BKM120 was analyzed in chronic lymphocytic leukemia primary samples in the context of B-cell receptor and microenvironment stimulation. NVP-BKM120 promoted mitochondrial apoptosis in most primary cells independently of common prognostic markers. NVP-BKM120 activity induced the blockage of phosphatidylinositol-3-kinase signaling, decreased Akt and FoxO3a phosphorylation leading to concomitant Mcl-1 downregulation and Bim induction. Accordingly, selective knockdown of BIM rescued cells from NVP-BKM120-induced apoptosis, while the kinase inhibitor synergistically enhanced the apoptosis induced by the BH3-mimetic ABT-263. We also found NVP-BKM120 to inhibit B-cell receptor- and stroma-dependent Akt pathway activation, thus sensitizing chronic lymphocytic leukemia cells to bendamustine and fludarabine. Furthermore, NVP-BKM120 down-regulated secretion of chemokines after B-cell receptor stimulation and inhibited cell chemotaxis and actin polymerization upon CXCR4 triggering by CXCL12. Our findings establish that NVP-BKM120 effectively inhibits the phosphatidylinositol-3-kinase signaling pathway and disturbs the protective effect of the tumor microenvironment with the subsequent apoptosis induction through the Akt/FoxO3a/Bim axis. We provide here a strong rationale for undertaking clinical trials of NVP-BKM120 in chronic lymphocytic leukemia patients alone or in combination therapies. PMID:23850807

  12. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  13. Oxytocin Increases Invasive Properties of Endometrial Cancer Cells Through Phosphatidylinositol 3-Kinase/AKT-Dependent Up-Regulation of Cyclooxygenase-1, -2, and X-Linked Inhibitor of Apoptosis Protein1

    PubMed Central

    Déry, Marie-Claude; Chaudhry, Parvesh; Leblanc, Valérie; Parent, Sophie; Fortier, Anne-Marie; Asselin, Eric

    2011-01-01

    Traditionally, oxytocin (OT) is well known to play a crucial role in the regulation of cyclic changes in the uterus, implantation of the embryo, and parturition. Recently, an additional role for OT has been identified in several types of cancer cells in which OT acts as a growth regulator. In endometrial cancer cells, OT is known to efficiently inhibit cellular proliferation. In the present study, we show that OT increases invasiveness of human endometrial carcinoma (HEC) cells, which are otherwise resistant to the growth-inhibiting effects of OT. Using pharmacological inhibitors, invasion assay, RNA interference, and immunofluorescence, we found that OT enhances the invasive properties of HEC cells through up-regulation of X-linked inhibitor of apoptosis protein (XIAP), matrix-metalloproteinase 2 (MMP2), and matrix-metalloproteinase 14 (MMP14). In addition, we show that OT-mediated invasion is both cyclooxygenase 1 (PTGS1) and cyclooxygenase-2 (PTGS2) dependent via the phosphatidylinositol 3-kinase/AKT (PIK3/AKT) pathway. PTGS2 knockdown by shRNA resulted in XIAP down-regulation. We also show that OT receptor is overexpressed in grade I to III endometrial cancer. Taken together, our results describe for the first time a novel role for OT in endometrial cancer cell invasion. PMID:21816851

  14. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

    NASA Astrophysics Data System (ADS)

    Novinec, Marko; Korenč, Matevž; Caflisch, Amedeo; Ranganathan, Rama; Lenarčič, Brigita; Baici, Antonio

    2014-02-01

    Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

  15. Allosteric modulators of the hERG K{sup +} channel

    SciTech Connect

    Yu, Zhiyi Klaasse, Elisabeth Heitman, Laura H. IJzerman, Adriaan P.

    2014-01-01

    Drugs that block the cardiac K{sup +} channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K{sup +} channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [{sup 3}H]astemizole and [{sup 3}H]dofetilide to the hERG K{sup +} channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC{sub 50} values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC{sub 50} values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K{sup +} channel, which is discussed in the light of findings on other ion channels. - Highlights: • Allosteric modulators on the hERG K{sup +} channel were evaluated in binding assays. • LUF6200 was identified as a potent allosteric inhibitor. • Potassium ions were found to behave as allosteric enhancers. • Positive cooperativity and distinct allosteric sites for them were proposed.

  16. Targeting the PI3K/AKT/mTOR pathway overcomes the stimulating effect of dabrafenib on the invasive behavior of melanoma cells with acquired resistance to the BRAF inhibitor.

    PubMed

    Caporali, Simona; Alvino, Ester; Lacal, Pedro Miguel; Levati, Lauretta; Giurato, Giorgio; Memoli, Domenico; Caprini, Elisabetta; Antonini Cappellini, Gian Carlo; D'Atri, Stefania

    2016-09-01

    BRAF inhibitors (BRAFi) have proven clinical benefits in patients with BRAF-mutant melanoma. However, acquired resistance eventually arises. The effects of BRAFi on melanoma cell proliferation and survival have been extensively studied, and several mechanisms involved in acquired resistance to the growth suppressive activity of these drugs have been identified. Much less is known about the impact of BRAFi, and in particular of dabrafenib, on the invasive potential of melanoma cells. In the present study, the BRAF-mutant human melanoma cell line A375 and its dabrafenib-resistant subline A375R were analyzed for invasive capacity, expression of vascular endothelial growth factor receptor (VEGFR)-2, and secretion of VEGF-A and matrix metalloproteinase (MMP)-9, under basal conditions or in response to dabrafenib. The consequences of inhibiting the PI3K/AKT/mTOR pathway on A375R cell responses to dabrafenib were also evaluated. We found that A375R cells were more invasive and secreted higher levels of VEGF-A and MMP-9 as compared with A375 cells. Dabrafenib reduced invasiveness, VEGFR-2 expression and VEGF-A secretion in A375 cells, whereas it increased invasiveness, VEGF-A and MMP-9 release in A375R cells. In these latter cells, the stimulating effects of dabrafenib on the invasive capacity were markedly impaired by the anti-VEGF‑A antibody bevacizumab, or by AKT1 silencing. A375R cells were not cross-resistant to the PI3K/mTOR inhibitor GSK2126458A. Moreover, this inhibitor given in combination with dabrafenib efficiently counteracted the stimulating effects of the BRAFi on invasiveness and VEGF-A and MMP-9 secretion. Our data demonstrate that melanoma cells with acquired resistance to dabrafenib possess a more invasive phenotype which is further stimulated by exposure to the drug. Substantial evidence indicates that continuing BRAFi therapy beyond progression produces a clinical benefit. Our results suggest that after the development of resistance, a regimen

  17. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  18. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.

    PubMed

    Burgess, Selena G; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W; Vernos, Isabelle; Matthews, David; Bayliss, Richard

    2016-07-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors. PMID:27411893

  19. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain

    PubMed Central

    Burgess, Selena G.; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W.; Vernos, Isabelle; Matthews, David

    2016-01-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors. PMID:27411893

  20. MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells

    PubMed Central

    Viola, Giampietro; Bortolozzi, Roberta; Hamel, Ernest; Moro, Stefano; Brun, Paola; Castagliuolo, Ignazio; Ferlin, Maria Grazia; Basso, Giuseppe

    2011-01-01

    We previously demonstrated that MG-2477 (3-cyclopropylmethyl-7-phenyl-3H-pyrrolo[3,2-f]quinolin-9(6H)-one) inhibits the growth of several cancer cell lines in vitro. Here we show that MG-2477 inhibited tubulin polymerization and caused cells to arrest in metaphase. The detailed mechanism of action of MG-2477 was investigated in a non-small cell lung carcinoma cell line (A549). Treatment of A549 cells with MG-2477 caused the cells to arrest in the G2/M phase of the cell cycle, with a concomitant accumulation of cyclin B. Moreover, the compound induced autophagy, which was followed at later times by apoptotic cell death. Autophagy was detected as early as 12 h by the conversion of microtubule associated protein 1 light chain 3 (LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. After 48 h of MG-2477 exposure, phosphatidylserine externalization on the cell membrane, caspase-3 activation, and PARP cleavage occurred, revealing that apoptotic cell death had begun. Pharmacological inhibition of autophagy with 3-methyladenine or bafilomycin A1 increased apoptotic cell death, suggesting that the autophagy caused by MG-2477 played a protective role and delayed apoptotic cell death. Additional studies revealed that MG-2477 inhibited survival signaling by blocking activation of Akt and its downstream targets, including mTOR, and FHKR. Treatment with MG-2477 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase and 4E-BP1. Overexpression of Akt by transfection with a Myr-Akt vector decreased MG-2477 induced autophagy, indicating that Akt is involved. Taken together, these results indicated that the autophagy induced by MG-2477 delayed apoptosis by exerting an adaptive response following microtubule damage. PMID:21964343

  1. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    NASA Astrophysics Data System (ADS)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  2. Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

    PubMed

    Costa, Barbara; Dangate, Milind; Vetro, Maria; Donvito, Giulia; Gabrielli, Luca; Amigoni, Loredana; Cassinelli, Giuliana; Lanzi, Cinzia; Ceriani, Michela; De Gioia, Luca; Filippi, Giulia; Cipolla, Laura; Zaffaroni, Nadia; Perego, Paola; Colombo, Diego

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

  3. Enzyme Inhibition by Allosteric Capture of an Inactive Conformation

    PubMed Central

    Lee, Gregory M.; Shahian, Tina; Baharuddin, Aida; Gable, Jonathan E.; Craik, Charles S.

    2011-01-01

    All members of the human herpesvirus protease family are active as weakly associating dimers, but inactive as monomers. A small molecule allosteric inhibitor of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low μM affinity. A 2.0 Å resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 Å from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease (HCMV Pr) via a similar mechanism. As all HHV proteases are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics which allosterically regulate enzymatic activity by disrupting protein-protein interactions. PMID:21723875

  4. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  5. Cardioprotective effects of the novel Na+/H+ exchanger-1 inhibitor KR-32560 in a perfused rat heart model of global ischemia and reperfusion: Involvement of the Akt-GSK-3beta cell survival pathway and antioxidant enzyme.

    PubMed

    Jung, In-Sang; Lee, Sung-Hun; Yang, Min-Kyu; Park, Jung-Woo; Yi, Kyu-Yang; Yoo, Sung-Eun; Kwon, Suk-Hyung; Chung, Hun-Jong; Choi, Wahn-Soo; Shin, Hwa-Sup

    2010-08-01

    To investigate the cardioprotective effects and mechanism of action of KR-32560 {[5-(2-methoxy-5-fluorophenyl)furan-2-ylcarbonyl]guanidine}, a newly synthesized NHE-1 inhibitor, we evaluated the effects of KR-32560 on cardiac function in a rat model of ischemia/reperfusion (I/R)-induced heart injury as well as the role antioxidant enzymes and pro-survival proteins play these observed effects. In isolated rat hearts subjected to 25 min of global ischemia followed by 30 min of reperfusion, KR-32560 (3 and 10 microM) significantly reversed the I/Rinduced decrease in left ventricular developed pressure and increase in left ventricular enddiastolic pressure. In rat hearts reperfused for 30 min, KR-32560 (10 microM) significantly decreased the malondialdehyde content while increasing the activities of both glutathione peroxidase and catalase, two important antioxidant enzymes. Western blotting analysis of left ventricles subjected to I/R showed that KR-32560 significantly increased phosphorylation of both Akt and GSK-3beta in a dose-dependent manner, with no effect on the phosphorylation of eNOS. These results suggest that KR-32560 exerts potent cardioprotective effects against I/Rinduced rat heart injury and that its mechanism involves antioxidant enzymes and the Akt-GSK-3beta cell survival pathway.

  6. Allosteric proteins as logarithmic sensors

    PubMed Central

    Olsman, Noah; Goentoro, Lea

    2016-01-01

    Many sensory systems, from vision and hearing in animals to signal transduction in cells, respond to fold changes in signal relative to background. Responding to fold change requires that the system senses signal on a logarithmic scale, responding identically to a change in signal level from 1 to 3, or from 10 to 30. It is an ongoing search in the field to understand the ways in which a logarithmic sensor can be implemented at the molecular level. In this work, we present evidence that logarithmic sensing can be implemented with a single protein, by means of allosteric regulation. Specifically, we find that mathematical models show that allosteric proteins can respond to stimuli on a logarithmic scale. Next, we present evidence from measurements in the literature that some allosteric proteins do operate in a parameter regime that permits logarithmic sensing. Finally, we present examples suggesting that allosteric proteins are indeed used in this capacity: allosteric proteins play a prominent role in systems where fold-change detection has been proposed. This finding suggests a role as logarithmic sensors for the many allosteric proteins across diverse biological processes. PMID:27410043

  7. Discovery of 4-Amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides As Selective, Orally Active Inhibitors of Protein Kinase B (Akt)†

    PubMed Central

    2010-01-01

    Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses. PMID:20151677

  8. Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR

    PubMed Central

    Verbrugge, Sue Ellen; Al, Marjon; Assaraf, Yehuda G.; Kammerer, Sarah; Chandrupatla, Durga M.S.H.; Honeywell, Richard; Musters, Rene P.J.; Giovannetti, Elisa; O'Toole, Tom; Scheffer, George L.; Krige, David; de Gruijl, Tanja D.; Niessen, Hans W.M.; Lems, Willem F.; Kramer, Pieternella A.; Scheper, Rik J.; Cloos, Jacqueline; Ossenkoppele, Gert J.; Peters, Godefridus J.; Jansen, Gerrit

    2016-01-01

    Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells. PMID:26496029

  9. The predominant protective effect of tianeptine over other antidepressants in models of neuronal apoptosis: the effect blocked by inhibitors of MAPK/ERK1/2 and PI3-K/Akt pathways.

    PubMed

    Jantas, D; Krawczyk, S; Lason, W

    2014-02-01

    Tianeptine (Tian) possesses neuroprotective potential, however, little is known about the effect of this drug in models of neuronal apoptosis. In the present study, we aimed (1) to compare the neuroprotective capacities of some antidepressants (ADs) in the models of staurosporine (St)- and doxorubicin (Dox)-evoked cell death, activating the intracellular and the extracellular apoptotic pathway, respectively; (2) to identify the Tian-modulated steps underlying its neuroprotective action; (3) to test the effect of various ADs against Dox-evoked cell damage in glia cells. Primary neuronal and glia cell cultures and retinoic acid-differentiated human neuroblastoma SH-SY5Y (RA-SH-SY5Y) cells were co-treated with imipramine, fluoxetine, citalopram, reboxetine, mirtazapine or Tian and St or Dox. The data showed the predominant neuroprotective effect of Tian over other tested ADs against St- and Dox-induced cell damage in primary neurons and in RA-SH-SY5Y cells. This effect was shown to be caspase-3-independent but connected with attenuation of DNA fragmentation. Moreover, neuroprotection elicited by Tian was blocked by pharmacological inhibitors of MAPK/ERK1/2 and PI3-K/Akt signaling pathways as well by inhibitor of necroptosis, necrostatin-1. Interestingly, the protective effects of all tested ADs were demonstrated in primary glia cells against the Dox-evoked cell damage. The obtained data suggests the glial cells as a common target for protective action of various ADs whereas in relation to neuronal cells only Tian possesses such properties, at least against St- and Dox-induced cell damage. Moreover, this neuroprotective effect of Tian is caspase-3-independent and engages the regulation of survival pathways (MAPK/ERK1/2 and PI3-K/Akt).

  10. The predominant protective effect of tianeptine over other antidepressants in models of neuronal apoptosis: the effect blocked by inhibitors of MAPK/ERK1/2 and PI3-K/Akt pathways.

    PubMed

    Jantas, D; Krawczyk, S; Lason, W

    2014-02-01

    Tianeptine (Tian) possesses neuroprotective potential, however, little is known about the effect of this drug in models of neuronal apoptosis. In the present study, we aimed (1) to compare the neuroprotective capacities of some antidepressants (ADs) in the models of staurosporine (St)- and doxorubicin (Dox)-evoked cell death, activating the intracellular and the extracellular apoptotic pathway, respectively; (2) to identify the Tian-modulated steps underlying its neuroprotective action; (3) to test the effect of various ADs against Dox-evoked cell damage in glia cells. Primary neuronal and glia cell cultures and retinoic acid-differentiated human neuroblastoma SH-SY5Y (RA-SH-SY5Y) cells were co-treated with imipramine, fluoxetine, citalopram, reboxetine, mirtazapine or Tian and St or Dox. The data showed the predominant neuroprotective effect of Tian over other tested ADs against St- and Dox-induced cell damage in primary neurons and in RA-SH-SY5Y cells. This effect was shown to be caspase-3-independent but connected with attenuation of DNA fragmentation. Moreover, neuroprotection elicited by Tian was blocked by pharmacological inhibitors of MAPK/ERK1/2 and PI3-K/Akt signaling pathways as well by inhibitor of necroptosis, necrostatin-1. Interestingly, the protective effects of all tested ADs were demonstrated in primary glia cells against the Dox-evoked cell damage. The obtained data suggests the glial cells as a common target for protective action of various ADs whereas in relation to neuronal cells only Tian possesses such properties, at least against St- and Dox-induced cell damage. Moreover, this neuroprotective effect of Tian is caspase-3-independent and engages the regulation of survival pathways (MAPK/ERK1/2 and PI3-K/Akt). PMID:24105645

  11. BGP-15, a PARP-inhibitor, prevents imatinib-induced cardiotoxicity by activating Akt and suppressing JNK and p38 MAP kinases.

    PubMed

    Sarszegi, Zsolt; Bognar, Eszter; Gaszner, Balazs; Kónyi, Attila; Gallyas, Ferenc; Sumegi, Balazs; Berente, Zoltan

    2012-06-01

    In this study, we investigate the cardiotoxic effects of the well-known cytostatic agent imatinib mesylate (Gleevec), and presented evidence for the cardioprotective effect of BGP-15 which is a novel insulin sensitizer. The cardiotoxic effect of imatinib mesylate was assessed in Langendorff rat heart perfusion system. The cardiac high-energy phosphate levels (creatine phosphate (PCr) and ATP) were monitored in situ by (31)P NMR spectroscopy. The protein oxidation, lipid peroxidation, and the activation of signaling pathways were determined from the freeze-clamped hearts. Prolonged treatment of the heart with imatinib mesylate (20 mg/kg) resulted in cardiotoxicity, which were characterized by the depletion of high-energy phosphates (PCr and ATP), and significantly increased protein oxidation and lipid peroxidation. Imatinib mesylate treatment-induced activation of MAP kinases (including ERK1/2, p38, and JNK) and the phosphorylation of Akt and GSK-3beta. BGP-15 (200 μM) prevented the imatinib mesylate-induced oxidative damages, attenuated the depletion of high-energy phosphates, altered the signaling effect of imatinib mesylate by preventing p38 MAP kinase and JNK activation, and induced the phosphorylation of Akt and GSK-3beta. The suppressive effect of BGP-15 on p38 and JNK activation could be significant because these kinases contribute to the cell death and inflammation in the isolated perfused heart.

  12. Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets.

    PubMed

    Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol; Chernoff, Jonathan; Kunapuli, Satya P

    2015-01-01

    Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated after translocation to the membrane through Gi signaling pathways by a phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared with adenosine 5'-diphosphate in platelets. This study investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor agonists caused translocation of Akt to the membrane rapidly, whereas phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq pathways. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3K/PIP3/Gi-independent mechanism. An Akt scaffolding protein, p21-activated kinase (PAK), translocates to the membrane after stimulation with protease-activated receptor agonists in a Gq-dependent manner, with the kinetics of translocation similar to that of Akt. Coimmunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a possible role of PAK in Akt translocation. These results show, for the first time, an important role of the Gq pathway in mediating Akt translocation to the membrane in a novel Gi/PI3K/PIP3-independent mechanism.

  13. Gq-mediated Akt translocation to the membrane: a novel PIP3-independent mechanism in platelets

    PubMed Central

    Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol; Chernoff, Jonathan

    2015-01-01

    Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated after translocation to the membrane through Gi signaling pathways by a phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared with adenosine 5′-diphosphate in platelets. This study investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor agonists caused translocation of Akt to the membrane rapidly, whereas phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq pathways. Interestingly, phosphatidylinositol 3-kinase (PI3K) inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3K/PIP3/Gi-independent mechanism. An Akt scaffolding protein, p21-activated kinase (PAK), translocates to the membrane after stimulation with protease-activated receptor agonists in a Gq-dependent manner, with the kinetics of translocation similar to that of Akt. Coimmunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a possible role of PAK in Akt translocation. These results show, for the first time, an important role of the Gq pathway in mediating Akt translocation to the membrane in a novel Gi/PI3K/PIP3-independent mechanism. PMID:25331114

  14. Akt phosphorylation is essential for nuclear translocation and retention in NGF-stimulated PC12 cells

    SciTech Connect

    Truong Le Xuan Nguyen; Choi, Joung Woo; Lee, Sang Bae; Ye, Keqiang; Woo, Soo-Dong; Lee, Kyung-Hoon; Ahn, Jee-Yin . E-mail: jyahn@med.skku.ac.kr

    2006-10-20

    Nerve growth factor (NGF) elicits Akt translocation into the nucleus, where it phosphorylates nuclear targets. Here, we describe that Akt phosphorylation can promote the nuclear translocation of Akt and is necessary for its nuclear retention. Overexpression of Akt-K179A, T308A, S473A-mutant failed to show either nuclear translocation or nuclear Akt phosphorylation, whereas expression of wild-type counterpart elicited profound Akt phosphorylation and induced nuclear translocation under NGF stimulation. Employing the PI3K inhibitor and a variety of mutants PI3K, we showed that nuclear translocation of Akt was mediated by activation of PI3K, and Akt phosphorylation status in the nucleus required PI3K activity. Thus the activity of PI3K might contribute to the nuclear translocation of Akt, and that Akt phosphorylation is essential for its nuclear retention under NGF stimulation conditions.

  15. Glutamate dehydrogenase: structure, allosteric regulation, and role in insulin homeostasis.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2014-01-01

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of L-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine and inhibitors include GTP, palmitoyl CoA, and ATP. Spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds blocked the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  16. The structure and allosteric regulation of mammalian glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2012-03-15

    Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate. Only in the animal kingdom is this enzyme heavily allosterically regulated by a wide array of metabolites. The major activators are ADP and leucine, while the most important inhibitors include GTP, palmitoyl CoA, and ATP. Recently, spontaneous mutations in the GTP inhibitory site that lead to the hyperinsulinism/hyperammonemia (HHS) syndrome have shed light as to why mammalian GDH is so tightly regulated. Patients with HHS exhibit hypersecretion of insulin upon consumption of protein and concomitantly extremely high levels of ammonium in the serum. The atomic structures of four new inhibitors complexed with GDH complexes have identified three different allosteric binding sites. Using a transgenic mouse model expressing the human HHS form of GDH, at least three of these compounds were found to block the dysregulated form of GDH in pancreatic tissue. EGCG from green tea prevented the hyper-response to amino acids in whole animals and improved basal serum glucose levels. The atomic structure of the ECG-GDH complex and mutagenesis studies is directing structure-based drug design using these polyphenols as a base scaffold. In addition, all of these allosteric inhibitors are elucidating the atomic mechanisms of allostery in this complex enzyme.

  17. ASBench: benchmarking sets for allosteric discovery.

    PubMed

    Huang, Wenkang; Wang, Guanqiao; Shen, Qiancheng; Liu, Xinyi; Lu, Shaoyong; Geng, Lv; Huang, Zhimin; Zhang, Jian

    2015-08-01

    Allostery allows for the fine-tuning of protein function. Targeting allosteric sites is gaining increasing recognition as a novel strategy in drug design. The key challenge in the discovery of allosteric sites has strongly motivated the development of computational methods and thus high-quality, publicly accessible standard data have become indispensable. Here, we report benchmarking data for experimentally determined allosteric sites through a complex process, including a 'Core set' with 235 unique allosteric sites and a 'Core-Diversity set' with 147 structurally diverse allosteric sites. These benchmarking sets can be exploited to develop efficient computational methods to predict unknown allosteric sites in proteins and reveal unique allosteric ligand-protein interactions to guide allosteric drug design.

  18. Structures of pyruvate kinases display evolutionarily divergent allosteric strategies.

    PubMed

    Morgan, Hugh P; Zhong, Wenhe; McNae, Iain W; Michels, Paul A M; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D

    2014-09-01

    The transition between the inactive T-state (apoenzyme) and active R-state (effector bound enzyme) of Trypanosoma cruzi pyruvate kinase (PYK) is accompanied by a symmetrical 8° rigid body rocking motion of the A- and C-domain cores in each of the four subunits, coupled with the formation of additional salt bridges across two of the four subunit interfaces. These salt bridges provide increased tetramer stability correlated with an enhanced specificity constant (k cat/S 0.5). A detailed kinetic and structural comparison between the potential drug target PYKs from the pathogenic protists T. cruzi, T. brucei and Leishmania mexicana shows that their allosteric mechanism is conserved. By contrast, a structural comparison of trypanosomatid PYKs with the evolutionarily divergent PYKs of humans and of bacteria shows that they have adopted different allosteric strategies. The underlying principle in each case is to maximize (k cat/S 0.5) by stabilizing and rigidifying the tetramer in an active R-state conformation. However, bacterial and mammalian PYKs have evolved alternative ways of locking the tetramers together. In contrast to the divergent allosteric mechanisms, the PYK active sites are highly conserved across species. Selective disruption of the varied allosteric mechanisms may therefore provide a useful approach for the design of species-specific inhibitors.

  19. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  20. Allosteric Modulation of Chemoattractant Receptors

    PubMed Central

    Allegretti, Marcello; Cesta, Maria Candida; Locati, Massimo

    2016-01-01

    Chemoattractants control selective leukocyte homing via interactions with a dedicated family of related G protein-coupled receptor (GPCR). Emerging evidence indicates that the signaling activity of these receptors, as for other GPCR, is influenced by allosteric modulators, which interact with the receptor in a binding site distinct from the binding site of the agonist and modulate the receptor signaling activity in response to the orthosteric ligand. Allosteric modulators have a number of potential advantages over orthosteric agonists/antagonists as therapeutic agents and offer unprecedented opportunities to identify extremely selective drug leads. Here, we resume evidence of allosterism in the context of chemoattractant receptors, discussing in particular its functional impact on functional selectivity and probe/concentration dependence of orthosteric ligands activities. PMID:27199992

  1. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA.

  2. Doing justice to allosteric regulation.

    PubMed

    Keller, Evelyn Fox

    2015-06-01

    Jacques Monod gave us not only our first regulatory system, but also our first smart molecules - i.e., he gave us allosteric proteins. But both of these contributions hung in a certain tension with his primary commitments. In particular, I focus here on the ways in which his ontological commitments constrained his thinking about the power of allostery. Although he wrote that "so far as regulation through allosteric interaction is concerned, everything is possible", for him, not everything was conceivable. In particular, what was not conceivable was a challenge to the primacy of DNA. PMID:25908117

  3. Akting up in the GABA hypothesis of schizophrenia: Akt1 deficiency modulates GABAergic functions and hippocampus-dependent functions.

    PubMed

    Chang, Chia-Yuan; Chen, Yi-Wen; Wang, Tsu-Wei; Lai, Wen-Sung

    2016-01-01

    Accumulating evidence implies that both AKT1 and GABAA receptor (GABAAR) subunit genes are involved in schizophrenia pathogenesis. Activated Akt promotes GABAergic neuron differentiation and increases GABAAR expression on the plasma membrane. To elucidate the role of Akt1 in modulating GABAergic functions and schizophrenia-related cognitive deficits, a set of 6 in vitro and in vivo experiments was conducted. First, an Akt1/2 inhibitor was applied to evaluate its effect on GABAergic neuron-like cell formation from P19 cells. Inhibiting Akt resulted in a reduction in parvalbumin-positive neuron-like cells. In Akt1(-/-) and wild-type mice, seizures induced using pentylenetetrazol (a GABAAR antagonist) were measured, and GABAAR expression and GABAergic interneuron abundance in the brain were examined. Female Akt1(-/-) mice, but not male Akt1(-/-) mice, exhibited less pentylenetetrazol-induced convulsive activity than their corresponding wild-type controls. Reduced parvalbumin-positive interneuron abundance and GABAAR subunit expression, especially in the hippocampus, were also observed in female Akt1(-/-) mice compared to female wild-type mice. Neuromorphometric analyses revealed significantly reduced neurite complexity in hippocampal pyramidal neurons. Additionally, female Akt1(-/-) mice displayed increased hippocampal oscillation power and impaired spatial memory compared to female wild-type mice. Our findings suggest that Akt1 deficiency modulates GABAergic interneurons and GABAAR expression, contributing to hippocampus-dependent cognitive functional impairment.

  4. Akting up in the GABA hypothesis of schizophrenia: Akt1 deficiency modulates GABAergic functions and hippocampus-dependent functions.

    PubMed

    Chang, Chia-Yuan; Chen, Yi-Wen; Wang, Tsu-Wei; Lai, Wen-Sung

    2016-01-01

    Accumulating evidence implies that both AKT1 and GABAA receptor (GABAAR) subunit genes are involved in schizophrenia pathogenesis. Activated Akt promotes GABAergic neuron differentiation and increases GABAAR expression on the plasma membrane. To elucidate the role of Akt1 in modulating GABAergic functions and schizophrenia-related cognitive deficits, a set of 6 in vitro and in vivo experiments was conducted. First, an Akt1/2 inhibitor was applied to evaluate its effect on GABAergic neuron-like cell formation from P19 cells. Inhibiting Akt resulted in a reduction in parvalbumin-positive neuron-like cells. In Akt1(-/-) and wild-type mice, seizures induced using pentylenetetrazol (a GABAAR antagonist) were measured, and GABAAR expression and GABAergic interneuron abundance in the brain were examined. Female Akt1(-/-) mice, but not male Akt1(-/-) mice, exhibited less pentylenetetrazol-induced convulsive activity than their corresponding wild-type controls. Reduced parvalbumin-positive interneuron abundance and GABAAR subunit expression, especially in the hippocampus, were also observed in female Akt1(-/-) mice compared to female wild-type mice. Neuromorphometric analyses revealed significantly reduced neurite complexity in hippocampal pyramidal neurons. Additionally, female Akt1(-/-) mice displayed increased hippocampal oscillation power and impaired spatial memory compared to female wild-type mice. Our findings suggest that Akt1 deficiency modulates GABAergic interneurons and GABAAR expression, contributing to hippocampus-dependent cognitive functional impairment. PMID:27615800

  5. Akting up in the GABA hypothesis of schizophrenia: Akt1 deficiency modulates GABAergic functions and hippocampus-dependent functions

    PubMed Central

    Chang, Chia-Yuan; Chen, Yi-Wen; Wang, Tsu-Wei; Lai, Wen-Sung

    2016-01-01

    Accumulating evidence implies that both AKT1 and GABAA receptor (GABAAR) subunit genes are involved in schizophrenia pathogenesis. Activated Akt promotes GABAergic neuron differentiation and increases GABAAR expression on the plasma membrane. To elucidate the role of Akt1 in modulating GABAergic functions and schizophrenia-related cognitive deficits, a set of 6 in vitro and in vivo experiments was conducted. First, an Akt1/2 inhibitor was applied to evaluate its effect on GABAergic neuron-like cell formation from P19 cells. Inhibiting Akt resulted in a reduction in parvalbumin-positive neuron-like cells. In Akt1−/− and wild-type mice, seizures induced using pentylenetetrazol (a GABAAR antagonist) were measured, and GABAAR expression and GABAergic interneuron abundance in the brain were examined. Female Akt1−/− mice, but not male Akt1−/− mice, exhibited less pentylenetetrazol-induced convulsive activity than their corresponding wild-type controls. Reduced parvalbumin-positive interneuron abundance and GABAAR subunit expression, especially in the hippocampus, were also observed in female Akt1−/− mice compared to female wild-type mice. Neuromorphometric analyses revealed significantly reduced neurite complexity in hippocampal pyramidal neurons. Additionally, female Akt1−/− mice displayed increased hippocampal oscillation power and impaired spatial memory compared to female wild-type mice. Our findings suggest that Akt1 deficiency modulates GABAergic interneurons and GABAAR expression, contributing to hippocampus-dependent cognitive functional impairment. PMID:27615800

  6. Phospho-tyrosine phosphatase inhibitor Bpv(Hopic) enhances C2C12 myoblast migration in vitro. Requirement of PI3K/AKT and MAPK/ERK pathways.

    PubMed

    Dimchev, Georgi A; Al-Shanti, Nasser; Stewart, Claire E

    2013-05-01

    Muscle progenitor cell migration is an important step in skeletal muscle myogenesis and regeneration. Migration is required for muscle precursors to reach the site of damage and for the alignment of myoblasts prior to their fusion, which ultimately contributes to muscle regeneration. Limited spreading and migration of donor myoblasts are reported problems of myoblast transfer therapy, a proposed therapeutic strategy for Duchenne Muscular Dystrophy, warranting further investigation into different approaches for improving the motility and homing of these cells. In this article, the effect of protein phospho-tyrosine phosphatase and PTEN inhibitor BpV(Hopic) on C2C12 myoblast migration and differentiation was investigated. Applying a wound healing migration model, it is reported that 1 μM BpV(Hopic) is capable of enhancing the migration of C2C12 myoblasts by approximately 40 % in the presence of myotube conditioned media, without significantly affecting their capacity to differentiate and fuse into multinucleated myotubes. Improved migration of myoblasts treated with 1 μM BpV(Hopic) was associated with activation of PI3K/AKT and MAPK/ERK pathways, while their inhibition with either LY294002 or UO126, respectively, resulted in a reduction of C2C12 migration back to control levels. These results propose that bisperoxovanadium compounds may be considered as potential tools for enhancing the migration of myoblasts, while not reducing their differentiation capacity and underpin the importance of PI3K/AKT and MAPK/ERK signalling for the process of myogenic progenitor migration. PMID:23553034

  7. Histone deacetylase inhibitor prevents cell growth in Burkitt's lymphoma by regulating PI3K/Akt pathways and leads to upregulation of miR-143, miR-145, and miR-101.

    PubMed

    Ferreira, Ana Carolina dos Santos; Robaina, Marcela Cristina; Rezende, Lídia Maria Magalhães de; Severino, Patricia; Klumb, Claudete Esteves

    2014-06-01

    Burkitt lymphoma (BL) is an aggressive B-cell lymphoma more common in children comprising one third of pediatric non-Hodgkin lymphoma cases. The recent discovery in BL pathogenesis highlighted the activation of PI3K pathway in cooperation with Myc in the development of BL. In this study, we demonstrated that PI3K/Akt pathway is a target to histone deacetylase inhibitor (HDACi) in BL cells. The combination of HDACi (sodium butyrate, NaB) and chemotherapy (VP-16) inhibited 51 % of the proliferation and enhanced the blockage of the cell cycle progression at G2/M with a concurrent decrease in the S phase. Microarray profiling showed a synergistic action of NaB/VP-16 combination through the differential regulation of 1,413 genes. Comparing VP-16 treatment with the NaB/VP-16 combination, 318 genes were deregulated: 250 genes were downregulated, and 68 were upregulated when compared with untreated cells. Among these genes, six (CDKN1A, CCND1, FAS, CHEK2, MDM4, and SESN2) belong to the p53-signaling pathway. The activation of this signaling pathway is usually induced by stress signals and ultimately leads to cell cycle arrest. Besides, the inhibition of the cell growth was related to reduced Akt phosphorylation, and decrease of c-Myc protein expression by about 60 % (p ≤ 0.005). Moreover, HDACi enhanced miR-101, miR-143, and miR-145 levels in BL cell line, which were inversely associated with the levels of miR-101, miR-143, and miR-145 found to be extremely downregulated in the sample of BL patients. We highlight the fact that effective combinations of HDACis with other target drugs could improve BL therapy in the future. PMID:24577510

  8. Akt2 Phosphorylates Ezrin to Trigger NHE3 Translocation and Activation*

    PubMed Central

    Shiue, Harn; Musch, Mark W.; Wang, Yingmin; Chang, Eugene B.; Turner, Jerrold R.

    2005-01-01

    Initiation of Na+-glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na+-glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na+-glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na+-glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation. PMID:15531580

  9. Akt2 phosphorylates ezrin to trigger NHE3 translocation and activation.

    PubMed

    Shiue, Harn; Musch, Mark W; Wang, Yingmin; Chang, Eugene B; Turner, Jerrold R

    2005-01-14

    Initiation of Na(+)-glucose cotransport in intestinal absorptive epithelia causes NHE3 to be translocated to the apical plasma membrane, leading to cytoplasmic alkalinization. We reported recently that this NHE3 translocation requires ezrin phosphorylation. However, the kinase that phosphorylates ezrin in this process has not been identified. Because Akt has also been implicated in NHE3 translocation, we investigated the hypothesis that Akt phosphorylates ezrin. After initiation of Na(+)-glucose cotransport, Akt is activated with kinetics that parallel those of ezrin phosphorylation. Inhibition of p38 MAP kinase, which blocks ezrin phosphorylation, also prevents Akt activation. Purified Akt directly phosphorylates recombinant ezrin at threonine 567 in vitro in an ATP-dependent manner. This in vitro phosphorylation can be prevented by Akt inhibitors. In intact cells, inhibition of either phosphoinositide 3-kinase, an upstream regulator of Akt, or inhibition of Akt itself using inhibitors validated in vitro prevents ezrin phosphorylation after initiation of Na(+)-glucose cotransport. Specific small interfering RNA knockdown of Akt2 prevented ezrin phosphorylation in intact cells. Pharmacological Akt inhibition or Akt2 knockdown also prevented NHE3 translocation and activation after initiation of Na(+)-glucose cotransport, confirming the functional role of Akt2. These studies therefore identify Akt2 as a critical kinase that regulates ezrin phosphorylation and activation. This Akt2-dependent ezrin phosphorylation leads to NHE3 translocation and activation.

  10. The investigational Aurora kinase A inhibitor alisertib (MLN8237) induces cell cycle G2/M arrest, apoptosis, and autophagy via p38 MAPK and Akt/mTOR signaling pathways in human breast cancer cells

    PubMed Central

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular

  11. Allosteric inhibition of the NS2B-NS3 protease from dengue virus.

    PubMed

    Yildiz, Muslum; Ghosh, Sumana; Bell, Jeffrey A; Sherman, Woody; Hardy, Jeanne A

    2013-12-20

    Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors. PMID:24164286

  12. Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma.

    PubMed

    Moore, Nathan F; Azarova, Anna M; Bhatnagar, Namrata; Ross, Kenneth N; Drake, Lauren E; Frumm, Stacey; Liu, Qinsong S; Christie, Amanda L; Sanda, Takaomi; Chesler, Louis; Kung, Andrew L; Gray, Nathanael S; Stegmaier, Kimberly; George, Rani E

    2014-09-30

    Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.

  13. AKT3 promotes prostate cancer proliferation cells through regulation of Akt, B-Raf & TSC1/TSC2

    PubMed Central

    Tseng, Jen-Chih; Jiang, Shih Sheng; Kuo, Ying-Yu; Chen, Shyh-Chang; Wang, Chih-Ting; Chan, Tzu-Min; Liou, Jun-Yang; Wang, John; Chang, Wun-Shaing Wayne; Chang, Chung-Ho; Kung, Hsing-Jien; Chuu, Chih-Pin

    2015-01-01

    The qRT-PCR analysis of 139 clinical samples and analysis of 150 on-line database clinical samples indicated that AKT3 mRNA expression level was elevated in primary prostate tumors. Immunohistochemical staining of 65 clinical samples revealed that AKT3 protein expression was higher in prostate tumors of stage I, II, III as compared to nearby normal tissues. Plasmid overexpression of AKT3 promoted cell proliferation of LNCaP, PC-3, DU-145, and CA-HPV-10 human prostate cancer (PCa) cells, while knockdown of AKT3 by siRNA reduced cell proliferation. Overexpression of AKT3 increased the protein expression of total AKT, phospho-AKT S473, phospho-AKT T308, B-Raf, c-Myc, Skp2, cyclin E, GSK3β, phospho-GSK3β S9, phospho-mTOR S2448, and phospho-p70S6K T421/S424, but decreased TSC1 (tuberous sclerosis 1) and TSC2 (tuberous Sclerosis Complex 2) proteins in PC-3 PCa cells. Overexpression of AKT3 also increased protein abundance of phospho-AKT S473, phospho-AKT T308, and B-Raf but decreased expression of TSC1 and TSC2 proteins in LNCaP, DU-145, and CA-HPV-10 PCa cells. Oncomine datasets analysis suggested that AKT3 mRNA level was positively correlated to BRAF. Knockdown of AKT3 in DU-145 cells with siRNA increased the sensitivity of DU-145 cells to B-Raf inhibitor treatment. Knockdown of TSC1 or TSC2 promoted the proliferation of PCa cells. Our observations implied that AKT3 may be a potential therapeutic target for PCa treatment. PMID:26318033

  14. Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

    Technology Transfer Automated Retrieval System (TEKTRAN)

    XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

  15. Emerging therapeutics for targeting Akt in cancer.

    PubMed

    Gdowski, Andrew; Panchoo, Marlyn; Treuren, Timothy Van; Basu, Alakananda

    2016-01-01

    The ultimate goal of cancer therapeutic research is to develop effective, targeted therapeutics that exploit the vulnerabilities of cancer cells. The three isoforms of Akt, also known as protein kinase B (PKB), are important mediators of various pathways that transmit mitogenic signals from the cell's exterior to the effector proteins of the cell's interior. Due to Akt\\\\\\\\\\\\\\'s importance in cell functions such as growth, proliferation and cell survival, many cancer cells rely on this pathway to aid in their survival. This dependence can lead to chemoresistance and selection of more adapted populations of cancer cells. Thus, it is important to understand the functional significance of isoform specificity and its relation to chemoresistance. In this review, we have summarized recent studies on Akt isoform specific regulation as well as each isoform's role in chemoresistance, emphasizing their potential as targets for cancer therapy. We have also condensed ongoing clinical studies involving various types of Akt inhibitors while highlighting the type of study, rationale and co-therapies involved in identifying Akt isoforms as promising therapeutic targets.

  16. Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter.

    PubMed

    Zhong, Huailing; Sánchez, Connie; Caron, Marc G

    2012-02-15

    The serotonin transporter (SERT) functions to transport serotonin (5-HT) from the extracellular space into neurons to maintain homeostatic control of 5-HT. It is the molecular target for selective serotonin reuptake inhibitor (SSRI) antidepressants. Preclinical research has shown that some SERT inhibitors can bind to two distinct binding sites on the SERT, a primary high affinity binding site and a low affinity allosteric binding site. Mutational studies of the SERT and computational modeling methods with escitalopram resulted in the identification of key amino acid residues important for the function of the allosteric binding site. While this allosteric binding site appears to influence the clinical efficacy of escitalopram under physiological conditions, the molecular mechanism of this effect is still poorly understood and may involve a large network of protein-protein interactions with the SERT. Dynamic interfaces between the SERT and the SERT interacting proteins (SIPs) potentially influence not only the SERT on its uptake function, its regulation, and trafficking, but also on known as well as yet to be identified non-canonical signaling pathways through SIPs. In this commentary, we outline approaches in the areas of selective small-molecule allosteric compound discovery, biochemistry, in vivo genetic knock-in mouse models, as well as computational and structural biology. These studies of the intra-molecular allosteric modulation of the SERT in the context of the myriad of potential inter-molecular signaling interactions with SIPs may help uncover unknown physiological functions of the SERT.

  17. Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli.

    PubMed

    Kim, J; Raushel, F M

    2001-09-18

    Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer

  18. Acetylcholine Receptor: An Allosteric Protein

    NASA Astrophysics Data System (ADS)

    Changeux, Jean-Pierre; Devillers-Thiery, Anne; Chemouilli, Phillippe

    1984-09-01

    The nicotine receptor for the neurotransmitter acetylcholine is an allosteric protein composed of four different subunits assembled in a transmembrane pentamer α 2β γ δ . The protein carries two acetylcholine sites at the level of the α subunits and contains the ion channel. The complete sequence of the four subunits is known. The membrane-bound protein undergoes conformational transitions that regulate the opening of the ion channel and are affected by various categories of pharmacologically active ligands.

  19. Co-administration of the mTORC1/TORC2 inhibitor INK128 and the Bcl-2/Bcl-xL antagonist ABT-737 kills human myeloid leukemia cells through Mcl-1 down-regulation and AKT inactivation

    PubMed Central

    Rahmani, Mohamed; Aust, Mandy Mayo; Hawkins, Elisa; Parker, Rebecca E.; Ross, Masey; Kmieciak, Maciej; Reshko, Leonid Borisovich; Rizzo, Kathryn A.; Dumur, Catherine I.; Ferreira-Gonzalez, Andrea; Grant, Steven

    2015-01-01

    Effects of concurrent inhibition of mTORC1/2 and Bcl-2/Bcl-xL in human acute myeloid leukemia cells were examined. Tetracycline-inducible Bcl-2/Bcl-xL dual knockdown markedly sensitized acute myeloid leukemia cells to the dual TORC1/2 inhibitor INK128 in vitro as well as in vivo. Moreover, INK128 co-administered with the Bcl-2/xL antagonist ABT-737 sharply induced cell death in multiple acute myeloid leukemia cell lines, including TKI-resistant FLT3-ITD mutants and primary acute myeloid leukemia blasts carrying various genetic aberrations e.g., FLT3, IDH2, NPM1, and Kras, while exerting minimal toxicity toward normal hematopoietic CD34+ cells. Combined treatment was particularly active against CD34+/CD38−/CD123+ primitive leukemic progenitor cells. The INK128/ABT-737 regimen was also effective in the presence of a protective stromal microenvironment. Notably, INK128 was more potent than the TORC1 inhibitor rapamycin in down-regulating Mcl-1, diminishing AKT and 4EBP1 phosphorylation, and potentiating ABT-737 activity. Mcl-1 ectopic expression dramatically attenuated INK128/ABT-737 lethality, indicating an important functional role for Mcl-1 down-regulation in INK128/ABT-737 actions. Immunoprecipitation analysis revealed that combined treatment markedly diminished Bax, Bak, and Bim binding to all major anti-apoptotic Bcl-2 members (Bcl-2/Bcl-xL/Mcl-1), while Bax/Bak knockdown reduced cell death. Finally, INK128/ABT-737 co-administration sharply attenuated leukemia growth and significantly prolonged survival in a systemic acute myeloid leukemia xenograft model. Analysis of subcutaneous acute myeloid leukemia-derived tumors revealed significant decrease in 4EBP1 phosphorylation and Mcl-1 protein level, consistent with results obtained in vitro. These findings demonstrate that co-administration of dual mTORC1/mTORC2 inhibitors and BH3-mimetics exhibits potent anti-leukemic activity in vitro and in vivo, arguing that this strategy warrants attention in acute myeloid

  20. Colocalized delivery of rapamycin and paclitaxel to tumors enhances synergistic targeting of the PI3K/Akt/mTOR pathway.

    PubMed

    Blanco, Elvin; Sangai, Takafumi; Wu, Suhong; Hsiao, Angela; Ruiz-Esparza, Guillermo U; Gonzalez-Delgado, Carlos A; Cara, Francisca E; Granados-Principal, Sergio; Evans, Kurt W; Akcakanat, Argun; Wang, Ying; Do, Kim-Anh; Meric-Bernstam, Funda; Ferrari, Mauro

    2014-07-01

    Ongoing clinical trials target the aberrant PI3K/Akt/mammalian target of rapamycin (mTOR) pathway in breast cancer through administration of rapamycin, an allosteric mTOR inhibitor, in combination with paclitaxel. However, synergy may not be fully exploited clinically because of distinct pharmacokinetic parameters of drugs. This study explores the synergistic potential of site-specific, colocalized delivery of rapamycin and paclitaxel through nanoparticle incorporation. Nanoparticle drug loading was accurately controlled, and synergistic drug ratios established in vitro. Precise drug ratios were maintained in tumors 48 hours after nanoparticle administration to mice, at levels twofold greater than liver and spleen, yielding superior antitumor activity compared to controls. Simultaneous and preferential in vivo delivery of rapamycin and paclitaxel to tumors yielded mechanistic insights into synergy involving suppression of feedback loop Akt phosphorylation and its downstream targets. Findings demonstrate that a same time, same place, and specific amount approach to combination chemotherapy by means of nanoparticle delivery has the potential to successfully translate in vitro synergistic findings in vivo. Predictive in vitro models can be used to determine optimum drug ratios for antitumor efficacy, while nanoparticle delivery of combination chemotherapies in preclinical animal models may lead to enhanced understanding of mechanisms of synergy, ultimately opening several avenues for personalized therapy.

  1. Colocalized Delivery of Rapamycin and Paclitaxel to Tumors Enhances Synergistic Targeting of the PI3K/Akt/mTOR Pathway

    PubMed Central

    Blanco, Elvin; Sangai, Takafumi; Wu, Suhong; Hsiao, Angela; Ruiz-Esparza, Guillermo U; Gonzalez-Delgado, Carlos A; Cara, Francisca E; Granados-Principal, Sergio; Evans, Kurt W; Akcakanat, Argun; Wang, Ying; Do, Kim-Anh; Meric-Bernstam, Funda; Ferrari, Mauro

    2014-01-01

    Ongoing clinical trials target the aberrant PI3K/Akt/mammalian target of rapamycin (mTOR) pathway in breast cancer through administration of rapamycin, an allosteric mTOR inhibitor, in combination with paclitaxel. However, synergy may not be fully exploited clinically because of distinct pharmacokinetic parameters of drugs. This study explores the synergistic potential of site-specific, colocalized delivery of rapamycin and paclitaxel through nanoparticle incorporation. Nanoparticle drug loading was accurately controlled, and synergistic drug ratios established in vitro. Precise drug ratios were maintained in tumors 48 hours after nanoparticle administration to mice, at levels twofold greater than liver and spleen, yielding superior antitumor activity compared to controls. Simultaneous and preferential in vivo delivery of rapamycin and paclitaxel to tumors yielded mechanistic insights into synergy involving suppression of feedback loop Akt phosphorylation and its downstream targets. Findings demonstrate that a same time, same place, and specific amount approach to combination chemotherapy by means of nanoparticle delivery has the potential to successfully translate in vitro synergistic findings in vivo. Predictive in vitro models can be used to determine optimum drug ratios for antitumor efficacy, while nanoparticle delivery of combination chemotherapies in preclinical animal models may lead to enhanced understanding of mechanisms of synergy, ultimately opening several avenues for personalized therapy. PMID:24569835

  2. Allosteric Inhibition of Macrophage Migration Inhibitory Factor Revealed by Ibudilast

    SciTech Connect

    Cho, Y.; Crichlow, G; Vermeire, J; Leng, L; Du, X; Hodsdon, M; Bucala, R; Cappello, M; Gross, M; et al.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells.

  3. Targeting PARP-1 allosteric regulation offers therapeutic potential against cancer.

    PubMed

    Steffen, Jamin D; Tholey, Renee M; Langelier, Marie-France; Planck, Jamie L; Schiewer, Matthew J; Lal, Shruti; Bildzukewicz, Nikolai A; Yeo, Charles J; Knudsen, Karen E; Brody, Jonathan R; Pascal, John M

    2014-01-01

    PARP-1 is a nuclear protein that has important roles in maintenance of genomic integrity. During genotoxic stress, PARP-1 recruits to sites of DNA damage where PARP-1 domain architecture initiates catalytic activation and subsequent poly(ADP-ribose)-dependent DNA repair. PARP-1 inhibition is a promising new way to selectively target cancers harboring DNA repair deficiencies. However, current inhibitors target other PARPs, raising important questions about long-term off-target effects. Here, we propose a new strategy that targets PARP-1 allosteric regulation as a selective way of inhibiting PARP-1. We found that disruption of PARP-1 domain-domain contacts through mutagenesis held no cellular consequences on recruitment to DNA damage or a model system of transcriptional regulation, but prevented DNA-damage-dependent catalytic activation. Furthermore, PARP-1 mutant overexpression in a pancreatic cancer cell line (MIA PaCa-2) increased sensitivity to platinum-based anticancer agents. These results not only highlight the potential of a synergistic drug combination of allosteric PARP inhibitors with DNA-damaging agents in genomically unstable cancer cells (regardless of homologous recombination status), but also signify important applications of selective PARP-1 inhibition. Finally, the development of a high-throughput PARP-1 assay is described as a tool to promote discovery of novel PARP-1 selective inhibitors.

  4. Allosteric inhibition of macrophage migration inhibitory factor revealed by ibudilast

    PubMed Central

    Cho, Yoonsang; Crichlow, Gregg V.; Vermeire, Jon J.; Leng, Lin; Du, Xin; Hodsdon, Michael E.; Bucala, Richard; Cappello, Michael; Gross, Matt; Gaeta, Federico; Johnson, Kirk; Lolis, Elias J.

    2010-01-01

    AV411 (ibudilast; 3-isobutyryl-2-isopropylpyrazolo-[1,5-a]pyridine) is an antiinflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a nonselective inhibitor of various phosphodiesterases (PDEs) and has varied antiinflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitor AV411 and its PDE-inhibition-compromised analog AV1013 inhibit the catalytic and chemotactic functions of the proinflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are noncompetitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF and an allosteric binding site of AV411 and AV1013 is detected by NMR. The allosteric inhibition mechanism is further elucidated by X-ray crystallography based on the MIF/AV1013 binary and MIF/AV1013/HPP ternary complexes. In addition, our antibody experiments directed against MIF receptors indicate that CXCR2 is the major receptor for MIF-mediated chemotaxis of peripheral blood mononuclear cells. PMID:20534506

  5. Allosteric Modulation of Purine and Pyrimidine Receptors

    PubMed Central

    Jacobson, Kenneth A.; Gao, Zhan-Guo; Göblyös, Anikó; IJzerman, Adriaan P.

    2011-01-01

    Among the purine and pyrimidine receptors, the discovery of small molecular allosteric modulators has been most highly advanced for the A1 and A3 ARs. These AR modulators have allosteric effects that are structurally separated from the orthosteric effects in SAR studies. The benzoylthiophene derivatives tend to act as allosteric agonists, as well as selective positive allosteric modulators (PAMs) of the A1 AR. A 2-amino-3-aroylthiophene derivative T-62 has been under development as a PAM of the A1 AR for the treatment of chronic pain. Several structurally distinct classes of allosteric modulators of the human A3 AR have been reported: 3-(2-pyridinyl)isoquinolines, 2,4-disubstituted quinolines, 1H-imidazo-[4,5-c]quinolin-4-amines, endocannabinoid 2-arachidonylglycerol and the food dye Brilliant Black BN. Site-directed mutagenesis of A1 and A3 ARs has identified residues associated with the allosteric effect, distinct from those that affect orthosteric binding. A few small molecular allosteric modulators have been reported for several of the P2X ligand-gated ion channels and the G protein-coupled P2Y receptor nucleotides. Metal ion modulation of the P2X receptors has been extensively explored. The allosteric approach to modulation of purine and pyrimidine receptors looks promising for development of drugs that are event-specific and site-specific in action. PMID:21586360

  6. SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways

    PubMed Central

    Zhou, T T; Quan, L L; Chen, L P; Du, T; Sun, K X; Zhang, J C; Yu, L; Li, Y; Wan, P; Chen, L L; Jiang, B H; Hu, L H; Chen, J; Shen, X

    2016-01-01

    Kv2.1 as a voltage-gated potassium (Kv) channel subunit has a pivotal role in the regulation of glucose-stimulated insulin secretion (GSIS) and pancreatic β-cell apoptosis, and is believed to be a promising target for anti-diabetic drug discovery, although the mechanism underlying the Kv2.1-mediated β-cell apoptosis is obscure. Here, the small molecular compound, ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2–a]pyrimidine-6-carboxylate (SP6616) was discovered to be a new Kv2.1 inhibitor. It was effective in both promoting GSIS and protecting β cells from apoptosis. Evaluation of SP6616 on either high-fat diet combined with streptozocin-induced type 2 diabetic mice or db/db mice further verified its efficacy in the amelioration of β-cell dysfunction and glucose homeostasis. SP6616 treatment efficiently increased serum insulin level, restored β-cell mass, decreased fasting blood glucose and glycated hemoglobin levels, and improved oral glucose tolerance. Mechanism study indicated that the promotion of SP6616 on β-cell survival was tightly linked to its regulation against both protein kinases C (PKC)/extracellular-regulated protein kinases 1/2 (Erk1/2) and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathways. To our knowledge, this may be the first report on the underlying pathway responsible for the Kv2.1-mediated β-cell protection. In addition, our study has also highlighted the potential of SP6616 in the treatment of type 2 diabetes. PMID:27148689

  7. A multi-pronged approach for compiling a global map of allosteric regulation in the apoptotic caspases

    PubMed Central

    Dagbay, Kevin; Eron, Scott J.; Serrano, Banyuhay P.; Velázquez-Delgado, Elih M.; Zhao, Yunlong; Lin, Di; Vaidya, Sravanti; Hardy, Jeanne A.

    2014-01-01

    One of the most promising and as yet underutilized means of regulating protein function is exploitation of allosteric sites. All caspases catalyze the same overall reaction, but they perform different biological roles and are differentially regulated. It is our hypothesis that many allosteric sites exist on various caspases and that understanding both the distinct and overlapping mechanisms by which each caspase can be allosterically controlled should ultimately enable caspase-specific inhibition. Here we describe the ongoing work and methods for compiling a comprehensive map of apoptotic caspase allostery. Central to this approach are the use of i) the embedded record of naturally evolved allosterically sites that are sensitive to zinc-medicated inhibition, phosphorylation and other post-translationally modifications, ii) structural and mutagenic approaches and iii) novel binding sites identified by both rationally-designed and screening-derived small-molecule inhibitors. PMID:24974292

  8. Unraveling structural mechanisms of allosteric drug action.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung

    2014-05-01

    Orthosteric drugs block the active site to obstruct function; allosteric drugs modify the population of the active state, to modulate function. Available data lead us to propose that allosteric drugs can constitute anchors and drivers. The anchor docks into an allosteric pocket. The conformation with which it interacts is unchanged during the transition between the inactive and active states. The anchor provides the foundation that allows the driver to exert a 'pull' and/or 'push' action that shifts the receptor population from the inactive to the active state. The presence or absence of driver atom in an allosteric drug can exert opposite agonism. We map a strategy for driver identification and expect the allosteric trigger concept to transform agonist/antagonist drug discovery.

  9. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    PubMed Central

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  10. Allosteric effects of carbamoyl phosphate synthetase from Escherichia coli are entropy-driven.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1996-09-10

    When catalyzing the formation of MgATP and carbamate from MgADP and carbamoyl phosphate, Escherichia coli carbamoyl phosphate synthetase (CPS) binds MgADP with a large negative change in heat capacity. The magnitude of this heat capacity change is not appreciably altered by the presence of a saturating concentration of either the allosteric activator ornithine or the inhibitor UMP despite the substantial and opposing effects these ligands have on the binding affinity for MgADP. By contrast, no detectable change in heat capacity is associated with the thermodynamic coupling between MgADP and either ornithine or UMP. The sign of the apparently constant enthalpic and entropic contributions to the coupling free energy for each of these ligands is opposite that of the coupling free energy, indicating that the observed allosteric phenomenology is in net opposed by the enthalpy of the interaction and instead arises from a change in entropy of the system. IMP produces only a very small allosteric effect as indicated by a near-zero value for the MgADP-IMP coupling free energy. However, the enthalpic and entropic contributions are individually larger in absolute value for the IMP coupling than for those pertaining to the other allosteric ligands, and entropy dominates the coupling free energy above 36 degrees C, causing IMP to become an activator at high temperature. In addition, the sign of the coupling enthalpy and entropy for IMP has the same sign as the coupling enthalpy and entropy produced by ornithine, suggesting that IMP and ornithine may similarly influence the enzyme at a molecular level despite binding to different allosteric sites on the enzyme. The data are consistent with a model in which the actions of the allosteric ligands arise primarily from changes in the conformational degeneracy introduced by each ligand. With this model, one can also rationalize the failure of these allosteric ligands to substantially influence kcat. PMID:8794775

  11. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase

    NASA Astrophysics Data System (ADS)

    Foda, Zachariah H.; Shan, Yibing; Kim, Eric T.; Shaw, David E.; Seeliger, Markus A.

    2015-01-01

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  12. AKT Pathway Genes Define 5 Prognostic Subgroups in Glioblastoma

    PubMed Central

    Smirnov, Ivan; Reiser, Mark; Misra, Anjan; Shapiro, William R.; Mills, Gordon B.; Kim, Seungchan; Feuerstein, Burt G.

    2014-01-01

    Activity of GFR/PI3K/AKT pathway inhibitors in glioblastoma clinical trials has not been robust. We hypothesized variations in the pathway between tumors contribute to poor response. We clustered GBM based on AKT pathway genes and discovered new subtypes then characterized their clinical and molecular features. There are at least 5 GBM AKT subtypes having distinct DNA copy number alterations, enrichment in oncogenes and tumor suppressor genes and patterns of expression for PI3K/AKT/mTOR signaling components. Gene Ontology terms indicate a different cell of origin or dominant phenotype for each subgroup. Evidence suggests one subtype is very sensitive to BCNU or CCNU (median survival 5.8 vs. 1.5 years; BCNU/CCNU vs other treatments; respectively). AKT subtyping advances previous approaches by revealing additional subgroups with unique clinical and molecular features. Evidence indicates it is a predictive marker for response to BCNU or CCNU and PI3K/AKT/mTOR pathway inhibitors. We anticipate Akt subtyping may help stratify patients for clinical trials and augment discovery of class-specific therapeutic targets. PMID:24984002

  13. Akt isoforms in vascular disease.

    PubMed

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-08-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease.

  14. Akt isoforms in vascular disease

    PubMed Central

    Yu, Haixiang; Littlewood, Trevor; Bennett, Martin

    2015-01-01

    The mammalian serine/threonine Akt kinases comprise three closely related isoforms: Akt1, Akt2 and Akt3. Akt activation has been implicated in both normal and disease processes, including in development and metabolism, as well as cancer and cardiovascular disease. Although Akt signalling has been identified as a promising therapeutic target in cancer, its role in cardiovascular disease is less clear. Importantly, accumulating evidence suggests that the three Akt isoforms exhibit distinct tissue expression profiles, localise to different subcellular compartments, and have unique modes of activation. Consistent with in vitro findings, genetic studies in mice show distinct effects of individual Akt isoforms on the pathophysiology of cardiovascular disease. This review summarises recent studies of individual Akt isoforms in atherosclerosis, vascular remodelling and aneurysm formation, to provide a comprehensive overview of Akt function in vascular disease. PMID:25929188

  15. Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

    PubMed

    Yu, Ker; Toral-Barza, Lourdes; Shi, Celine; Zhang, Wei-Guo; Lucas, Judy; Shor, Boris; Kim, Jamie; Verheijen, Jeroen; Curran, Kevin; Malwitz, David J; Cole, Derek C; Ellingboe, John; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Gibbons, James J; Abraham, Robert T; Nowak, Pawel; Zask, Arie

    2009-08-01

    The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

  16. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin

    PubMed Central

    Verespy III, Stephen; Mehta, Akul Y.; Afosah, Daniel; Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. PMID:27053426

  17. 13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase*

    PubMed Central

    Kudalkar, Shalley N.; Nikas, Spyros P.; Kingsley, Philip J.; Xu, Shu; Galligan, James J.; Rouzer, Carol A.; Banerjee, Surajit; Ji, Lipin; Eno, Marsha R.; Makriyannis, Alexandros; Marnett, Lawrence J.

    2015-01-01

    Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism. PMID:25648895

  18. AKT/GSK3β signaling pathway is critically involved in human pluripotent stem cell survival

    PubMed Central

    Romorini, Leonardo; Garate, Ximena; Neiman, Gabriel; Luzzani, Carlos; Furmento, Verónica Alejandra; Guberman, Alejandra Sonia; Sevlever, Gustavo Emilio; Scassa, María Elida; Miriuka, Santiago Gabriel

    2016-01-01

    Human embryonic and induced pluripotent stem cells are self-renewing pluripotent stem cells (PSC) that can differentiate into a wide range of specialized cells. Basic fibroblast growth factor is essential for PSC survival, stemness and self-renewal. PI3K/AKT pathway regulates cell viability and apoptosis in many cell types. Although it has been demonstrated that PI3K/AKT activation by bFGF is relevant for PSC stemness maintenance its role on PSC survival remains elusive. In this study we explored the molecular mechanisms involved in the regulation of PSC survival by AKT. We found that inhibition of AKT with three non-structurally related inhibitors (GSK690693, AKT inhibitor VIII and AKT inhibitor IV) decreased cell viability and induced apoptosis. We observed a rapid increase in phosphatidylserine translocation and in the extent of DNA fragmentation after inhibitors addition. Moreover, abrogation of AKT activity led to Caspase-9, Caspase-3, and PARP cleavage. Importantly, we demonstrated by pharmacological inhibition and siRNA knockdown that GSK3β signaling is responsible, at least in part, of the apoptosis triggered by AKT inhibition. Moreover, GSK3β inhibition decreases basal apoptosis rate and promotes PSC proliferation. In conclusion, we demonstrated that AKT activation prevents apoptosis, partly through inhibition of GSK3β, and thus results relevant for PSC survival. PMID:27762303

  19. Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation.

    PubMed

    Vojtek, Anne B; Taylor, Jennifer; DeRuiter, Stacy L; Yu, Jenn-Yah; Figueroa, Claudia; Kwok, Roland P S; Turner, David L

    2003-07-01

    Neural basic helix-loop-helix (bHLH) transcription factors regulate neurogenesis in vertebrates. Signaling by peptide growth factors also plays critical roles in regulating neuronal differentiation and survival. Many peptide growth factors activate phosphatidylinositol 3-kinase (PI3K) and subsequently the Akt kinases, raising the possibility that Akt may impact bHLH protein function during neurogenesis. Here we demonstrate that reducing expression of endogenous Akt1 and Akt2 by RNA interference (RNAi) reduces neuron generation in P19 cells transfected with a neural bHLH expression vector. The reduction in neuron generation from decreased Akt expression is not solely due to decreased cell survival, since addition of the caspase inhibitor z-VAD-FMK rescues cell death associated with loss of Akt function but does not restore neuron formation. This result indicates that Akt1 and Akt2 have additional functions during neuronal differentiation that are separable from neuronal survival. We show that activated Akt1 enhances complex formation between bHLH proteins and the transcriptional coactivator p300. Activated Akt1 also significantly augments the transcriptional activity of the bHLH protein neurogenin 3 in complex with the coactivators p300 or CBP. In addition, inhibition of endogenous Akt activity by the PI3K/Akt inhibitor LY294002 abolishes transcriptional cooperativity between the bHLH proteins and p300. We propose that Akt regulates the assembly and activity of bHLH-coactivator complexes to promote neuronal differentiation.

  20. mTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms

    PubMed Central

    Martinelli, Serena; Tozzi, Lorenzo; Guglielmelli, Paola; Bosi, Alberto; Vannucchi, Alessandro M.

    2013-01-01

    Background Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with JAK2V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects in vitro of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells. Findings Mouse and human JAK2V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of JAK2V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera

  1. Molecular mechanism underlying Akt activation in zinc-induced cardioprotection

    PubMed Central

    Lee, SungRyul; Chanoit, Guillaume; McIntosh, Rachel; Zvara, David A.; Xu, Zhelong

    2009-01-01

    Our previous study demonstrated that zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via Akt and glycogen synthetase kinase 3β (GSK-3β). We aimed to address the mechanism by which zinc activates Akt. Treatment of H9c2 cells with ZnCl2 (10 μM) in the presence of the zinc ionophore pyrithione (4 μM) for 20 min enhanced Akt phosphorylation (Ser473), indicating that zinc can rapidly activate Akt. Zinc did not alter either phosphatase and tensin homolog deleted on chromosome 10 (PTEN) phosphorylation and total PTEN protein levels or PTEN oxidation, implying that PTEN may not play a role in the action of zinc. However, zinc-induced Akt phosphorylation was blocked by both the nonselective receptor tyrosine kinase (RTK) inhibitor genistein and the selective insulin-like growth factor-1 RTK (IGF-1RTK) inhibitor AG1024, indicating that zinc activates Akt via IGF-1RTK. Zinc-induced phosphorylation of protein tyrosine and Ser/Thr was also abolished by AG1024. In addition, zinc markedly enhanced phosphorylation of IGF-1 receptor (IGF-1R), which was again reversed by genistein and AG1024. A confocal imaging study revealed that AG1024 abolished the preventive effect of zinc on oxidant-induced mPTP opening, confirming that IGF-1RTK plays a role in zinc-induced cardioprotection. Furthermore, zinc decreased the activity of protein phosphatase 2A (PP2A), a major protein Ser/Thr phosphatase, implying that protein Ser/Thr phosphatases may also play a role in the action of zinc on Akt activity. Taken together, these findings demonstrate that exogenous zinc activates Akt via IGF-1RTK and prevents the mPTP opening in cardiac cells. Inactivation of Ser/Thr protein phosphatases may also contribute to zinc-induced Akt activation. PMID:19525380

  2. Rational design of allosteric-inhibition sites in classical protein tyrosine phosphatases

    PubMed Central

    Chio, Cynthia M.; Yu, Xiaoling; Bishop, Anthony C.

    2015-01-01

    Protein tyrosine phosphatases (PTPs), which catalyze the dephosphorylation of phosphotyrosine in protein substrates, are critical regulators of metazoan cell signaling and have emerged as potential drug targets for a range of human diseases. Strategies for chemically targeting the function of individual PTPs selectively could serve to elucidate the signaling roles of these enzymes and would potentially expedite validation of the therapeutic promise of PTP inhibitors. Here we report a novel strategy for the design of non-natural allosteric-inhibition sites in PTPs; these sites, which can be introduced into target PTPs through protein engineering, serve to sensitize target PTPs to potent and selective inhibition by a biarsenical small molecule. Building on the recent discovery of a naturally occurring cryptic allosteric site in wild-type Src-homology-2 domain containing PTP (Shp2) that can be targeted by biarsenical compounds, we hypothesized that Shp2’s unusual sensitivity to biarsenicals could be strengthened through rational design and that the Shp2-specific site could serve as a blueprint for the introduction of non-natural inhibitor sensitivity in other PTPs. Indeed, we show here that the strategic introduction of a cysteine residue at a position removed from the Shp2 active site can serve to increase the potency and selectivity of the interaction between Shp2’s allosteric site and the biarsenical inhibitor. Moreover, we find that “Shp2-like” allosteric sites can be installed de novo in PTP enzymes that do not possess naturally occurring sensitivity to biarsenical compounds. Using primary-sequence alignments to guide our enzyme engineering, we have successfully introduced allosteric-inhibition sites in four classical PTPs—PTP1B, PTPH-1, FAP-1, and HePTP—from four different PTP subfamilies, suggesting that our sensitization approach can likely be applied widely across the classical PTP family to generate biarsenical-responsive PTPs. PMID:25828055

  3. Supramolecular Allosteric Cofacial Porphyrin Complexes

    SciTech Connect

    Oliveri, Christopher G.; Gianneschi, Nathan C.; Nguyen, Son Binh T.; Mirkin, Chad A.; Stern, Charlotte L.; Wawrzak, Zdzislaw; Pink, Maren

    2008-04-12

    Nature routinely uses cooperative interactions to regulate cellular activity. For years, chemists have designed synthetic systems that aim toward harnessing the reactivity common to natural biological systems. By learning how to control these interactions in situ, one begins to allow for the preparation of man-made biomimetic systems that can efficiently mimic the interactions found in Nature. To this end, we have designed a synthetic protocol for the preparation of flexible metal-directed supramolecular cofacial porphyrin complexes which are readily obtained in greater than 90% yield through the use of new hemilabile porphyrin ligands with bifunctional ether-phosphine or thioether-phosphine substituents at the 5 and 15 positions on the porphyrin ring. The resulting architectures contain two hemilabile ligand-metal domains (Rh{sup I} or Cu{sup I} sites) and two cofacially aligned porphyrins (Zn{sup II} sites), offering orthogonal functionalities and allowing these multimetallic complexes to exist in two states, 'condensed' or 'open'. Combining the ether-phosphine ligand with the appropriate Rh{sup I} or Cu{sup I} transition-metal precursors results in 'open' macrocyclic products. In contrast, reacting the thioether-phosphine ligand with RhI or CuI precursors yields condensed structures that can be converted into their 'open' macrocyclic forms via introduction of additional ancillary ligands. The change in cavity size that occurs allows these structures to function as allosteric catalysts for the acyl transfer reaction between X-pyridylcarbinol (where X = 2, 3, or 4) and 1-acetylimidazole. For 3- and 4-pyridylcarbinol, the 'open' macrocycle accelerates the acyl transfer reaction more than the condensed analogue and significantly more than the porphyrin monomer. In contrast, an allosteric effect was not observed for 2-pyridylcarbinol, which is expected to be a weaker binder and is unfavorably constrained inside the macrocyclic cavity.

  4. Allosteric modulators of the extracellular calcium receptor.

    PubMed

    Nemeth, E F

    2013-01-01

    The extracellular calcium receptor (CaR) is a Family C G protein-coupled receptor that controls systemic Ca2+ homeostasis, largely by regulating the secretion of parathyroid hormone (PTH). Ligands that activate the CaR have been termed calcimimetics and are classified as either Type I (agonists) or Type II (allosteric activators) and effectively inhibit the secretion of PTH. CaR antagonists have been termed calcilytics and all act allosterically to stimulate secretion of PTH. The calcimimetic cinacalcet has been approved for treating parathyroid cancer and secondary hyperparathyroidism in patients on renal replacement therapy. Cinacalcet was the first allosteric modulator of a G proteincoupled receptor to achieve regulatory approval. This review will focus on the technologies used to discover and develop allosterically acting calcimimetics and calcilytics as novel therapies for bone and mineral-related disorders. PMID:24050279

  5. Molecular and functional interactions between AKT and SOX2 in breast carcinoma

    PubMed Central

    Mir, Perihan; Konantz, Martina; Pereboom, Tamara C.; Paczulla, Anna M.; Merz, Britta; Fehm, Tanja; Perner, Sven; Rothfuss, Oliver C.; Kanz, Lothar; Schulze-Osthoff, Klaus; Lengerke, Claudia

    2015-01-01

    The transcription factor SOX2 is a key regulator of pluripotency in embryonic stem cells and plays important roles in early organogenesis. Recently, SOX2 expression was documented in various cancers and suggested as a cancer stem cell (CSC) marker. Here we identify the Ser/Thr-kinase AKT as an upstream regulator of SOX2 protein turnover in breast carcinoma (BC). SOX2 and pAKT are co-expressed and co-regulated in breast CSCs and depletion of either reduces clonogenicity. Ectopic SOX2 expression restores clonogenicity and in vivo tumorigenicity of AKT-inhibited cells, suggesting that SOX2 acts as a functional downstream AKT target. Mechanistically, we show that AKT physically interacts with the SOX2 protein to modulate its subcellular distribution. AKT kinase inhibition results in enhanced cytoplasmic retention of SOX2, presumably via impaired nuclear import, and in successive cytoplasmic proteasomal degradation of the protein. In line, blockade of either nuclear transport or proteasomal degradation rescues SOX2 expression in AKT-inhibited BC cells. Finally, AKT inhibitors efficiently suppress the growth of SOX2-expressing putative cancer stem cells, whereas conventional chemotherapeutics select for this population. Together, our results suggest the AKT/SOX2 molecular axis as a regulator of BC clonogenicity and AKT inhibitors as promising drugs for the treatment of SOX2-positive BC. PMID:26498353

  6. Temperature-induced inversion of allosteric phenomena.

    PubMed

    Braxton, B L; Tlapak-Simmons, V L; Reinhart, G D

    1994-01-01

    Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation. In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand. Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate. The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner. These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography. PMID:8276837

  7. Disentangling the web of allosteric communication in a homotetramer: heterotropic inhibition of phosphofructokinase from Bacillus stearothermophilus.

    PubMed

    Ortigosa, Allison D; Kimmel, Jennifer L; Reinhart, Gregory D

    2004-01-20

    A strategy for isolating each of the four potentially unique heterotropic pairwise allosteric interactions that exist in the homotetramer phosphofructokinase from Bacillus stearothermophilus is described. The strategy involves the construction of hybrid tetramers containing one wild-type subunit and three mutant subunits that have been modified to block binding of both the substrate, fructose 6-phosphate (Fru-6-P), and the allosteric inhibitor, phospho(enol)pyruvate (PEP). Each type of binding site occurs at a subunit interface, and mutations on either side of the interface have been identified that will greatly diminish binding at the respective site. Consequently, four different types of mutant subunits have been created, each containing a different active site and allosteric site modification. The corresponding 1:3 hybrids isolate a different pair of unmodified substrate and allosteric sites with a unique structural disposition located 22, 30, 32, and 45 A apart, respectively. The allosteric inhibition exhibited by the unmodified sites in each of these four hybrids has been quantitatively evaluated in terms of a coupling free energy. Each of the coupling free energies is unique in magnitude, and their relative magnitudes vary with pH. Importantly, the sum of these coupling free energies at each pH is equal to the total heterotropic coupling free energy associated with the tetrameric enzyme. The latter quantity was assessed from the overall inhibition of a control hybrid that removed the homotropic interactions in PEP binding. The results do not agree with either the concerted or sequential models that are often invoked to explain allosteric behavior in oligomeric enzymes.

  8. CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

    PubMed

    Mortensen, Deborah S; Fultz, Kimberly E; Xu, Shuichan; Xu, Weiming; Packard, Garrick; Khambatta, Godrej; Gamez, James C; Leisten, Jim; Zhao, Jingjing; Apuy, Julius; Ghoreishi, Kamran; Hickman, Matt; Narla, Rama Krishna; Bissonette, Rene; Richardson, Samantha; Peng, Sophie X; Perrin-Ninkovic, Sophie; Tran, Tam; Shi, Tao; Yang, Wen Qing; Tong, Zeen; Cathers, Brian E; Moghaddam, Mehran F; Canan, Stacie S; Worland, Peter; Sankar, Sabita; Raymon, Heather K

    2015-06-01

    mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

  9. The AKT-mTOR signalling pathway in kidney cancer tissues

    NASA Astrophysics Data System (ADS)

    Spirina, L. V.; Usynin, Y. A.; Kondakova, I. V.; Yurmazov, Z. A.; Slonimskaya, E. M.; Kolegova, E. S.

    2015-11-01

    An increased expression of phospho-AKT, m-TOR, glycogen regulator GSK-3-beta and transcription inhibitor 4E-BP1 was observed in kidney cancer tissues. Tumor size growth was associated with a high level of c-Raf and low content of phospho-m-TOR. Cancer metastasis development led to a decreased PTEN and phospho-AKT expression.

  10. Inhibition of PTEN and activation of Akt by menadione.

    PubMed

    Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

    2007-04-01

    Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

  11. The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

    SciTech Connect

    Dormond-Meuwly, Anne; Roulin, Didier; Dufour, Marc; Benoit, Michael; Demartines, Nicolas; Dormond, Olivier

    2011-04-22

    Highlights: {yields} Targeting mTOR in endothelial cell activates MAPK. {yields} Blocking MAPK enhances the anti-angiogenic effects of mTOR inhibitors. {yields} The anti-angiogenic efficacy of ATP-competitive inhibitors of mTOR is superior to that of rapamycin. -- Abstract: The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.

  12. An IκBα phosphorylation inhibitor induces heme oxygenase-1(HO-1) expression through the activation of reactive oxygen species (ROS)-Nrf2-ARE signaling and ROS-PI3K/Akt signaling in an NF-κB-independent mechanism.

    PubMed

    Min, Kyoung-jin; Lee, Jung Tae; Joe, Eun-hye; Kwon, Taeg Kyu

    2011-09-01

    Reactive oxygen species (ROS) are important signaling molecules in cells. Excessive ROS induce expression of inflammatory mediators, such as iNOS and COX2. Antioxidant enzymes, such as, heme oxygenase-1 (HO-1), tightly regulate ROS levels within cells. Here, we show that Bay 11-7082 (Bay) increased HO-1 mRNA and protein expression in human colon cancer HT29 cells. Bay induced translocation of NF-E2-related factor 2 (Nrf2) into nuclei and increased the binding activity of the antioxidant response element (ARE). In addition, PI3K/Akt inhibitor (LY294002) blocked Bay-induced HO-1 expression. Pretreatment with anti-oxidants (N-acetylcysteine (NAC) or glutathione) significantly reduced Bay-induced HO-1 mRNA/protein expression, nuclear translocation of Nrf2 and phosphorylation of Akt. However, PI3K/Akt signaling was independent of Bay-induced Nrf2 translocation and ARE binding activity. Furthermore, other NF-κB inhibitors, such as pyrrolidine dithiocarbamate (PDTC) and MG132, also increased HO-1 mRNA and protein expression. However, although overexpression of dominant negative inhibitory κB (IκB) reduced NF-κB-driven transcriptional activity, IκB overexpression did not increase HO-1 expression. Taken together, our results suggest that in human colon cancer HT29 cells, Bay induces HO-1 expression by increasing ROS production in an Nrf2-ARE and PI3K dependent manner, but Bay acts independently of NF-κB.

  13. In Vivo Structure-Activity Relationship Studies Support Allosteric Targeting of a Dual Specificity Phosphatase

    PubMed Central

    Korotchenko, Vasiliy N.; Saydmohammed, Manush; Vollmer, Laura L.; Bakan, Ahmet; Sheetz, Kyle; Debiec, Karl T.; Greene, Kristina A.; Agliori, Christine S.; Bahar, Ivet; Day, Billy W.; Vogt, Andreas; Tsang, Michael

    2014-01-01

    Dual specificity phosphatase 6 (DUSP6) functions as a feedback attenuator of Fibroblast Growth Factor signaling during development. In vitro high throughput chemical screening attempts to discover DUSP6 inhibitors have yielded limited success. Yet, in vivo whole organism screens using zebrafish identified 1 (BCI) as an allosteric inhibitor of DUSP6. Here we designed and synthesized a panel of analogs to define structure-activity relationship (SAR) of DUSP6 inhibition. In vivo, high-content analysis in transgenic zebrafish coupled with cell-based chemical complementation assays identified structural features of the 1 pharmacophore that were essential for biological activity. In vitro assays of DUSP hyperactivation corroborated the results from in vivo and cellular SAR. The results reinforce the notion that DUSPs are druggable through allosteric mechanisms, and illustrate the utility of zebrafish as a model organism for in vivo SAR analyses. PMID:24909879

  14. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  15. Toward understanding the molecular basis for chemical allosteric modulator design.

    PubMed

    Wang, Qi; Zheng, Mingyue; Huang, Zhimin; Liu, Xinyi; Zhou, Huchen; Chen, Yingyi; Shi, Ting; Zhang, Jian

    2012-09-01

    Among the regulation mechanisms of cellular function, allosteric regulation is the most direct, rapid and efficient. Due to the wider receptor selectivity and lower target-based toxicity, compared with orthosteric ligands, allosteric modulators are expected to play a larger role in pharmaceutical research and development. However, current difficulties, such as a low affinity and unknown structural features of potential allosteric small-molecules, usually obstruct the discovery of allosteric modulators. In this study, we compared known allosteric modulators with various compounds from different databases to unveil the structural and qualitative characteristics of allosteric modulators. The results show that allosteric modulators generally contain more hydrophobic scaffolds and have a higher structural rigidity, i.e., less rotatable bonds and more rings. Based on this analysis, an empirical rule was defined to determine the structural requirements for an allosteric modulator. It was found that a large proportion of allosteric modulators (80%) can be successfully retrieved by this "allosteric-like" filter, which shows good discriminatory power in identifying allosteric modulators. Therefore, the study provides deeper insight into the chemical properties of allosteric modulators and has a good potential for the design or optimization of allosteric compounds. PMID:23085171

  16. MECHANISMS OF SPHINGOSINE-1-PHOSPHATE INDUCED AKT DEPENDENT SMOOTH MUSCLE CELL MIGRATION

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid released from activated platelets, which stimulates migration of vascular smooth muscle cells (VSMC) in vitro. S-1-P will activate akt, which can regulate multiple cellular functions including cell migration. Akt activation is downstream of phosphatidyl-inositol 3′ kinase (PI3-K) and Phosphoinositide-dependent protein kinase-1 (PDK1). Objective To examine the regulation of akt signaling during smooth muscle cell migration in response to S-1-P. Methods Murine arterial SMCs were cultured in vitro. Linear wound and Boyden microchemotaxis assays of migration were performed in the presence of S-1-P with and without an akt inhibitor (aktI). Assays were performed for PI3-K, PDK1, akt and GSK3β activation in the presence of various inhibitors and after transfection with the Gβγ inhibitor. βARKCT. Results S-1-P induced time dependent PI3-K, PDK1 and akt activation. The migratory responses in both assays to S-1-P were blocked by akt inhibitor (aktI). Activation of akt and dephosphorylation of its downstream kinase, GSK3 β, were inhibited by aktI. Inhibition of PI3-K with LY294002 significantly reduced both PI3-K and akt activation. Inhibition of G βγ inhibited akt activation through a reduction in both PI3-K and PDK1 activation. While inhibition of the ras with manumycin A had no effect, inhibition of rho with C3 limited both PI3K and akt activation. PDK1 responses were unchanged by inhibition of GTPases. Inhibition of reactive oxygen species generation with N-acetylcysteine and of EGFR with AG1478 inhibited PDK1 activation in response to S-1-P. Conclusion S-1-P mediated migration is akt dependent. S-1-P mediated akt phosphorylation is controlled by G βγ dependent, PI3-K activation, which requires the GTPase rho and Gβγ. PDK1 activation requires Gβγ reactive oxygen species generation and EGFR activation. PMID:19081473

  17. FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

    PubMed Central

    Tiraidis, Costas; Alexacou, Kyra-Melinda; Zographos, Spyros E.; Leonidas, Demetres D.; Gimisis, Thanasis; Oikonomakos, Nikos G.

    2007-01-01

    FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b–FR258900 complex and refined it to 2.2 Å resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45′ (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy-phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site. PMID:17600143

  18. Allosteric modulation of glycine receptors

    PubMed Central

    Yevenes, Gonzalo E; Zeilhofer, Hanns Ulrich

    2011-01-01

    Inhibitory (or strychnine sensitive) glycine receptors (GlyRs) are anion-selective transmitter-gated ion channels of the cys-loop superfamily, which includes among others also the inhibitory γ-aminobutyric acid receptors (GABAA receptors). While GABA mediates fast inhibitory neurotransmission throughout the CNS, the action of glycine as a fast inhibitory neurotransmitter is more restricted. This probably explains why GABAA receptors constitute a group of extremely successful drug targets in the treatment of a wide variety of CNS diseases, including anxiety, sleep disorders and epilepsy, while drugs specifically targeting GlyRs are virtually lacking. However, the spatially more restricted distribution of glycinergic inhibition may be advantageous in situations when a more localized enhancement of inhibition is sought. Inhibitory GlyRs are particularly relevant for the control of excitability in the mammalian spinal cord, brain stem and a few selected brain areas, such as the cerebellum and the retina. At these sites, GlyRs regulate important physiological functions, including respiratory rhythms, motor control, muscle tone and sensory as well as pain processing. In the hippocampus, RNA-edited high affinity extrasynaptic GlyRs may contribute to the pathology of temporal lobe epilepsy. Although specific modulators have not yet been identified, GlyRs still possess sites for allosteric modulation by a number of structurally diverse molecules, including alcohols, neurosteroids, cannabinoids, tropeines, general anaesthetics, certain neurotransmitters and cations. This review summarizes the present knowledge about this modulation and the molecular bases of the interactions involved. PMID:21557733

  19. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

    PubMed

    Al-Horani, Rami A; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K; Desai, Umesh R

    2016-01-01

    Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants. PMID:27467511

  20. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile

    PubMed Central

    Al-Horani, Rami A.; Karuturi, Rajesh; Lee, Michael; Afosah, Daniel K.

    2016-01-01

    Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants. PMID:27467511

  1. Biochemical and Cellular Evidence Demonstrating AKT-1 as a Binding Partner for Resveratrol Targeting Protein NQO2

    PubMed Central

    Hsieh, Tze-chen; Lin, Chia-Yi; Bennett, Dylan John; Wu, Erxi; Wu, Joseph M.

    2014-01-01

    Background AKT plays an important role in the control of cell proliferation and survival. Aberrant activation of AKT frequently occurs in human cancers making it an attractive drug targets and leading to the synthesis of numerous AKT inhibitors as therapeutic candidates. Less is known regarding proteins that control AKT. We recently reported that quinone reductase 2 (NQO2) inhibited AKT activity, by unknown mechanisms. Methodology/Principal Findings In this study, molecular modeling was used to query interaction between NQO2 and AKT. We found that pleckstrin homology (PH) and kinase domains of AKT bind to chains A and B of NQO2. Pull-down and deletion assays revealed that PH domain of AKT is essential for interaction with NQO2. Modeling analysis further revealed that kinase domain of AKT binds NQO2 in the vicinity of asparagine 161 located in the resveratrol-binding domain of NQO2. In studies to test whether exposure to resveratrol potentiates or diminishes AKT binding to NQO2, we showed that pre-binding by resveratrol in wild type but not histidine-161 (N161H) mutant NQO2 significantly affected this interaction. To obtain information on interplay between resveratrol and AKT, resveratrol affinity chromatography was performed. AKT binds with high affinity to the column suggesting that it is a target of resveratrol. The half-life of AKT mRNA decreased from ∼4 h in control cells to ∼1 h in NQO2-knockdown cells. The inhibition of AKT by resveratrol was attenuated in NQO2-expressing relative to NQO2-knockdown cells. Conclusion/Significance Both NQO2 and AKT are targets of resveratrol; NQO2:AKT interaction is a novel physiological regulator of AKT activation/function. PMID:24968355

  2. Akt is translocated to the mitochondria during etoposide-induced apoptosis of HeLa cells.

    PubMed

    Park, Byoungduck; Je, Young-Tae; Chun, Kwang-Hoon

    2015-11-01

    Akt, or protein kinase B, is a key serine-threonine kinase, which exerts anti-apoptotic effects and promotes cell proliferation in response to various stimuli. Recently, however, it was demonstrated that Akt exhibits a proapoptotic role in certain contexts. During etoposide‑induced apoptosis of HeLa cells, Akt enhances the interaction of second mitochondria‑derived activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO) and X‑linked inhibitor of apoptosis protein by phosphorylating Smac at serine 67, and thus promotes apoptosis. However, the detailed mechanisms underlying Akt regulation in etoposide‑mediated apoptosis remain to be determined. The present study investigated whether etoposide triggers the translocation of Akt into the mitochondria. It was found that Akt activity was increased and sustained during apoptosis triggered by etoposide in HeLa cells. During apoptosis, Akt was translocated from the cytoplasm into the mitochondria in a phosphoinositide 3‑kinase-dependent manner at the early and late stages of apoptosis. Concomitantly, the depletion of Akt in the nuclear fraction was observed after etoposide treatment from analysis of confocal microscopy. The results suggest that etoposide‑stimulated Akt is translocated into the mitochondria, thereby possibly enhancing its interaction with Smac and promoting apoptosis in HeLa cells. These results indicate that Akt may be a promising candidate for a pro-apoptotic approach in cancer treatment.

  3. AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

    PubMed

    Hamilton, Garth; Abraham, Aswin G; Morton, Jennifer; Sampson, Oliver; Pefani, Dafni E; Khoronenkova, Svetlana; Grawenda, Anna; Papaspyropoulos, Angelos; Jamieson, Nigel; McKay, Colin; Sansom, Owen; Dianov, Grigory L; O'Neill, Eric

    2014-08-15

    Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.

  4. AKT regulates NPM dependent ARF localization and p53mut stability in tumors

    PubMed Central

    Morton, Jennifer; Sampson, Oliver; Pefani, Dafni E.; Khoronenkova, Svetlana; Grawenda, Anna; Papaspyropoulos, Angelos; Jamieson, Nigel; McKay, Colin; Sansom, Owen; Dianov, Grigory L.; O'Neill, Eric

    2014-01-01

    Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53mut stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53mut status. PMID:25071014

  5. Allosteric Inhibition Through Core Disruption

    SciTech Connect

    Horn, James R.; Shoichet, Brian K.

    2010-03-05

    Although inhibitors typically bind pre-formed sites on proteins, it is theoretically possible to inhibit by disrupting the folded structure of a protein or, in the limit, to bind preferentially to the unfolded state. Equilibria defining how such molecules act are well understood, but structural models for such binding are unknown. Two novel inhibitors of {beta}-lactamase were found to destabilize the enzyme at high temperatures, but at lower temperatures showed no preference for destabilized mutant enzymes versus stabilized mutants. X-ray crystal structures showed that both inhibitors bound to a cryptic site in {beta}-lactamase, which the inhibitors themselves created by forcing apart helixes 11 and 12. This opened up a portion of the hydrophobic core of the protein, into which these two inhibitors bind. Although this binding site is 16 {angstrom} from the center of the active site, the conformational changes were transmitted through a sequence of linked motions to a key catalytic residue, Arg244, which in the complex adopts conformations very different from those in catalytically competent enzyme conformations. These structures offer a detailed view of what has heretofore been a theoretical construct, and suggest the possibility for further design against this novel site.

  6. Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis, and Biological Evaluation.

    PubMed

    Nguyen, Thuy; Coover, Robert A; Verghese, Jenson; Moran, Richard G; Ellis, Keith C

    2014-05-01

    Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3β cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.

  7. Enzyme-substrate complexes of allosteric citrate synthase: evidence for a novel intermediate in substrate binding.

    PubMed

    Duckworth, Harry W; Nguyen, Nham T; Gao, Yin; Donald, Lynda J; Maurus, Robert; Ayed, Ayeda; Bruneau, Brigitte; Brayer, Gary D

    2013-12-01

    The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.

  8. Detecting Allosteric Networks Using Molecular Dynamics Simulation.

    PubMed

    Bowerman, S; Wereszczynski, J

    2016-01-01

    Allosteric networks allow enzymes to transmit information and regulate their catalytic activities over vast distances. In principle, molecular dynamics (MD) simulations can be used to reveal the mechanisms that underlie this phenomenon; in practice, it can be difficult to discern allosteric signals from MD trajectories. Here, we describe how MD simulations can be analyzed to reveal correlated motions and allosteric networks, and provide an example of their use on the coagulation enzyme thrombin. Methods are discussed for calculating residue-pair correlations from atomic fluctuations and mutual information, which can be combined with contact information to identify allosteric networks and to dynamically cluster a system into highly correlated communities. In the case of thrombin, these methods show that binding of the antagonist hirugen significantly alters the enzyme's correlation landscape through a series of pathways between Exosite I and the catalytic core. Results suggest that hirugen binding curtails dynamic diversity and enforces stricter venues of influence, thus reducing the accessibility of thrombin to other molecules. PMID:27497176

  9. An Allosteric Circuit in Caspase-1

    SciTech Connect

    Datta, D.; Scheer, J.M.; Romanowski, M.J.; Wells, J.A.

    2009-05-14

    Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface approximately 15 A from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them-Arg286 and Glu390, which form a salt bridge-have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k(cat)/K(m)). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k(cat)/K(m). X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or 'hot wire' that promotes site-to-site coupling.

  10. Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

    SciTech Connect

    Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won; Bae, Jin Ung; Kim, Gyu Hee; Park, So Youn; Kim, Chi Dae

    2012-04-01

    Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.

  11. Modifying akt signaling in B-cell chronic lymphocytic leukemia cells.

    PubMed

    Hofbauer, Sebastian W; Piñón, Josefina D; Brachtl, Gabriele; Haginger, Lucia; Wang, Wei; Jöhrer, Karin; Tinhofer, Ingeborg; Hartmann, Tanja Nicole; Greil, Richard

    2010-09-15

    Emerging evidence suggests that the survival of B-cell chronic lymphocytic leukemia (CLL) cells is dependent on microenvironmental influences such as antigenic stimulation and support by stromal cells. Akt, also known as protein kinase B, is a central component in prosurvival signaling downstream of these events. We investigated the role of Akt and its modulation by the protooncogene T-cell leukemia 1a (Tcl1a) in the survival pathways of primary CLL samples and CLL-derived prolymphocytic cell lines MEC-1 and MEC-2. Akt activation was increased by the protective presence of human bone marrow stromal cells and B-cell receptor mimicking signals but antagonized by direct Akt blockade with the novel specific inhibitor AiX, with preferential apoptosis induction in CLL cells with an unmutated immunoglobulin status, which predicts poor clinical outcome. In addition, we found a direct interaction of Akt with Tcl1a in an endogenous coimmunoprecipitation assay. Confirming the critical role of Tcl1a in modulating Akt signaling, Akt activation was enhanced by overexpressing Tcl1a in CLL. In contrast, decreasing Tcl1a levels by small interfering RNA reduced Akt activation in the fludarabine-insensitive CLL cell line MEC-2 and sensitized the malignant cells to fludarabine treatment. In summary, our data reveal a significant role for the Akt-Tcl1a axis in CLL survival and propose a further evaluation of this interplay for targeting chemoresistance phenomena.

  12. Nitric Oxide Synthase and Breast Cancer: Role of TIMP-1 in NO-mediated Akt Activation

    PubMed Central

    Ridnour, Lisa A.; Barasch, Kimberly M.; Windhausen, Alisha N.; Dorsey, Tiffany H.; Lizardo, Michael M.; Yfantis, Harris G.; Lee, Dong H.; Switzer, Christopher H.; Cheng, Robert Y. S.; Heinecke, Julie L.; Brueggemann, Ernst; Hines, Harry B.; Khanna, Chand; Glynn, Sharon A.; Ambs, Stefan; Wink, David A.

    2012-01-01

    Prediction of therapeutic response and cancer patient survival can be improved by the identification of molecular markers including tumor Akt status. A direct correlation between NOS2 expression and elevated Akt phosphorylation status has been observed in breast tumors. Tissue inhibitor matrix metalloproteinase-1 (TIMP-1) has been proposed to exert oncogenic properties through CD63 cell surface receptor pathway initiation of pro-survival PI3k/Akt signaling. We employed immunohistochemistry to examine the influence of TIMP-1 on the functional relationship between NOS2 and phosphorylated Akt in breast tumors and found that NOS2-associated Akt phosphorylation was significantly increased in tumors expressing high TIMP-1, indicating that TIMP-1 may further enhance NO-induced Akt pathway activation. Moreover, TIMP-1 silencing by antisense technology blocked NO-induced PI3k/Akt/BAD phosphorylation in cultured MDA-MB-231 human breast cancer cells. TIMP-1 protein nitration and TIMP-1/CD63 co-immunoprecipitation was observed at NO concentrations that induced PI3k/Akt/BAD pro-survival signaling. In the survival analysis, elevated tumor TIMP-1 predicted poor patient survival. This association appears to be mainly restricted to tumors with high NOS2 protein. In contrast, TIMP-1 did not predict poor survival in patient tumors with low NOS2 expression. In summary, our findings suggest that tumors with high TIMP-1 and NOS2 behave more aggressively by mechanisms that favor Akt pathway activation. PMID:22957045

  13. Lysophosphatidic acid induces cell migration through the selective activation of Akt1

    PubMed Central

    Kim, Eun Kyoung; Yun, Sung Ji; Do, Kee Hun; Kim, Min Sung; Cho, Mong; Suh, Dong-Soo; Kim, Chi Dae; Kim, Jae Ho; Birnbaum, Morris J.

    2008-01-01

    Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration. PMID:18779657

  14. Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements.

    PubMed

    Cisneros, David A; Montero-Morán, Gabriela M; Lara-González, Samuel; Calcagno, Mario L

    2004-01-01

    Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.

  15. Vitamin E Facilitates the Inactivation of the Kinase Akt by the Phosphatase PHLPP1

    PubMed Central

    Huang, Po-Hsien; Chuang, Hsiao-Ching; Chou, Chih-Chien; Wang, Huiling; Lee, Su-Lin; Yang, Hsiao-Ching; Chiu, Hao-Chieh; Kapuriya, Naval; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

    2014-01-01

    rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors. PMID:23512990

  16. PP2A inhibition results in hepatic insulin resistance despite Akt2 activation.

    PubMed

    Galbo, Thomas; Perry, Rachel J; Nishimura, Erica; Samuel, Varman T; Quistorff, Bjørn; Shulman, Gerald I

    2013-10-01

    In the liver, insulin suppresses hepatic gluconeogenesis by activating Akt, which inactivates the key gluconeogenic transcription factor FoxO1 (Forkhead Box O1). Recent studies have implicated hyperactivity of the Akt phosphatase Protein Phosphatase 2A (PP2A) and impaired Akt signaling as a molecular defect underlying insulin resistance. We therefore hypothesized that PP2A inhibition would enhance insulin-stimulated Akt activity and decrease glucose production. PP2A inhibitors increased hepatic Akt phosphorylation and inhibited FoxO1in vitro and in vivo, and suppressed gluconeogenesis in hepatocytes. Paradoxically, PP2A inhibition exacerbated insulin resistance in vivo. This was explained by phosphorylation of both hepatic glycogen synthase (GS) (inactivation) and phosphorylase (activation) resulting in impairment of glycogen storage. Our findings underline the significance of GS and Phosphorylase as hepatic PP2A substrates and importance of glycogen metabolism in acute plasma glucose regulation. PMID:24150286

  17. Design of an allosterically regulated retroaldolase

    PubMed Central

    Raymond, Elizabeth A; Mack, Korrie L; Yoon, Jennifer H; Moroz, Olesia V; Moroz, Yurii S; Korendovych, Ivan V

    2015-01-01

    We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca2+ ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue. PMID:25516403

  18. Molecular Basis of Allosteric Transitions: GroEL

    NASA Astrophysics Data System (ADS)

    Horovitz, Amnon

    Chaperonins such as GroEL from Escherichia coli are molecular machines that facilitate protein folding by undergoing energy (ATP)-dependent movements that are coordinated in time and space owing to complex allosteric regulation. Here, we describe some of the various functional (allosteric) states of GroEL, the pathways by which they inter-convert and the coupling between allosteric transitions and protein folding reactions.

  19. MYOCARDIAL AKT: THE OMNIPRESENT NEXUS

    PubMed Central

    Sussman, Mark A.; Völkers, Mirko; Fischer, Kimberlee; Bailey, Brandi; Cottage, Christopher T.; Din, Shabana; Gude, Natalie; Avitabile, Daniele; Alvarez, Roberto; Sundararaman, Balaji; Quijada, Pearl; Mason, Matt; Konstandin, Mathias H.; Malhowski, Amy; Cheng, Zhaokang; Khan, Mohsin; McGregor, Michael

    2013-01-01

    One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses. PMID:21742795

  20. Pemetrexed Induces S-Phase Arrest and Apoptosis via a Deregulated Activation of Akt Signaling Pathway

    PubMed Central

    Chen, Kun-Chieh; Yang, Tsung-Ying; Wu, Chun-Chi; Cheng, Chi-Chih; Hsu, Shih-Lan; Hung, Hsiao-Wen

    2014-01-01

    Pemetrexed is approved for first-line and maintenance treatment of patients with advanced or metastatic non-small-cell lung cancer (NSCLC). The protein kinase Akt/protein kinase B is a well-known regulator of cell survival which is activated by pemetrexed, but its role in pemetrexed-mediated cell death and its molecular mechanisms are unclear. This study showed that stimulation with pemetrexed induced S-phase arrest and cell apoptosis and a parallel increase in sustained Akt phosphorylation and nuclear accumulation in the NSCLC A549 cell line. Inhibition of Akt expression by Akt specific siRNA blocked S-phase arrest and protected cells from apoptosis, indicating an unexpected proapoptotic role of Akt in the pemetrexed-mediated toxicity. Treatment of A549 cells with pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and Ly294002, similarly inhibited pemetrexed-induced S-phase arrest and apoptosis and Akt phosphorylation, indicating that PI3K is an upstream mediator of Akt and is involved in pemetrexed-mediated cell death. Previously, we identified cyclin A-associated cyclin-dependent kinase 2 (Cdk2) as the principal kinase that was required for pemetrexed-induced S-phase arrest and apoptosis. The current study showed that inhibition of Akt function and expression by pharmacological inhibitors as well as Akt siRNA drastically inhibited cyclin A/Cdk2 activation. These pemetrexed-mediated biological and molecular events were also observed in a H1299 cell line. Overall, our results indicate that, in contrast to its normal prosurvival role, the activated Akt plays a proapoptotic role in pemetrexed-mediated S-phase arrest and cell death through a mechanism that involves Cdk2/cyclin A activation. PMID:24847863

  1. Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

    PubMed

    Rodina, Anna; Patel, Pallav D; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J H; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R; Patel, Hardik J; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T; Young, Jason C; Chiosis, Gabriela

    2013-12-19

    Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  2. Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein

    PubMed Central

    Rodina, Anna; Patel, Pallav D.; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J.H.; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R.; Patel, Hardik J.; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T.; Young, Jason C.; Chiosis, Gabriela

    2014-01-01

    SUMMARY Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  3. Identification of an allosteric pocket on human hsp70 reveals a mode of inhibition of this therapeutically important protein.

    PubMed

    Rodina, Anna; Patel, Pallav D; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J H; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R; Patel, Hardik J; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T; Young, Jason C; Chiosis, Gabriela

    2013-12-19

    Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70.

  4. Novel Allosteric Modulators of G Protein-coupled Receptors*

    PubMed Central

    Gentry, Patrick R.; Sexton, Patrick M.; Christopoulos, Arthur

    2015-01-01

    G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization. PMID:26100627

  5. Universal allosteric mechanism for Gα activation by GPCRs

    PubMed Central

    Flock, Tilman; Venkatakrishnan, A. J.; Kayikci, Melis; Tate, Christopher G.; Veprintsev, Dmitry B.; Babu, M. Madan

    2016-01-01

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ~800 human GPCRs and 16 different Gα proteins, does a universal allosteric mechanism govern Gα activation? Here we show that different GPCRs interact and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that can undergo disorder-order transitions decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, whilst conserving the allosteric activation mechanism. PMID:26147082

  6. Universal allosteric mechanism for Gα activation by GPCRs.

    PubMed

    Flock, Tilman; Ravarani, Charles N J; Sun, Dawei; Venkatakrishnan, A J; Kayikci, Melis; Tate, Christopher G; Veprintsev, Dmitry B; Babu, M Madan

    2015-08-13

    G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism. PMID:26147082

  7. Identification of novel allosteric regulators of human-erythrocyte pyruvate kinase.

    PubMed

    Kharalkar, Shilpa S; Joshi, Gajanan S; Musayev, Faik N; Fornabaio, Micaela; Abraham, Donald J; Safo, Martin K

    2007-11-01

    Erythrocyte pyruvate kinase (PK) is an important glycolytic enzyme, and manipulation of its regulatory behavior by allosteric modifiers is of interest for medicinal purposes. Human-erythrocyte PK was expressed in Rosetta cells and purified on an Ni-NTA column. A search of the small-molecules database of the National Cancer Institute (NCI), using the UNITY software, led to the identification of several compounds with similar pharmacophores as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the human kinases. The compounds were subsequently docked into the FBP binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates, compounds 1-7, were obtained from the NCI, and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). The allosteric effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction.

  8. Allosteric inhibition of g-protein coupled receptor oligomerization: strategies and challenges for drug development.

    PubMed

    Hurevich, Mattan; Talhami, Alaa; Shalev, Deborah E; Gilon, Chaim

    2014-01-01

    G-protein coupled receptors (GPCRs) mediate a large number of biological pathways and are major therapeutic targets. One of the most exiting phenomena of GPCRs is their ability to interact with other GPCRs. GPCRGPCR interactions, also known as GPCR oligomerization, may create various functional entities such as homo- and heterodimers and also form complex multimeric GPCR clusters. In many biological systems, GPCR-GPCR interactions are crucial for signal regulation. The interaction with other receptors results in allosteric modifications of GPCRs through conformational changes. Allosteric inhibition of GPCRs is considered an attractive strategy for drug development and does not involve targeting the orthosteric site. Understanding the nature of GPCR-GPCR interactions is mandatory for developing allosteric inhibitors. Studying GPCR-GPCR interactions is a challenging task and many methods have been developed to analyze these events. This review will highlight some of the methods developed to study GPCR-GPCR interactions and will describe pivotal studies that provided the basic understanding of the importance of GPCR oligomerization. We will also describe the significance of GPCR interaction networks for drug development. Recent studies will be reviewed to illustrate the use of state-of-the-art biophysical and spectroscopic methods for the discovery of GPCR oligomerization modulators.

  9. Blockade of Glioma Proliferation Through Allosteric Inhibition of JAK2

    PubMed Central

    He, Kunyan; Qi, Qi; Chan, Chi-Bun; Xiao, Ge; Liu, Xia; Tucker-Burden, Carol; Wang, Liya; Mao, Hui; Lu, Xiang; McDonald, Frank E.; Luo, Hongbo; Fan, Qi-Wen; Weiss, William A.; Sun, Shi-Yong; Brat, Daniel J.; Ye, Keqiang

    2016-01-01

    The gene that encodes the epidermal growth factor receptor (EGFR) is frequently overexpressed or mutated in human cancers, including glioblastoma. However, the efficacy of EGFR-targeted small-molecule inhibitors or monoclonal antibodies in glioblastomas that also have mutation or deletion of the gene encoding phosphatase and tensin homolog (PTEN) has been modest. We found that EGFR signaling was blocked by a small molecule (G5-7) that selectively inhibited Janus kinase 2 (JAK2)–mediated phosphorylation and activation of EGFR and STAT3 (signal transducer and activator of transcription 3) by binding to JAK2, thereby decreasing the activity of downstream signaling by mTOR (mammalian target of rapamycin) and inducing cell cycle arrest. G5-7 inhibited the proliferation of PTEN-deficient glioblastoma cell lines harboring a constitutively active variant of EGFR (U87MG/EGFRvIII) and human glioblastoma explant neurosphere cultures, but the drug only weakly inhibited the proliferation of either glioblastoma cell lines that were wild type for EGFR and stably transfected with PTEN (U87MG/PTEN) or normal neural progenitor cells and astrocytes. Additionally, G5-7 reduced vascular endothelial growth factor (VEGF) secretion and endothelial cell migration and induced apoptosis in glioblastoma xenografts, thereby suppressing glioblastoma growth in vivo. Furthermore, G5-7 was more potent than EGFR or JAK2 inhibitors that interfere with either ligand or adenosine 5′-triphosphate (ATP) binding at impeding glioblastoma cell proliferation, demonstrating that this allosteric JAK2 inhibitor may be an effective clinical strategy. PMID:23838182

  10. Vitamin E facilitates the inactivation of the kinase Akt by the phosphatase PHLPP1.

    PubMed

    Huang, Po-Hsien; Chuang, Hsiao-Ching; Chou, Chih-Chien; Wang, Huiling; Lee, Su-Lin; Yang, Hsiao-Ching; Chiu, Hao-Chieh; Kapuriya, Naval; Wang, Dasheng; Kulp, Samuel K; Chen, Ching-Shih

    2013-03-19

    Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain-dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.

  11. Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

    PubMed

    Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

    2003-12-12

    Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt. PMID:14522978

  12. PI3K-Akt pathway: its functions and alterations in human cancer.

    PubMed

    Osaki, M; Oshimura, M; Ito, H

    2004-11-01

    Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and generates phosphatidylinositol-3,4,5-trisphosphate (PI(3, 4, 5)P3). PI(3, 4, 5)P3 is a second messenger essential for the translocation of Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase (PDK) 1 and PDK2. Activation of Akt plays a pivotal role in fundamental cellular functions such as cell proliferation and survival by phosphorylating a variety of substrates. In recent years, it has been reported that alterations to the PI3K-Akt signaling pathway are frequent in human cancer. Constitutive activation of the PI3K-Akt pathway occurs due to amplification of the PIK3C gene encoding PI3K or the Akt gene, or as a result of mutations in components of the pathway, for example PTEN (phosphatase and tensin homologue deleted on chromosome 10), which inhibit the activation of Akt. Several small molecules designed to specifically target PI3K-Akt have been developed, and induced cell cycle arrest or apoptosis in human cancer cells in vitro and in vivo . Moreover, the combination of an inhibitor with various cytotoxic agents enhances the anti-tumor efficacy. Therefore, specific inhibition of the activation of Akt may be a valid approach to treating human malignancies and overcoming the resistance of cancer cells to radiation or chemotherapy. PMID:15505410

  13. Glutaredoxin exerts an antiapoptotic effect by regulating the redox state of Akt.

    PubMed

    Murata, Hiroaki; Ihara, Yoshito; Nakamura, Hajime; Yodoi, Junji; Sumikawa, Koji; Kondo, Takahito

    2003-12-12

    Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt.

  14. Low-power laser irradiation inhibits Aβ25-35-induced cell apoptosis through Akt activation

    NASA Astrophysics Data System (ADS)

    Zhang, Zhigang; Tang, Yonghong

    2009-08-01

    Low-power laser irradiation (LPLI) can modulate various cellular processes such as proliferation, differentiation and apoptosis. Recently, LPLI has been applied to moderate Alzheimer's disease (AD), but the underlying mechanism remains unknown. The protective role of LPLI against the amyloid beta peptide (Aβ), a major constituent of AD plaques, has not been studied. PI3K/Akt pathway is extremely important in protecting cells from apoptosis caused by diverse stress stimuli. However, whether LPLI can inhibit Aβ-induced apoptosis through Akt activation is still unclear. In current study, using FRET (fluorescence resonance energy transfer) technique, we investigated the activity of Akt in response to LPLI treatment. B kinase activity reporter (BKAR), a recombinant FRET probe of Akt, was utilized to dynamically detect the activation of Akt after LPLI treatment. The results show that LPLI promoted the activation of Akt. Moreover, LPLI inhibits apoptosis induced by Aβ25-35 and the apoptosis inhibition can be abolished by wortmannin, a specific inhibitor of PI3K/Akt. Taken together, these results suggest that LPLI can inhibit Aβ25-35-induced cell apoptosis through Akt activation.

  15. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition

    PubMed Central

    Shi, Hubing; Hong, Aayoung; Kong, Xiangju; Koya, Richard C.; Song, Chunying; Moriceau, Gatien; Hugo, Willy; Yu, Clarissa C.; Ng, Charles; Chodon, Thinle; Scolyer, Richard A.; Kefford, Richard F.; Ribas, Antoni; Long, Georgina V.; Lo, Roger S.

    2013-01-01

    BRAF inhibitor (BRAFi) therapy leads to remarkable anti-melanoma responses, but the initial tumor shrinkage is commonly incomplete, providing a nidus for subsequent disease progression. Adaptive signaling may underlie early BRAFi resistance and influence the selection pattern for genetic variants causing late, acquired resistance. We show here that BRAFi (or BRAFi+MEKi) therapy in patients frequently led to rebound p-AKT levels in their melanomas early on treatment. In cell lines, BRAFi treatment led to rebound levels of RTKs (including PDGFRβ), PIP3, pleckstrin homology domain (PHD) recruitment, and p-AKT. PTEN expression limited this BRAFi-elicited PI3K-AKT signaling, which could be rescued by introduction of a mutant AKT1 (Q79K) kown to confer acquired BRAFi resistance. Functionally, AKT1 Q79K conferred BRAFi resistance via amplifying BRAFi-elicited PI3K-AKT signaling. Additionally, MAPK pathway inhibition enhanced clonogenic growth dependency on PI3K or AKT. Thus, adaptive or genetic upregulation of AKT critically participates in melanoma survival during BRAFi therapy. PMID:24265152

  16. Low Phosphorylated AKT Expression in Laryngeal Cancer: Indications for a Higher Metastatic Risk

    SciTech Connect

    Nijkamp, Monique M.; Span, Paul N.; Stegeman, Hanneke; Grénman, Reidar; Kaanders, Johannes H.A.M.; Bussink, Johan

    2013-10-01

    Purpose: To validate the association of phosphorylated (p)AKT with lymph node metastasis in an independent, homogeneous cohort of patients with larynx cancer. Methods and Materials: Seventy-eight patients with laryngeal cancer were included. Epidermal growth factor receptor, pAKT, vimentin, E-cadherin, hypoxia, and blood vessels were visualized in biopsy material using immunohistochemistry. Positive tumor areas and spatial relationships between markers were assessed by automated image analysis. In 6 laryngeal cancer cell lines, E-cadherin and vimentin messenger RNA was quantified by real-time polymerase chain reaction and by immunohistochemistry before and after treatment with the pAKT inhibitor MK-2206. Results: A significant correlation was found between low pAKT in the primary tumor and positive lymph node status (P=.0005). Tumors with lymph node metastases had an approximately 10-fold lower median pAKT value compared with tumors without lymph node metastases, albeit with large intertumor variations, validating our previous results. After inhibition of pAKT in laryngeal cancer cells with MK-2206, up-regulation of vimentin and a downregulation of E-cadherin occurred, consistent with epithelial–mesenchymal transition. Conclusion: Low pAKT expression in larynx tumors is associated with lymph node metastases. Further, inhibition of pAKT in laryngeal cancer induces epithelial–mesenchymal transition, predisposing for an increased metastatic risk.

  17. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

    PubMed

    Alvarez-Añorve, Laura I; Alonzo, Diego A; Mora-Lugo, Rodrigo; Lara-González, Samuel; Bustos-Jaimes, Ismael; Plumbridge, Jacqueline; Calcagno, Mario L

    2011-12-01

    The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

  18. ATP-competitive inhibitors of mTOR: an update.

    PubMed

    Schenone, S; Brullo, C; Musumeci, F; Radi, M; Botta, M

    2011-01-01

    mTOR (mammalian target of rapamycin) is a serine-threonine kinase belonging to the PI3K/Akt/mTOR signalling pathway that is involved in several cell functions, including growth, proliferation, apoptosis and autophagy. mTOR hyperactivation has been detected in several human cancers, thus representing, together with its upstream effectors, an important target for cancer therapy. mTOR exists in two different complexes in cells, mTORC1 and mTORC2 which could both be targeted by potential anticancer agents. Rapamycin, the selective and allosteric inhibitor of mTOR, inhibits the enzyme in mTORC1, but not in mTORC2. In the last few years a number of mTOR ATP-competitive inhibitors has been reported acting on mTOR in both complexes and possessing a more complete anticancer activity in comparison with that of rapamycin and its derivatives. mTOR shares high sequence homology in the hinge-region with PI3K that is a lipid kinase upstream to mTOR in the same signaling pathway; for this reason some compounds originally developed as PI3K inhibitors later showed to also target mTOR. As indicated by preclinical and clinical studies, compounds acting on more than one target could result in a better biological response and in enhanced therapeutic potential and also dual PI3K/mTOR inhibitors result of great interest as potential antitumor agents. This review mainly reports the recently discovered mTOR ATP-competitive inhibitors in terms of medicinal chemistry, classified by their chemical structures, focusing on SAR and modelling studies that led to the discovery of very potent and selective agents, such as AZD-8055, OSI-027 and INK128, already entered clinical trials, or WYE-132, Torin1 and others in preclinical studies. Also some examples of dual PI3K/mTOR inhibitors, including PI-103, GNE477, WJD008 and GSK2126458 are reported together with their biological and clinical data.

  19. The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia.

    PubMed

    Mendes, Rui D; Canté-Barrett, Kirsten; Pieters, Rob; Meijerink, Jules P P

    2016-09-01

    The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia. PMID:27582570

  20. The relevance of PTEN-AKT in relation to NOTCH1-directed treatment strategies in T-cell acute lymphoblastic leukemia

    PubMed Central

    Mendes, Rui D.; Canté-Barrett, Kirsten; Pieters, Rob; Meijerink, Jules P.P.

    2016-01-01

    The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycolysis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia. PMID:27582570

  1. Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2.

    PubMed

    Yu, Ker; Shi, Celine; Toral-Barza, Lourdes; Lucas, Judy; Shor, Boris; Kim, Jae Eun; Zhang, Wei-Guo; Mahoney, Robert; Gaydos, Christine; Tardio, Luanna; Kim, Sung Kyoo; Conant, Roger; Curran, Kevin; Kaplan, Joshua; Verheijen, Jeroen; Ayral-Kaloustian, Semiramis; Mansour, Tarek S; Abraham, Robert T; Zask, Arie; Gibbons, James J

    2010-01-15

    The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC(50): 0.19 +/- 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy.

  2. Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor.

    PubMed

    Burford, Neil T; Clark, Mary J; Wehrman, Tom S; Gerritz, Samuel W; Banks, Martyn; O'Connell, Jonathan; Traynor, John R; Alt, Andrew

    2013-06-25

    μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin-recruitment assay. PMID:23754417

  3. Neurobiological Insights from mGlu Receptor Allosteric Modulation

    PubMed Central

    O’Brien, Daniel E

    2016-01-01

    Allosteric modulation of metabotropic glutamate (mGlu) receptors offers a promising pharmacological approach to normalize neural circuit dysfunction associated with various psychiatric and neurological disorders. As mGlu receptor allosteric modulators progress through discovery and clinical development, both technical advances and novel tool compounds are providing opportunities to better understand mGlu receptor pharmacology and neurobiology. Recent advances in structural biology are elucidating the structural determinants of mGlu receptor–negative allosteric modulation and supplying the means to resolve active, allosteric modulator-bound mGlu receptors. The discovery and characterization of allosteric modulators with novel pharmacological profiles is uncovering the biological significance of their intrinsic agonist activity, biased mGlu receptor modulation, and novel mGlu receptor heterodimers. The development and exploitation of optogenetic and optopharmacological tools is permitting a refined spatial and temporal understanding of both mGlu receptor functions and their allosteric modulation in intact brain circuits. Together, these lines of research promise to provide a more refined understanding of mGlu receptors and their allosteric modulation that will inform the development of mGlu receptor allosteric modulators as neurotherapeutics in the years to come. PMID:26647381

  4. The structural basis of ATP as an allosteric modulator.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-09-01

    Adenosine-5'-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  5. The Structural Basis of ATP as an Allosteric Modulator

    PubMed Central

    Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

    2014-01-01

    Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

  6. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells.

    PubMed

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5' AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression of

  7. Danusertib, a potent pan-Aurora kinase and ABL kinase inhibitor, induces cell cycle arrest and programmed cell death and inhibits epithelial to mesenchymal transition involving the PI3K/Akt/mTOR-mediated signaling pathway in human gastric cancer AGS and NCI-N78 cells

    PubMed Central

    Yuan, Chun-Xiu; Zhou, Zhi-Wei; Yang, Yin-Xue; He, Zhi-Xu; Zhang, Xueji; Wang, Dong; Yang, Tianxing; Pan, Si-Yuan; Chen, Xiao-Wu; Zhou, Shu-Feng

    2015-01-01

    Gastric cancer is the second leading cause of cancer-related death worldwide, with a poor response to current chemotherapy. Danusertib is a pan-inhibitor of the Aurora kinases and a third-generation Bcr-Abl tyrosine kinase inhibitor with potent anticancer effects, but its antitumor effect and underlying mechanisms in the treatment of human gastric cancer are unknown. This study aimed to investigate the effects of danusertib on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition and the molecular mechanisms involved in human gastric cancer AGS and NCI-N78 cells. The results showed that danusertib had potent growth-inhibitory, apoptosis-inducing, and autophagy-inducing effects on AGS and NCI-N78 cells. Danusertib arrested AGS and NCI-N78 cells in G2/M phase, with downregulation of expression of cyclin B1 and cyclin-dependent kinase 1 and upregulation of expression of p21 Waf1/Cip1, p27 Kip1, and p53. Danusertib induced mitochondria-mediated apoptosis, with an increase in expression of proapoptotic protein and a decrease in antiapoptotic proteins in both cell lines. Danusertib induced release of cytochrome c from the mitochondria to the cytosol and triggered activation of caspase 9 and caspase 3 in AGS and NCI-N78 cells. Further, danusertib induced autophagy, with an increase in expression of beclin 1 and conversion of microtubule-associated protein 1A/1B-light chain 3 (LC3-I) to LC3-II in both cell lines. Inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways as well as activation of 5′ AMP-activated protein kinase contributed to the proautophagic effect of danusertib in AGS and NCI-N78 cells. SB202191 and wortmannin enhanced the autophagy-inducing effect of danusertib in AGS and NCI-N78 cells. In addition, danusertib inhibited epithelial to mesenchymal transition with an increase in expression of E-cadherin and a decrease in expression

  8. Molecular Insights into Metabotropic Glutamate Receptor Allosteric Modulation

    PubMed Central

    Gregory, Karen J.

    2015-01-01

    The metabotropic glutamate (mGlu) receptors are a group of eight family C G protein–coupled receptors that are expressed throughout the central nervous system (CNS) and periphery. Within the CNS the different subtypes are found in neurons, both pre- and/or postsynaptically, where they mediate modulatory roles and in glial cells. The mGlu receptor family provides attractive targets for numerous psychiatric and neurologic disorders, with the majority of discovery programs focused on targeting allosteric sites, with allosteric ligands now available for all mGlu receptor subtypes. However, the development of allosteric ligands remains challenging. Biased modulation, probe dependence, and molecular switches all contribute to the complex molecular pharmacology exhibited by mGlu receptor allosteric ligands. In recent years we have made significant progress in our understanding of this molecular complexity coupled with an increased understanding of the structural basis of mGlu allosteric modulation. PMID:25808929

  9. Detection of DNA methyltransferase activity using allosteric molecular beacons.

    PubMed

    Zhang, Weiting; Zu, Xiaolong; Song, Yanling; Zhu, Zhi; Yang, Chaoyong James

    2016-01-21

    Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity. When the specific site in an aMB is methylated by Dam MTase, the probe can be cut by the restriction nuclease DpnI to release a fluorophore labeled aptamer specific for streptavidin (SA) which will bind to SA beads to generate highly fluorescent beads for easy signal readout by a microscope or flow cytometer. However, aMBs maintain a hairpin structure without the binding ability to SA beads in the absence of Dam MTase, leading to weakly fluorescent SA beads. Unlike the existing signal amplified assays, our method is simpler and more convenient. The high performance of the aptamer and the easy bead separation process make this probe superior to other methods for the detection of MTase in complex biological systems. Overall, the proposed method with a detection limit of 0.57 U mL(-1) for Dam MTase shows great potential for further applications in the detection of other MTases, screening of MTase inhibitors, and early diagnosis of cancer.

  10. Viewpoint: Discriminating between noncompetitive and allosteric interactions.

    PubMed

    Ochs, Raymond S; Ashby, Charles R

    2008-03-01

    Understanding inhibition modes other than competitive is of clear importance in neuropharmacology. However, there appears to be some confusion concerning modes of inhibition that are not of the competitive type. It is critical to make a distinction between "not competitive" and "noncompetitive," as the later is a particular mode of inhibition. Further, there appears to be confusion between noncompetitive and allosteric behavior. Our purpose in this contribution is to explore the basis of these terms so that insight into pharmacological systems has a firmer mechanistic basis.

  11. Chemical, Target, and Bioactive Properties of Allosteric Modulation

    PubMed Central

    van Westen, Gerard J. P.; Gaulton, Anna; Overington, John P.

    2014-01-01

    Allosteric modulators are ligands for proteins that exert their effects via a different binding site than the natural (orthosteric) ligand site and hence form a conceptually distinct class of ligands for a target of interest. Here, the physicochemical and structural features of a large set of allosteric and non-allosteric ligands from the ChEMBL database of bioactive molecules are analyzed. In general allosteric modulators are relatively smaller, more lipophilic and more rigid compounds, though large differences exist between different targets and target classes. Furthermore, there are differences in the distribution of targets that bind these allosteric modulators. Allosteric modulators are over-represented in membrane receptors, ligand-gated ion channels and nuclear receptor targets, but are underrepresented in enzymes (primarily proteases and kinases). Moreover, allosteric modulators tend to bind to their targets with a slightly lower potency (5.96 log units versus 6.66 log units, p<0.01). However, this lower absolute affinity is compensated by their lower molecular weight and more lipophilic nature, leading to similar binding efficiency and surface efficiency indices. Subsequently a series of classifier models are trained, initially target class independent models followed by finer-grained target (architecture/functional class) based models using the target hierarchy of the ChEMBL database. Applications of these insights include the selection of likely allosteric modulators from existing compound collections, the design of novel chemical libraries biased towards allosteric regulators and the selection of targets potentially likely to yield allosteric modulators on screening. All data sets used in the paper are available for download. PMID:24699297

  12. International Union of Basic and Clinical Pharmacology. XC. multisite pharmacology: recommendations for the nomenclature of receptor allosterism and allosteric ligands.

    PubMed

    Christopoulos, Arthur; Changeux, Jean-Pierre; Catterall, William A; Fabbro, Doriano; Burris, Thomas P; Cidlowski, John A; Olsen, Richard W; Peters, John A; Neubig, Richard R; Pin, Jean-Philippe; Sexton, Patrick M; Kenakin, Terry P; Ehlert, Frederick J; Spedding, Michael; Langmead, Christopher J

    2014-10-01

    Allosteric interactions play vital roles in metabolic processes and signal transduction and, more recently, have become the focus of numerous pharmacological studies because of the potential for discovering more target-selective chemical probes and therapeutic agents. In addition to classic early studies on enzymes, there are now examples of small molecule allosteric modulators for all superfamilies of receptors encoded by the genome, including ligand- and voltage-gated ion channels, G protein-coupled receptors, nuclear hormone receptors, and receptor tyrosine kinases. As a consequence, a vast array of pharmacologic behaviors has been ascribed to allosteric ligands that can vary in a target-, ligand-, and cell-/tissue-dependent manner. The current article presents an overview of allostery as applied to receptor families and approaches for detecting and validating allosteric interactions and gives recommendations for the nomenclature of allosteric ligands and their properties.

  13. HDAC Inhibition Elicits Myocardial Protective Effect through Modulation of MKK3/Akt-1

    PubMed Central

    Zhao, Ting C.; Du, Jianfeng; Zhuang, Shugang; Liu, Paul; Zhang, Ling X.

    2013-01-01

    We and others have demonstrated that HDAC inhibition protects the heart against myocardial injury. It is known that Akt-1 and MAP kinase play an essential role in modulation of myocardial protection and cardiac preconditioning. Our recent observations have shown that Akt-1 was activated in post-myocardial infarction following HDAC inhibition. However, it remains unknown whether MKK3 and Akt-1 are involved in HDAC inhibition-induced myocardial protection in acute myocardial ischemia and reperfusion injury. We sought to investigate whether the genetic disruption of Akt-1 and MKK3 eliminate cardioprotection elicited by HDAC inhibition and whether Akt-1 is associated with MKK3 to ultimately achieve protective effects. Adult wild type and MKK3−/−, Akt-1−/− mice received intraperitoneal injections of trichostatin A (0.1mg/kg), a potent inhibitor of HDACs. The hearts were subjected to 30 min myocardial ischemia/30 min reperfusion in the Langendorff perfused heart after twenty four hours to elicit pharmacologic preconditioning. Left ventricular function was measured, and infarct size was determined. Acetylation and phosphorylation of MKK3 were detected and disruption of Akt-1 abolished both acetylation and phosphorylation of MKK3. HDAC inhibition produces an improvement in left ventricular functional recovery, but these effects were abrogated by disruption of either Akt-1 or MKK3. Disruption of Akt-1 or MKK3 abolished the effects of HDAC inhibition-induced reduction of infarct size. Trichostatin A treatment resulted in an increase in MKK3 phosphorylation or acetylation in myocardium. Taken together, these results indicate that stimulation of the MKK3 and Akt-1 pathway is a novel approach to HDAC inhibition -induced cardioprotection. PMID:23762381

  14. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  15. Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

    PubMed Central

    Sadidi, Mahdieh; Lentz, Stephen I.; Feldman, Eva L.

    2009-01-01

    Reactive oxygen species such as hydrogen peroxide (H2O2) are involved in many cellular processes that positively and negatively regulate cell fate. H2O2, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H2O2 was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H2O2-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery. PMID:19278624

  16. Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.

    PubMed

    Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N; Braus, Gerhard H

    2002-05-14

    The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.

  17. mTOR inhibitor temsirolimus and MEK1/2 inhibitor U0126 promote chromosomal instability and cell type-dependent phenotype changes of glioblastoma cells.

    PubMed

    Stepanenko, A A; Andreieva, S V; Korets, K V; Mykytenko, D O; Baklaushev, V P; Chekhonin, V P; Dmitrenko, V V

    2016-03-15

    The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and the RAF/mitogen-activated and extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are frequently deregulated in cancer. Temsirolimus (TEM) and its primary active metabolite rapamycin allosterically block mTOR complex 1 substrate recruitment. The context-/experimental setup-dependent opposite effects of rapamycin on the multiple centrosome formation, aneuploidy, DNA damage/repair, proliferation, and invasion were reported. Similarly, the context-dependent either tumor-promoting or suppressing effects of RAF-MEK-ERK pathway and its inhibitors were demonstrated. Drug treatment-mediated stress may promote chromosomal instability (CIN), accelerating changes in the genomic landscape and phenotype diversity. Here, we characterized the genomic and phenotypic changes of U251 and T98G glioblastoma cell lines long-term treated with TEM or U0126, an inhibitor of MEK1/2. TEM significantly increased clonal and non-clonal chromosome aberrations. Both TEM and U0126 affected copy number alterations (CNAs) pattern. A proliferation rate of U251TEM and U251U0126 cells was lower and higher, respectively, than control cells. Colony formation efficiency of U251TEM significantly decreased, whereas U251U0126 did not change. U251TEM and U251U0126 cells decreased migration. In contrast, T98GTEM and T98GU0126 cells did not change proliferation, colony formation efficiency, and migration. Changes in the sensitivity of inhibitor-treated cells to the reduction of the glucose concentration were observed. Our results suggest that CIN and adaptive reprogramming of signal transduction pathways may be responsible for the cell type-dependent phenotype changes of long-term TEM- or U0126-treated tumor cells. PMID:26748241

  18. Recent computational advances in the identification of allosteric sites in proteins.

    PubMed

    Lu, Shaoyong; Huang, Wenkang; Zhang, Jian

    2014-10-01

    Allosteric modulators have the potential to fine-tune protein functional activity. Therefore, the targeting of allosteric sites, as a strategy in drug design, is gaining increasing attention. Currently, it is not trivial to find and characterize new allosteric sites by experimental approaches. Alternatively, computational approaches are useful in helping researchers analyze and select potential allosteric sites for drug discovery. Here, we review state-of-the-art computational approaches directed at predicting putative allosteric sites in proteins, along with examples of successes in identifying allosteric sites utilizing these methods. We also discuss the challenges in developing reliable methods for predicting allosteric sites and tactics to resolve demanding tasks. PMID:25107670

  19. The inhibition of Akt-Pdpk1 interaction efficiently suppresses the growth of murine primary liver tumor cells.

    PubMed

    Mäemets-Allas, Kristina; Belitškin, Denis; Jaks, Viljar

    2016-05-20

    The lack of primary liver tumor cells has hampered testing of potential chemotherapeutic agents in vitro. To overcome this issue we developed a primary mouse liver tumor cell line K07074. The K07074 cells were immortal, exhibited a biliary phenotype, formed colonies in soft agar and displayed an increase in Hedgehog, Notch and Akt signaling. To study the effect of single and combined inhibition of the liver tumor-related pathways on the growth of K07074 cells we treated these with small-molecule antitumor agents. While the inhibition of Akt and Notch pathways strongly inhibited the growth of K07074 cells the inhibition of Wnt and Hedgehog pathways was less efficient in cell growth suppression. Interestingly, the inhibition of Akt pathway at the level of Akt-Pdpk1 interaction was sufficient to suppress the growth of tumor cells and no significant additive effect could be detected when co-treated with the inhibitors of Wnt, Hedgehog or Notch pathways. Only when suboptimal doses of Akt-Pdpk1 interaction inhibitor NSC156529 were used an additive effect with Notch inhibition was seen. We conclude that the Akt pathway inhibitor NSC156529 is potentially useful as single treatment for liver tumors with hyperactivated Akt signaling. PMID:27103434

  20. Allosteric dominance in carbamoyl phosphate synthetase.

    PubMed

    Braxton, B L; Mullins, L S; Raushel, F M; Reinhart, G D

    1999-02-01

    A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E. coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration. All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP. Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP. IMP causes only a slight inhibition at 25 degreesC. When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously. The same result was obtained with ornithine and IMP. By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site. When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound. The relationship of this observation to the structure of CPS is discussed. PMID:9931004

  1. Allosteric Modulators for mGlu Receptors

    PubMed Central

    Gasparini, F; Spooren, W

    2007-01-01

    The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson’s disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors. PMID:19305801

  2. Functional anatomy of an allosteric protein

    NASA Astrophysics Data System (ADS)

    Purohit, Prasad; Gupta, Shaweta; Jadey, Snehal; Auerbach, Anthony

    2013-12-01

    Synaptic receptors are allosteric proteins that switch on and off to regulate cell signalling. Here, we use single-channel electrophysiology to measure and map energy changes in the gating conformational change of a nicotinic acetylcholine receptor. Two separated regions in the α-subunits—the transmitter-binding sites and αM2-αM3 linkers in the membrane domain—have the highest ϕ-values (change conformation the earliest), followed by the extracellular domain, most of the membrane domain and the gate. Large gating-energy changes occur at the transmitter-binding sites, α-subunit interfaces, the αM1 helix and the gate. We hypothesize that rearrangements of the linkers trigger the global allosteric transition, and that the hydrophobic gate unlocks in three steps. The mostly local character of side-chain energy changes and the similarly high ϕ-values of separated domains, both with and without ligands, suggest that gating is not strictly a mechanical process initiated by the affinity change for the agonist.

  3. Expression, purification and characterization of human glutamate dehydrogenase (GDH) allosteric regulatory mutations.

    PubMed Central

    Fang, Jie; Hsu, Betty Y L; MacMullen, Courtney M; Poncz, Mortimer; Smith, Thomas J; Stanley, Charles A

    2002-01-01

    Glutamate dehydrogenase (GDH) catalyses the reversible oxidative deamination of l-glutamate to 2-oxoglutarate in the mitochondrial matrix. In mammals, this enzyme is highly regulated by allosteric effectors. The major allosteric activator and inhibitor are ADP and GTP, respectively; allosteric activation by leucine may play an important role in amino acid-stimulated insulin secretion. The physiological significance of this regulation has been highlighted by the identification of children with an unusual hyperinsulinism/hyperammonaemia syndrome associated with dominant mutations in GDH that cause a loss in GTP inhibition. In order to determine the effects of these mutations on the function of the human GDH homohexamer, we studied the expression, purification and characterization of two of these regulatory mutations (H454Y, which affects the putative GTP-binding site, and S448P, which affects the antenna region) and a mutation designed to alter the putative binding site for ADP (R463A). The sensitivity to GTP inhibition was impaired markedly in the purified H454Y (ED(50), 210 microM) and S448P (ED(50), 3.1 microM) human GDH mutants compared with the wild-type human GDH (ED(50), 42 nM) or GDH isolated from heterozygous patient cells (ED(50), 290 and 280 nM, respectively). Sensitivity to ADP or leucine stimulation was unaffected by these mutations, confirming that they interfere specifically with the inhibitory GTP-binding site. Conversely, the R463A mutation completely eliminated ADP activation of human GDH, but had little effect on either GTP inhibition or leucine activation. The effects of these three mutations on ATP regulation indicated that this nucleotide inhibits human GDH through binding of its triphosphate tail to the GTP site and, at higher concentrations, activates the enzyme through binding of the nucleotide to the ADP site. These data confirm the assignment of the GTP and ADP allosteric regulatory sites on GDH based on X-ray crystallography and provide

  4. Emerging Computational Methods for the Rational Discovery of Allosteric Drugs

    PubMed Central

    2016-01-01

    Allosteric drug development holds promise for delivering medicines that are more selective and less toxic than those that target orthosteric sites. To date, the discovery of allosteric binding sites and lead compounds has been mostly serendipitous, achieved through high-throughput screening. Over the past decade, structural data has become more readily available for larger protein systems and more membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets. In parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms. As a result of these advances, the field of predictive allosteric drug development is now on the cusp of a new era of rational structure-based computational methods. Here, we review algorithms that predict allosteric sites based on sequence data and molecular dynamics simulations, describe tools that assess the druggability of these pockets, and discuss how Markov state models and topology analyses provide insight into the relationship between protein dynamics and allosteric drug binding. In each section, we first provide an overview of the various method classes before describing relevant algorithms and software packages. PMID:27074285

  5. TEIF associated centrosome activity is regulated by EGF/PI3K/Akt signaling.

    PubMed

    Zhao, Jing; Zou, Yongxin; Liu, Haijing; Wang, Huali; Zhang, Hong; Hou, Wei; Li, Xin; Jia, Xinying; Zhang, Jing; Hou, Lin; Zhang, Bo

    2014-09-01

    Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis. PMID:24769208

  6. Calculated pKa Variations Expose Dynamic Allosteric Communication Networks.

    PubMed

    Lang, Eric J M; Heyes, Logan C; Jameson, Geoffrey B; Parker, Emily J

    2016-02-17

    Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically driven regulation mechanisms for which no major conformational changes are observed. To identify these dynamic allosteric communication networks, we have developed an approach that monitors the pKa variations of ionizable residues over the course of molecular dynamics simulations performed in the presence and absence of an allosteric regulator. As the pKa of ionizable residues depends on their environment, it represents a simple metric to monitor changes in several complex factors induced by binding an allosteric effector. These factors include Coulombic interactions, hydrogen bonding, and solvation, as well as backbone motions and side chain fluctuations. The predictions that can be made with this method concerning the roles of ionizable residues for allosteric communication can then be easily tested experimentally by changing the working pH of the protein or performing single point mutations. To demonstrate the method's validity, we have applied this approach to the subtle dynamic regulation mechanism observed for Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, the first enzyme of aromatic biosynthesis. We were able to identify key communication pathways linking the allosteric binding site to the active site of the enzyme and to validate these findings experimentally by reestablishing the catalytic activity of allosterically inhibited enzyme via modulation of the working pH, without compromising the binding affinity of the allosteric regulator.

  7. Evidence of Allosteric Enzyme Regulation via Changes in Conformational Dynamics: A Hydrogen/Deuterium Exchange Investigation of Dihydrodipicolinate Synthase.

    PubMed

    Sowole, Modupeola A; Simpson, Sarah; Skovpen, Yulia V; Palmer, David R J; Konermann, Lars

    2016-09-27

    Dihydrodipicolinate synthase is a tetrameric enzyme of the diaminopimelate pathway in bacteria and plants. The protein catalyzes the condensation of pyruvate (Pyr) and aspartate semialdehyde en route to the end product lysine (Lys). Dihydrodipicolinate synthase from Campylobacter jejuni (CjDHDPS) is allosterically inhibited by Lys. CjDHDPS is a promising antibiotic target, as highlighted by the recent development of a potent bis-lysine (bisLys) inhibitor. The mechanism whereby Lys and bisLys allosterically inhibit CjDHDPS remains poorly understood. In contrast to the case for other allosteric enzymes, crystallographically detectable conformational changes in CjDHDPS upon inhibitor binding are very minor. Also, it is difficult to envision how Pyr can access the active site; the available X-ray data seemingly imply that each turnover step requires diffusion-based mass transfer through a narrow access channel. This study employs hydrogen/deuterium exchange mass spectrometry for probing the structure and dynamics of CjDHDPS in a native solution environment. The deuteration kinetics reveal that the most dynamic protein regions are in the direct vicinity of the substrate access channel. This finding is consistent with the view that transient opening/closing fluctuations facilitate access of the substrate to the active site. Under saturating conditions, both Lys and bisLys cause dramatically reduced dynamics in the inhibitor binding region. In addition, rigidification extends to regions close to the substrate access channel. This finding strongly suggests that allosteric inhibitors interfere with conformational fluctuations that are required for CjDHDPS substrate turnover. In particular, our data imply that Lys and bisLys suppress opening/closing events of the access channel, thereby impeding diffusion of the substrate into the active site. Overall, this work illustrates why allosteric control does not have to be associated with crystallographically detectable large

  8. Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation

    PubMed Central

    Salazar, M; Lorente, M; García-Taboada, E; Pérez Gómez, E; Dávila, D; Zúñiga-García, P; María Flores, J; Rodríguez, A; Hegedus, Z; Mosén-Ansorena, D; Aransay, A M; Hernández-Tiedra, S; López-Valero, I; Quintanilla, M; Sánchez, C; Iovanna, J L; Dusetti, N; Guzmán, M; Francis, S E; Carracedo, A; Kiss-Toth, E; Velasco, G

    2015-01-01

    Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis. PMID:25168244

  9. An allosteric model for the functional plasticity of olfactory chemoreceptors

    NASA Astrophysics Data System (ADS)

    Colosimo, Alfredo

    2000-12-01

    A simple allosteric model may describe the relatively (a)specific behaviour of olfactory chemoreceptors (OCs) and their functional plasticity with a minimum number of parameters. Allosteric, heterotropic effectors are suggested as a possible cause of variable responses documented, in particular, in frog OCs. As an immediate spinoff of the continuously increasing amount of structural information available on natural OCs, development of appropriate allosteric models is foreseen to provide plausible molecular mechanisms for their complex functional performance. This may also have implications in the design of artificial olfaction systems.

  10. Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

    PubMed

    Ostrakhovitch, Elena A; Lordnejad, Mohammad Reza; Schliess, Freimut; Sies, Helmut; Klotz, Lars-Oliver

    2002-01-15

    Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a

  11. Inhibition of Rb Phosphorylation Leads to mTORC2-Mediated Activation of Akt.

    PubMed

    Zhang, Jinfang; Xu, Kai; Liu, Pengda; Geng, Yan; Wang, Bin; Gan, Wenjian; Guo, Jianping; Wu, Fei; Chin, Y Rebecca; Berrios, Christian; Lien, Evan C; Toker, Alex; DeCaprio, James A; Sicinski, Piotr; Wei, Wenyi

    2016-06-16

    The retinoblastoma (Rb) protein exerts its tumor suppressor function primarily by inhibiting the E2F family of transcription factors that govern cell-cycle progression. However, it remains largely elusive whether the hyper-phosphorylated, non-E2F1-interacting form of Rb has any physiological role. Here we report that hyper-phosphorylated Rb directly binds to and suppresses the function of mTORC2 but not mTORC1. Mechanistically, Rb, but not p107 or p130, interacts with Sin1 and blocks the access of Akt to mTORC2, leading to attenuated Akt activation and increased sensitivity to chemotherapeutic drugs. As such, inhibition of Rb phosphorylation by depleting cyclin D or using CDK4/6 inhibitors releases Rb-mediated mTORC2 suppression. This, in turn, leads to elevated Akt activation to confer resistance to chemotherapeutic drugs in Rb-proficient cells, which can be attenuated with Akt inhibitors. Therefore, our work provides a molecular basis for the synergistic usage of CDK4/6 and Akt inhibitors in treating Rb-proficient cancer. PMID:27237051

  12. Akt regulates Progesterone Receptor B dependent transcription and angiogenesis in endometrial cancer cells

    PubMed Central

    Lee, Irene I.; Maniar, Kruti; Lydon, John P.; Kim, J. Julie

    2016-01-01

    Progestins have long been used clinically for the treatment of endometrial cancers, however, the response rates to progestin therapy vary and the molecular mechanisms behind progestin insensitivity are poorly understood. We hypothesized that in PTEN mutated endometrial cancers, hyperactive Akt signaling downregulates Progesterone Receptor B (PRB) transcriptional activity, leading to overall impaired progestin responses. We report that inhibition of Akt with the Akt inhibitor, MK-2206 (MK), in conjunction with progestin (R5020) treatment, is sufficient to upregulate a subset of PRB target genes in Ishikawa cells stably expressing PRB (PRB-Ishikawa). Through gene ontology analysis of Akt-regulated PRB target genes, angiogenesis was found to be the principle process regulated by Akt-PRB. To further interrogate the mechanism by which Akt modulates PRB transcriptional activity, ChIP-Mass Spectrometry was performed to identify potential cofactors that differentially interact with PRB in the presence of the R5020 and MK+R5020. 14-3-3σ was identified as a protein enriched in the MK+R5020 dataset, and it was demonstrated that 14-3-3σ is required for the upregulation in PRB target gene expression following inhibition of Akt. In order to determine the ramifications of MK+R5020 treatment on angiogenesis, in vitro assays were performed and combinatorial MK+R5020 treatment significantly decreased endothelial cell invasion and tube formation more than MK or R5020 treatment alone. Furthermore, we found that combinatorial MK-2206+Progesterone treatments decreased angiogenesis and proliferation in the Ptend/d conditional mouse model of endometrial cancer. Taken together, these findings suggest that a combinatorial therapeutic approach utilizing Akt inhibitors with progestins may improve the efficacy of progestin therapy for the treatment of endometrial cancer. PMID:26996671

  13. Pharmacological Modulation of NMDA Receptor Activity and the Advent of Negative and Positive Allosteric Modulators

    PubMed Central

    Monaghan, Daniel T.; Irvine, Mark W.; Costa, Blaise Mathias; Fang, Guangyu; Jane, David E.

    2012-01-01

    The NMDA receptor (NMDAR) family of L-glutamate receptors are well known to have diverse roles in CNS function as well as in various neuropathological and psychiatric conditions. Until recently, the types of agents available to pharmacologically regulate NMDAR function have been quite limited in terms of mechanism of action and subtype selectivity. This has changed significantly in the past two years. The purpose of this review is to summarize the many drug classes now available for modulating NMDAR activity. Previously, this included competitive antagonists at the L-glutamate and glycine binding sites, high and low affinity channel blockers, and GluN2B-selective N-terminal domain binding site antagonists. More recently, we and others have identifed new classes of NMDAR agents that are either positive or negative allosteric modulators (PAMs and NAMs, respectively). These compounds include the pan potentiator UBP646, the GluN2A-selective potentiator/GluN2C & GluN2D inhibitor UBP512, the GluN2D-selective potentiator UBP551, the GluN2C/GluN2D-selective potentiator CIQ as well as the new NMDAR-NAMs such as the pan-inhibitor UBP618, the GluN2C/GluN2D-selective inhibitor QZN46 and the GluN2A inhibitors UBP608 and TCN201. These new agents do not bind within the L-glutamate or glycine binding sites, the ion channel pore or the N-terminal regulatory domain. Collectively, these new allosteric modulators appear to be acting at multiple novel sites on the NMDAR complex. Importantly, these agents display improved subtype-selectivity and as NMDAR PAMs and NAMs, they represent a new generation of potential NMDAR therapeutics. PMID:22269804

  14. PI3K/Akt/mTOR signaling pathway in cancer stem cells: from basic research to clinical application

    PubMed Central

    Xia, Pu; Xu, Xiao-Yan

    2015-01-01

    Cancer stem cells (CSCs) are a subpopulation of tumor cells that possess unique self-renewal activity and mediate tumor initiation and propagation. The PI3K/Akt/mTOR signaling pathway can be considered as a master regulator for cancer. More and more recent studies have shown the links between PI3K/Akt/mTOR signaling pathway and CSC biology. Herein, we provide a comprehensive review on the role of signaling components upstream and downstream of PI3K/Akt/mTOR signaling in CSC. In addition, we also summarize various classes of small molecule inhibitors of PI3K/Akt/mTOR signaling pathway and their clinical potential in CSC. Overall, the current available data suggest that the PI3K/Akt/mTOR signaling pathway could be a promising target for development of CSC-target drugs. PMID:26175931

  15. Bimatoprost protects retinal neuronal damage via Akt pathway.

    PubMed

    Takano, Norihito; Tsuruma, Kazuhiro; Ohno, Yuta; Shimazawa, Masamitsu; Hara, Hideaki

    2013-02-28

    Worldwide, prostaglandin analogs, such as bimatoprost, have become the major therapeutic class for medical treatment of glaucoma because of their efficacy and generally well tolerated systemic safety profile. However, the detailed mechanism of the direct action of bimatoprost on retinal ganglion cells (RGC) has rarely been understood. Thus, in this study, we elucidated the mechanism of the protective effects of bimatoprost on RGC against oxidative stress. To examine the protective effects of bimatoprost, cultured RGC with various concentrations of bimatoprost (in both free acid and amide form) were exposed to l-buthionin-(S,R)-sulfoximine (BSO) plus glutamate or serum depletion in vitro and intravitreal injection of N-methyl-D-aspartate (NMDA) was used to induce retinal damage in vivo. To elucidate the protective mechanism of bimatoprost, we used western blot analysis to investigate the phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Bimatoprost significantly reduced BSO plus glutamate- and serum deprivation-induced death in concentration-dependent manners. Bimatoprost induced activation of Akt and ERK, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the protective effect of bimatoprost. On the other hand, a mitogen-activated protein kinase kinase inhibitor, U0126, exhibited protective effect unexpectedly. Moreover, ERK was more phosphorylated by attenuation of Akt activity in cultured RGC. In an in vivo study, bimatoprost reduced NMDA-induced RGC death. Taken together, these findings indicate that bimatoprost has protective effects on in vitro and in vivo retinal damage, suggesting that the mechanism underlying may be via the Akt pathway, which may modulate the ERK pathway.

  16. Untangling the glutamate dehydrogenase allosteric nightmare.

    PubMed

    Smith, Thomas J; Stanley, Charles A

    2008-11-01

    Glutamate dehydrogenase (GDH) is found in all living organisms, but only animal GDH is regulated by a large repertoire of metabolites. More than 50 years of research to better understand the mechanism and role of this allosteric network has been frustrated by its sheer complexity. However, recent studies have begun to tease out how and why this complex behavior evolved. Much of GDH regulation probably occurs by controlling a complex ballet of motion necessary for catalytic turnover and has evolved concomitantly with a long antenna-like feature of the structure of the enzyme. Ciliates, the 'missing link' in GDH evolution, might have created the antenna to accommodate changing organelle functions and was refined in humans to, at least in part, link amino acid catabolism with insulin secretion.

  17. Allosteric sodium in class A GPCR signaling

    PubMed Central

    Katritch, Vsevolod; Fenalti, Gustavo; Abola, Enrique E.; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2014-01-01

    Despite their functional and structural diversity, G protein-coupled receptors (GPCRs) share a common mechanism of signal transduction via conformational changes in the seven-transmembrane (7TM) helical domain. New major insights into this mechanism come from the recent crystallographic discoveries of a partially hydrated sodium ion that is specifically bound in the middle of the 7TM bundle of multiple class A GPCRs. This review discusses the remarkable structural conservation and distinct features of the Na+ pocket in this most populous GPCR class, as well as the conformational collapse of the pocket on receptor activation. New insights help to explain allosteric effects of sodium on GPCR agonist binding and activation, and sodium’s role as a potential co-factor in class A GPCR function. PMID:24767681

  18. Akt1-mediated fast/glycolytic skeletal muscle growth attenuates renal damage in experimental kidney disease.

    PubMed

    Hanatani, Shinsuke; Izumiya, Yasuhiro; Araki, Satoshi; Rokutanda, Taku; Kimura, Yuichi; Walsh, Kenneth; Ogawa, Hisao

    2014-12-01

    Muscle wasting is frequently observed in patients with kidney disease, and low muscle strength is associated with poor outcomes in these patients. However, little is known about the effects of skeletal muscle growth per se on kidney diseases. In this study, we utilized a skeletal muscle-specific, inducible Akt1 transgenic (Akt1 TG) mouse model that promotes the growth of functional skeletal muscle independent of exercise to investigate the effects of muscle growth on kidney diseases. Seven days after Akt1 activation in skeletal muscle, renal injury was induced by unilateral ureteral obstruction (UUO) in Akt1 TG and wild-type (WT) control mice. The expression of atrogin-1, an atrophy-inducing gene in skeletal muscle, was upregulated 7 days after UUO in WT mice but not in Akt1 TG mice. UUO-induced renal interstitial fibrosis, tubular injury, apoptosis, and increased expression of inflammatory, fibrosis-related, and adhesion molecule genes were significantly diminished in Akt1 TG mice compared with WT mice. An increase in the activating phosphorylation of eNOS in the kidney accompanied the attenuation of renal damage by myogenic Akt1 activation. Treatment with the NOS inhibitor L-NAME abolished the protective effect of skeletal muscle Akt activation on obstructive kidney disease. In conclusion, Akt1-mediated muscle growth reduces renal damage in a model of obstructive kidney disease. This improvement appears to be mediated by an increase in eNOS signaling in the kidney. Our data support the concept that loss of muscle mass during kidney disease can contribute to renal failure, and maintaining muscle mass may improve clinical outcome. PMID:25012168

  19. Leishmania promastigotes activate PI3K/Akt signalling to confer host cell resistance to apoptosis.

    PubMed

    Ruhland, Aaron; Leal, Nicole; Kima, Peter E

    2007-01-01

    Previous reports have shown that cells infected with promastigotes of some Leishmania species are resistant to the induction of apoptosis. This would suggest that either parasites elaborate factors that block signalling from apoptosis inducers or that parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter scenario, we determined whether Leishmania infection results in the activation of signalling pathways that have been shown to mediate resistance to apoptosis in other infection models. First, we showed that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania amazonensis activates signalling through p38 mitogen-activated protein kinase (MAPK), NFkappaB and PI3K/Akt. Then we found that inhibition of signalling through the PI3K/Akt pathway with LY294002 and Akt IV inhibitor reversed resistance of infected bone marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover, reduction of Akt levels with small interfering RNAs to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the role of PI3K/Akt signalling in the promotion of cell survival by infected cells was obtained with the finding that Bad, which is a substrate of Akt, becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt signalling, inhibition of p38 MAPK signalling with SB202190 or NFkappaB signalling with wedelolactone had limited effect on parasite-induced resistance to apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to the infected cell, the capacity to resist death from activators of apoptosis.

  20. AKT (protein kinase B) is implicated in meiotic maturation of porcine oocytes.

    PubMed

    Kalous, Jaroslav; Kubelka, Michal; Solc, Petr; Susor, Andrej; Motlík, Jan

    2009-10-01

    The aim of this study was to investigate the involvement of the serine/threonine protein kinase AKT (also called protein kinase B) in the control of meiosis of porcine denuded oocytes (DOs) matured in vitro. Western blot analysis revealed that the two principal AKT phosphorylation sites, Ser473 and Thr308, are phosphorylated at different stages of meiosis. In freshly isolated germinal vesicle (GV)-stage DOs, Ser473 was already phosphorylated. After the onset of oocyte maturation, the intensity of the Ser473 phosphorylation increased, however, which declined sharply when DOs underwent GV breakdown (GVBD) and remained at low levels in metaphase I- and II-stage (MI- and MII-stage). In contrast, phosphorylation of Thr308 was increased by the time of GVBD and reached maximum at MI-stage. A peak of AKT activity was noticed around GVBD and activity of AKT declined at MI-stage. To assess the role of AKT during meiosis, porcine DOs were cultured in 50 microM SH-6, a specific inhibitor of AKT. In SH-6-treated DOs, GVBD was not inhibited; on the contrary, a significant acceleration of meiosis resumption was observed. The dynamics of the Ser473 phosphorylation was not affected; however, phosphorylation of Thr308 was reduced, AKT activity was diminished at the time of GVBD, and meiotic progression was arrested in early MI-stage. Moreover, the activity of the cyclin-dependent kinase 1 (CDK1) and MAP kinase declined when SH-6-treated DOs underwent GVBD, indicating that AKT activity is involved in the regulation of CDK1 and MAP kinase. These results suggest that activity of AKT is not essential for induction of GVBD in porcine oocytes but plays a substantial role during progression of meiosis to MI/MII-stage.

  1. pAKT Expression and Response to Sorafenib in Differentiated Thyroid Cancer.

    PubMed

    Yarchoan, Mark; Ma, Changqing; Troxel, Andrea B; Stopenski, Stephen J; Tang, Waixing; Cohen, Aaron B; Pappas-Paxinos, Marina; Johnson, Burles A; Chen, Emerson Y; Feldman, Michael D; Brose, Marcia S

    2016-06-01

    Sorafenib has an antitumor activity in patients with radioactive iodine-refractory differentiated thyroid carcinoma (RAIR-DTC). Prior research has implicated signaling through the MAPK and AKT/PI3K pathways in the progression of DTC. To assess whether the activity of these pathways is predictive of response to sorafenib, we retrospectively studied molecular tumor markers from these two pathways from a phase 2 study of sorafenib in RAIR-DTC. Tumor samples from 40 of 53 DTC subjects obtained prior to initiation of sorafenib were immunostained with DAB-labeled antibodies to phospho-AKT (pAKT), phospho-ERK (pERK), and phospho-S6 (pS6). BRAFV600E genetic mutation analysis was performed on all samples. Expression levels and mutational status were compared to response and progression-free survival (PFS) for each patient. Low tumor expression of nuclear pAKT was associated with partial response to sorafenib (p < 0.01). Patients with nuclear pAKT expression that was below the median for our sample were more than three times as likely to have a partial response as patients with equal to or above median expression. There was no correlation between tumor expression of nuclear pERK or pS6 and response. Endothelial cell and pericyte expression of pERK, pAKT, and pS6 were not predictive of response. There was no correlation between BRAFV600E mutation status and partial response. No correlation was observed between either the expression of pAKT, pERK, or pS6, or the presence of the BRAFV600E mutation, and PFS. In conclusion, lower tumor expression of nuclear pAKT was associated with higher rate of response to sorafenib. This observation justifies evaluation of combination therapy with sorafenib and an inhibitor of the PI3K/AKT signaling pathway in RAIR-DTC. PMID:26994002

  2. Akt1-mediated fast/glycolytic skeletal muscle growth attenuates renal damage in experimental kidney disease.

    PubMed

    Hanatani, Shinsuke; Izumiya, Yasuhiro; Araki, Satoshi; Rokutanda, Taku; Kimura, Yuichi; Walsh, Kenneth; Ogawa, Hisao

    2014-12-01

    Muscle wasting is frequently observed in patients with kidney disease, and low muscle strength is associated with poor outcomes in these patients. However, little is known about the effects of skeletal muscle growth per se on kidney diseases. In this study, we utilized a skeletal muscle-specific, inducible Akt1 transgenic (Akt1 TG) mouse model that promotes the growth of functional skeletal muscle independent of exercise to investigate the effects of muscle growth on kidney diseases. Seven days after Akt1 activation in skeletal muscle, renal injury was induced by unilateral ureteral obstruction (UUO) in Akt1 TG and wild-type (WT) control mice. The expression of atrogin-1, an atrophy-inducing gene in skeletal muscle, was upregulated 7 days after UUO in WT mice but not in Akt1 TG mice. UUO-induced renal interstitial fibrosis, tubular injury, apoptosis, and increased expression of inflammatory, fibrosis-related, and adhesion molecule genes were significantly diminished in Akt1 TG mice compared with WT mice. An increase in the activating phosphorylation of eNOS in the kidney accompanied the attenuation of renal damage by myogenic Akt1 activation. Treatment with the NOS inhibitor L-NAME abolished the protective effect of skeletal muscle Akt activation on obstructive kidney disease. In conclusion, Akt1-mediated muscle growth reduces renal damage in a model of obstructive kidney disease. This improvement appears to be mediated by an increase in eNOS signaling in the kidney. Our data support the concept that loss of muscle mass during kidney disease can contribute to renal failure, and maintaining muscle mass may improve clinical outcome.

  3. PTEN and PI3K/AKT in non-small-cell lung cancer.

    PubMed

    Pérez-Ramírez, Cristina; Cañadas-Garre, Marisa; Molina, Miguel Ángel; Faus-Dáder, María José; Calleja-Hernández, Miguel Ángel

    2015-11-01

    Non-small-cell lung cancer (NSCLC) is the leading cause of cancer deaths worldwide. In the last years, the identification of activating EGFR mutations, conferring increased sensitivity and disease response to tyrosine kinase inhibitors, has changed the prospect of NSCLC patients. The PTEN/PI3K/AKT pathway regulates multiple cellular functions, including cell growth, differentiation, proliferation, survival, motility, invasion and intracellular trafficking. Alterations in this pathway, mainly PTEN inactivation, have been associated with resistance to EGFR-tyrosine kinase inhibitor therapy and lower survival in NSCLC patients. In this review, we will briefly discuss the main PTEN/PI3K/AKT pathway alterations found in NSCLC, as well as the cell processes regulated by PTEN/PI3K/AKT leading to tumorigenesis.

  4. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  5. Ligand Binding to Macromolecules: Allosteric and Sequential Models of Cooperativity.

    ERIC Educational Resources Information Center

    Hess, V. L.; Szabo, Attila

    1979-01-01

    A simple model is described for the binding of ligands to macromolecules. The model is applied to the cooperative binding by hemoglobin and aspartate transcarbamylase. The sequential and allosteric models of cooperative binding are considered. (BB)

  6. The Akt switch model: Is location sufficient?

    PubMed

    Gray, Catheryn W; Coster, Adelle C F

    2016-06-01

    Akt/PKB is a biochemical regulator that functions as an important cross-talk node between several signalling pathways in the mammalian cell. In particular, Akt is a key mediator of glucose transport in response to insulin. The phosphorylation (activation) of only a small percentage of the Akt pool of insulin-sensitive cells results in maximal translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This enables the diffusion of glucose into the cell. The dysregulation of Akt signalling is associated with the development of diabetes, cancer and cardiovascular disease. Akt is synthesised in the cytoplasm in the inactive state. Under the influence of insulin, it moves to the PM, where it is phosphorylated to form pAkt. Although phosphorylation occurs only at the PM, pAkt is found in many cellular locations, including the PM, the cytoplasm, and the nucleus. Indeed, the spatial distribution of pAkt within the cell appears to be an important determinant of downstream regulation. Here we present a simple, linear, four-compartment ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and the physical location of Akt. This model embodies the main features of the activation of this important cross-talk node and is consistent with the experimental data. In particular, it allows different downstream signalling motifs without invoking separate feedback pathways. Moreover, the model is computationally tractable, readily analysed, and elucidates some of the apparent anomalies in insulin signalling via Akt. PMID:26992575

  7. MMP Inhibitors: Past, present and future.

    PubMed

    Cathcart, Jillian M; Cao, Jian

    2015-01-01

      Development of inhibitors of matrix metalloproteinases (MMPs) has been fraught with challenges. Early compounds largely failed due to poor selectivity and bioavailability. Dose-limiting side effects, off-target interactions, and improperly designed clinical trials significantly impeded clinical success. As information becomes available and technology evolves, tools to combat these obstacles have been developed. Improved methods for high throughput screening and drug design have led to identification of compounds exhibiting high potency, binding affinity, and favorable pharmacokinetic profiles. Current research into MMP inhibitors employs innovative approaches for drug delivery methods and allosteric inhibitors. Such innovation is key for development of clinically successful compounds.

  8. Upregulation of p‑Akt by glial cell line‑derived neurotrophic factor ameliorates cell apoptosis in the hippocampus of rats with streptozotocin‑induced diabetic encephalopathy.

    PubMed

    Cui, Weigang; Zhang, Yinghua; Lu, Derong; Ren, Mingxin; Yuan, Guoyan

    2016-01-01

    The loss of neurotrophic factor support has been shown to contribute to the development of the central nervous system. Glial cell line‑derived neurotrophic factor (GDNF), a potent neurotrophic factor, is closely associated with apoptosis and exerts neuroprotective effects on numerous populations of cells. However, the underlying mechanisms of these protective effects remain unknown. In the present study, a significant increase in Bax levels and DNA fragmentation was observed in the hippocampus obtained from the brains of diabetic rats 60 days after diabetes had been induced. The apoptotic changes were correlated with the loss of GDNF/Akt signaling. GDNF administration was found to reverse the diabetes‑induced Bax and DNA fragmentation changes. This was associated with an improvement in the level of p‑Akt/Akt. In addition, combination of GDNF with a specific inhibitor of the phosphoinositide 3‑kinase (PI3K)/Akt pathway, Wortmannin, significantly abrogated the effects of GDNF on the levels of p‑Akt/Akt, Bax and DNA fragmentation. However, a p38 mitogen‑activated proten kinase (MAPK) inhibitor, SB203580, had no effect on the expression of p‑Akt/Akt, Bax or DNA fragmentation. These results demonstrate the pivotal role of GDNF as well as the PI3K/Akt pathway, but not the MAPK pathway, in the prevention of diabetes‑induced neuronal apoptosis in the hippocampus. PMID:26549420

  9. Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site.

    PubMed

    Adams, Julian; Chen, Zhi-Ping; Van Denderen, Bryce J W; Morton, Craig J; Parker, Michael W; Witters, Lee A; Stapleton, David; Kemp, Bruce E

    2004-01-01

    AMP-activated protein kinase (AMPK) is a alphabetagamma heterotrimer that is activated in response to both hormones and intracellular metabolic stress signals. AMPK is regulated by phosphorylation on the alpha subunit and by AMP allosteric control previously thought to be mediated by both alpha and gamma subunits. Here we present evidence that adjacent gamma subunit pairs of CBS repeat sequences (after Cystathionine Beta Synthase) form an AMP binding site related to, but distinct from the classical AMP binding site in phosphorylase, that can also bind ATP. The AMP binding site of the gamma(1) CBS1/CBS2 pair, modeled on the structures of the CBS sequences present in the inosine monophosphate dehydrogenase crystal structure, contains three arginine residues 70, 152, and 171 and His151. The yeast gamma homolog, snf4 contains a His151Gly substitution, and when this is introduced into gamma(1), AMP allosteric control is substantially lost and explains why the yeast snf1p/snf4p complex is insensitive to AMP. Arg70 in gamma(1) corresponds to the site of mutation in human gamma(2) and pig gamma(3) genes previously identified to cause an unusual cardiac phenotype and glycogen storage disease, respectively. Mutation of any of AMP binding site Arg residues to Gln substantially abolishes AMP allosteric control in expressed AMPK holoenzyme. The Arg/Gln mutations also suppress the previously described inhibitory properties of ATP and render the enzyme constitutively active. We propose that ATP acts as an intrasteric inhibitor by bridging the alpha and gamma subunits and that AMP functions to derepress AMPK activity.

  10. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  11. Differential regulation of mTOR signaling determines sensitivity to AKT inhibition in diffuse large B cell lymphoma.

    PubMed

    Ezell, Scott A; Wang, Suping; Bihani, Teeru; Lai, Zhongwu; Grosskurth, Shaun E; Tepsuporn, Suprawee; Davies, Barry R; Huszar, Dennis; Byth, Kate F

    2016-02-23

    Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL. PMID:26824321

  12. Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

    SciTech Connect

    Varma, Shailly; Shrivastav, Anuraag; Changela, Sheena; Khandelwal, Ramji L.

    2008-04-01

    Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3{beta} (GSK-3{beta}) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-{alpha} (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20-30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20-50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40-60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

  13. Tetrandrine suppresses metastatic phenotype of prostate cancer cells by regulating Akt/mTOR/MMP-9 signaling pathway.

    PubMed

    Kou, Bo; Liu, Wei; He, Wenbo; Zhang, Yuanyuan; Zheng, Jianjie; Yan, Yang; Zhang, Yongjian; Xu, Suochun; Wang, Haichen

    2016-05-01

    Tetrandrine (TET), a bisbenzylisoquinoline alkaloid found in traditional Chinese medicines, exerts anticancer activity in vitro and in vivo. However, its potential role in the prostate cancer metastatic process has not yet been elucidated. Thus, we investigated the inhibition effect of tetrandrine on prostate cancer migration and invasion and the corresponding molecular basis underlying its anticancer activity. Cell migration and invasion were determined using the Transwell chamber model. The protein expression of Akt, phosphorylated Akt, the mammalian target of rapamycin (mTOR), phosphorylated mTOR and matrix metalloproteinases 9 (MMP-9) was detected by western blot in the presence or absence of tetrandrine or in the group tetrandrine combination with LY294002 (inhibitor of Akt) and rapamycin (inhibitor of mTOR). Our studies showed that excluding the effect of tetrandrine on cell proliferation, tetrandrine significantly inhibited cell migration and invasion in prostate cancer DU145 and PC3 cells. Furthermore, tetrandrine decreased the protein levels of p-Akt, p-mTOR, and MMP-9. While the inhibition of Akt or mTOR by the respective inhibitors could potentiate this effect of tetrandrine on prostate cancer cells, the studies indicate that tetrandrine inhibits the metastasis process by negatively regulating the Akt/mTOR/MMP-9 signaling pathway. These results suggest that tetrandrine might serve as a potential metastasis suppressor to treat cancer cells that have escaped surgical removal or that have disseminated widely. PMID:26935264

  14. Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants.

    PubMed

    Al-Horani, Rami A; Gailani, David; Desai, Umesh R

    2015-08-01

    Recent development of sulfated non-saccharide glycosaminoglycan mimetics, especially sulfated pentagalloyl glucopyranoside (SPGG), as potent inhibitors of factor XIa (FXIa) (J. Med. Chem. 2013; 56:867-878 and J. Med. Chem. 2014; 57:4805-4818) has led to a strong possibility of developing a new line of factor XIa-based anticoagulants. In fact, SPGG represents the first synthetic, small molecule inhibitor that appears to bind in site remote from the active site. Considering that allosteric inhibition of FXIa is a new mechanism for developing a distinct line of anticoagulants, we have studied SPGG's interaction with FXIa with a goal of evaluating its pre-clinical relevance. Comparative inhibition studies with several glycosaminoglycans revealed the importance of SPGG's non-saccharide backbone. SPGG did not affect the activity of plasma kallikrein, activated protein C and factor XIIIa suggesting that SPGG-based anticoagulation is unlikely to affect other pathways connected with coagulation factors. SPGG's effect on APTT of citrated human plasma was also not dependent on antithrombin or heparin cofactor II. Interestingly, SPGG's anticoagulant potential was diminished by serum albumin as well as factor XI, while it could be reversed by protamine or polybrene, which implies possible avenues for developing antidote strategy. Studies with FXIa mutants indicated that SPGG engages Lys529, Arg530 and Arg532, but not Arg250, Lys252, Lys253 and Lys255. Finally, SPGG competes with unfractionated heparin, but not with polyphosphates and/or glycoprotein Ibα, for binding to FXIa. These studies enhance understanding on the first allosteric inhibitor of FXIa and highlight its value as a promising anticoagulant. PMID:25935648

  15. Na+/Ca2+ exchanger 1 (NCX-1) mediates the anti-apoptotic effect of Akt1 in neonatal rat cardiomyocytes during ischemia/reperfusion

    PubMed Central

    Huang, Manman; Pan, Defeng; Du, Yinping; Zhu, Hong; Zhang, Lin; Xu, Tongda; Luo, Yuanyuan; Li, Dongye

    2016-01-01

    The purpose of this study was to investigate the anti-apoptotic role of Akt1 gene in neonatal rat cardiomyocytes and the relationship with Na+/Ca2+ exchanger 1 (NCX1) during ischemia/reperfusion (IR). The cultured original rat cardiomyocytes were randomly divided into five groups: normal control group (C group), hypoxia/reoxygenation group (HR group), the control vector pLVX-EGFP-3FLAG group (CV group), the gene pLVX-EGFP-3FLAG-Akt1 transfection group (A group), and Akt1 inhibitor LY294002 group (LY group). Cardiomyocyte vitality was determined using MTT, and the apoptosis was determined by TUNEL to verify the anti-apoptotic role of Akt1. The mRNA levels of Akt1 and NCX1 were determined by RT-PCR, the protein expression of Akt1, p-Akt1, NCX1 and the apoptotic proteins of mitochondrial pathway cytochrome C (Cyto C) and caspase-9 were measured by Western blot. As a result, transfected Akt1 (A group) showed increased myocardial cell viability and reduced apoptosis, with increase in Akt1 expression and decrease in NCX1 expression. The levels of apoptotic proteins Cyto C and caspase-9 also declined. This study demonstrated that lentivirus-mediated transfection of Akt1 played an anti-apoptotic role during IR of rat cardiomyocytes, via inhibition of NCX1 and other mitochondrial proteins. PMID:27186265

  16. Cross regulation between cGMP-dependent protein kinase and Akt in vasodilatation of porcine pulmonary artery.

    PubMed

    Liu, Juan; Liu, Huixia; Li, Yanjing; Xu, Xiaojian; Chen, Zhengju; Liu, Limei; Yu, Xiaoxing; Gao, Yuansheng; Dou, Dou

    2014-11-01

    cGMP-dependent protein kinase (PKG) plays a crucial role in vasodilatation induced by cGMP-elevating agents. Akt has been demonstrated to be involved in modulating vasoreactivity. The present study was to determine the interaction between PKG and Akt and their influences on nitric oxide (NO)-induced vasodilatation. Isolated fourth-generation porcine pulmonary arteries were dissected from the lung and cut into rings in ice-cold modified Krebs-Ringer bicarbonate buffer. The relaxant responses of vessels were determined by organ chamber technique, cGMP was assayed by using enzyme-linked immunosorbent assay kit, the protein levels of phosphorylated Akt were examined by Western blotting, and the activity of phosphodiesterase type 5 (PDE5) was assayed by measuring the rate of cGMP degradation. Incubation with DETA NONOate (a stable NO donor) and 8-Br-cGMP (a cell membrane permeable analog of cGMP) attenuated Akt phosphorylation at Ser-473, which was prevented by Rp-8-Br-PET-cGMPS (a specific inhibitor of PKG) and calyculin A (an inhibitor of protein phosphatase 1 and 2A) but not by okadaic acid (a selective inhibitor of protein phosphatase 2A). Inhibition of Akt enhanced the relaxation and cGMP elevation of porcine pulmonary arteries induced by DETA NONOate or sodium nitroprusside, which was prevented by zaprinast, a specific inhibitor of PDE5. Incubation with LY294002 or Akt inhibitor reduced PDE5 activity in porcine pulmonary arteries. The present study indicates that PKG may attenuate Akt phosphorylation through protein phosphatase 1, which leads to an augmented cGMP elevation by inhibition of PDE5. The increased cGMP in turn activates PKG. Such a positive feedback may play an important role in NO-induced pulmonary vasodilatation.

  17. Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Sorescu, George; Boyd, Nolan; Shiojima, Ichiro; Walsh, Kenneth; Du, Jie; Jo, Hanjoong

    2002-01-01

    Recently, we have shown that shear stress stimulates NO(*) production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91, 1574-1581). Akt has been believed to regulate shear-dependent production of NO(*) by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S(1179)), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S(1179). Here, we show that shear-dependent phosphorylation of eNOS-S(1179) is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S(1179) if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO(*) production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S(1179) and NO(*) production. In contrast, H89 did not inhibit phosphorylation of eNOS-S(1179) induced by expressing a constitutively active Akt mutant (Akt(Myr)) in BAEC, showing that the inhibitor did not affect the Akt pathway. 8-Bromo-cAMP alone phosphorylated eNOS-S(1179) within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S(1179) in a PKA-dependent, but Aktindependent manner, whereas the NO(*) production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction

  18. Dual Targeting of Akt and mTORC1 Impairs Repair of DNA Double-Strand Breaks and Increases Radiation Sensitivity of Human Tumor Cells

    PubMed Central

    Holler, Marina; Grottke, Astrid; Mueck, Katharina; Manes, Julia; Jücker, Manfred

    2016-01-01

    Inhibition of mammalian target of rapamycin-complex 1 (mTORC1) induces activation of Akt. Because Akt activity mediates the repair of ionizing radiation-induced DNA double-strand breaks (DNA-DSBs) and consequently the radioresistance of solid tumors, we investigated whether dual targeting of mTORC1 and Akt impairs DNA-DSB repair and induces radiosensitization. Combining mTORC1 inhibitor rapamycin with ionizing radiation in human non-small cell lung cancer (NSCLC) cells (H661, H460, SK-MES-1, HTB-182, A549) and in the breast cancer cell line MDA-MB-231 resulted in radiosensitization of H661 and H460 cells (responders), whereas only a very slight effect was observed in A549 cells, and no effect was observed in SK-MES-1, HTB-182 or MDA-MB-231 cells (non-responders). In responder cells, rapamycin treatment did not activate Akt1 phosphorylation, whereas in non-responders, rapamycin mediated PI3K-dependent Akt activity. Molecular targeting of Akt by Akt inhibitor MK2206 or knockdown of Akt1 led to a rapamycin-induced radiosensitization of non-responder cells. Compared to the single targeting of Akt, the dual targeting of mTORC1 and Akt1 markedly enhanced the frequency of residual DNA-DSBs by inhibiting the non-homologous end joining repair pathway and increased radiation sensitivity. Together, lack of radiosensitization induced by rapamycin was associated with rapamycin-mediated Akt1 activation. Thus, dual targeting of mTORC1 and Akt1 inhibits repair of DNA-DSB leading to radiosensitization of solid tumor cells. PMID:27137757

  19. Akt mediated phosphorylation of LARP6; critical step in biosynthesis of type I collagen

    PubMed Central

    Zhang, Yujie; Stefanovic, Branko

    2016-01-01

    La ribonucleoprotein domain family, member 6 (LARP6) is the RNA binding protein, which regulates translation of collagen mRNAs and synthesis of type I collagen. Posttranslational modifications of LARP6 and how they affect type I collagen synthesis have not been studied. We show that in lung fibroblasts LARP6 is phosphorylated at 8 serines, 6 of which are located within C-terminal domain. Phosphorylation of LARP6 follows a hierarchical order; S451 phosphorylation being a prerequisite for phosphorylations of other serines. Inhibition of PI3K/Akt pathway reduced the phosphorylation of LARP6, but had no effect on the S451A mutant, suggesting that PI3K/Akt pathway targets S451 and we have identified Akt as the responsible kinase. Overexpression of S451A mutant had dominant negative effect on collagen biosynthesis; drastically reduced secretion of collagen and induced hyper-modifications of collagen α2 (I) polypeptides. This indicates that LARP6 phosphorylation at S451 is critical for regulating translation and folding of collagen polypeptides. Akt inhibitor, GSK-2141795, which is in clinical trials for treatment of solid tumors, reduced collagen production by human lung fibroblasts with EC50 of 150 nM. This effect can be explained by inhibition of LARP6 phosphorylation and suggests that Akt inhibitors may be effective in treatment of various forms of fibrosis. PMID:26932461

  20. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress.

    PubMed

    Wang, Kaijun; Jiang, Yiqian; Wang, Wei; Ma, Jian; Chen, Min

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H2O2) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H2O2-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H2O2 were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H2O2. Reversely, escin was more potent against H2O2 damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H2O2 was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling. PMID:26505797

  1. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    PubMed Central

    Jacoby, D B; Gleich, G J; Fryer, A D

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma. Images PMID:8473484

  2. The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus.

    PubMed

    Whitaker, Amy M; Reinhart, Gregory D

    2016-10-01

    The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP) for phosphofructokinase-1 (PFK) from the moderate thermophile, Bacillus stearothermophilus (BsPFK), results from compensating enthalpy and entropy components. In BsPFK the positive coupling free energy that defines inhibition is opposite in sign from the negative enthalpy term and is therefore determined by the larger absolute value of the negative entropy term. Variants of BsPFK were made to determine the effect of adding small cavities to the structure on the allosteric function of the enzyme. The BsPFK Ile → Val (cavity containing) mutants have varied values for the coupling free energy between PEP and Fru-6-P, indicating that the modifications altered the effectiveness of PEP as an inhibitor. Notably, the mutation I153V had a substantial positive impact on the magnitude of inhibition by PEP. Van't Hoff analysis determined that this is the result of decreased entropy-enthalpy compensation with a larger change in the enthalpy term compared to the entropy term. PMID:27477958

  3. Propofol inhibits SIRT2 deacetylase through a conformation-specific, allosteric site.

    PubMed

    Weiser, Brian P; Eckenhoff, Roderic G

    2015-03-27

    meta-Azi-propofol (AziPm) is a photoactive analog of the general anesthetic propofol. We photolabeled a myelin-enriched fraction from rat brain with [(3)H]AziPm and identified the sirtuin deacetylase SIRT2 as a target of the anesthetic. AziPm photolabeled three SIRT2 residues (Tyr(139), Phe(190), and Met(206)) that are located in a single allosteric protein site, and propofol inhibited [(3)H]AziPm photolabeling of this site in myelin SIRT2. Structural modeling and in vitro experiments with recombinant human SIRT2 determined that propofol and [(3)H]AziPm only bind specifically and competitively to the enzyme when co-equilibrated with other substrates, which suggests that the anesthetic site is either created or stabilized in enzymatic conformations that are induced by substrate binding. In contrast to SIRT2, specific binding of [(3)H]AziPm or propofol to recombinant human SIRT1 was not observed. Residues that line the propofol binding site on SIRT2 contact the sirtuin co-substrate NAD(+) during enzymatic catalysis, and assays that measured SIRT2 deacetylation of acetylated α-tubulin revealed that propofol inhibits enzymatic function. We conclude that propofol inhibits the mammalian deacetylase SIRT2 through a conformation-specific, allosteric protein site that is unique from the previously described binding sites of other inhibitors. This suggests that propofol might influence cellular events that are regulated by protein acetylation state.

  4. Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists.

    PubMed

    Chigaev, Alexandre; Wu, Yang; Williams, D Bart; Smagley, Yelena; Sklar, Larry A

    2011-02-18

    Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ∼2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies. PMID:21131351

  5. Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis.

    PubMed

    Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H; Hickson, Gilles R; Archambault, Vincent

    2014-10-27

    Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks.

  6. Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug-Binding Site Promotes Islet Insulin Release.

    PubMed

    Scott, John W; Galic, Sandra; Graham, Kate L; Foitzik, Richard; Ling, Naomi X Y; Dite, Toby A; Issa, Samah M A; Langendorf, Chris G; Weng, Qing Ping; Thomas, Helen E; Kay, Thomas W; Birnberg, Neal C; Steinberg, Gregory R; Kemp, Bruce E; Oakhill, Jonathan S

    2015-06-18

    The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis. Pharmacological inhibition of AMPK is regarded as a therapeutic strategy in some disease settings including obesity and cancer; however, the broadly used direct AMPK inhibitor compound C suffers from poor selectivity. We have discovered a dihydroxyquinoline drug (MT47-100) with novel AMPK regulatory properties, being simultaneously a direct activator and inhibitor of AMPK complexes containing the β1 or β2 isoform, respectively. Allosteric inhibition by MT47-100 was dependent on the β2 carbohydrate-binding module (CBM) and determined by three non-conserved CBM residues (Ile81, Phe91, Ile92), but was independent of β2-Ser108 phosphorylation. Whereas MT47-100 regulation of total cellular AMPK activity was determined by β1/β2 expression ratio, MT47-100 augmented glucose-stimulated insulin secretion from isolated mouse pancreatic islets via a β2-dependent mechanism. Our findings highlight the therapeutic potential of isoform-specific AMPK allosteric inhibitors. PMID:26091167

  7. Inhibition of AMP-Activated Protein Kinase at the Allosteric Drug-Binding Site Promotes Islet Insulin Release.

    PubMed

    Scott, John W; Galic, Sandra; Graham, Kate L; Foitzik, Richard; Ling, Naomi X Y; Dite, Toby A; Issa, Samah M A; Langendorf, Chris G; Weng, Qing Ping; Thomas, Helen E; Kay, Thomas W; Birnberg, Neal C; Steinberg, Gregory R; Kemp, Bruce E; Oakhill, Jonathan S

    2015-06-18

    The AMP-activated protein kinase (AMPK) is a metabolic stress-sensing αβγ heterotrimer responsible for energy homeostasis. Pharmacological inhibition of AMPK is regarded as a therapeutic strategy in some disease settings including obesity and cancer; however, the broadly used direct AMPK inhibitor compound C suffers from poor selectivity. We have discovered a dihydroxyquinoline drug (MT47-100) with novel AMPK regulatory properties, being simultaneously a direct activator and inhibitor of AMPK complexes containing the β1 or β2 isoform, respectively. Allosteric inhibition by MT47-100 was dependent on the β2 carbohydrate-binding module (CBM) and determined by three non-conserved CBM residues (Ile81, Phe91, Ile92), but was independent of β2-Ser108 phosphorylation. Whereas MT47-100 regulation of total cellular AMPK activity was determined by β1/β2 expression ratio, MT47-100 augmented glucose-stimulated insulin secretion from isolated mouse pancreatic islets via a β2-dependent mechanism. Our findings highlight the therapeutic potential of isoform-specific AMPK allosteric inhibitors.

  8. Combined AKT and MEK Pathway Blockade in Pre-Clinical Models of Enzalutamide-Resistant Prostate Cancer

    PubMed Central

    Toren, Paul; Kim, Soojin; Johnson, Fraser; Zoubeidi, Amina

    2016-01-01

    Despite recent improvements in patient outcomes using newer androgen receptor (AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer (CRPC) continues to remain a clinical problem. Co-targeting alternate resistance pathways are of significant interest to treat CRPC and delay the onset of resistance. Both the AKT and MEK signaling pathways become activated as prostate cancer develops resistance to AR-targeted therapies. This pre-clinical study explores co-targeting these pathways in AR-positive prostate cancer models. Using various in vitro models of prostate cancer disease states including androgen dependent (LNCaP), CRPC (V16D and 22RV1) and ENZ-resistant prostate cancer (MR49C and MR49F), we evaluate the relevance of targeting both AKT and MEK pathways. Our data reveal that AKT inhibition induces apoptosis and inhibits cell growth in PTEN null cell lines independently of their sensitivity to hormone therapy; however, AKT inhibition had no effect on the PTEN positive 22RV1 cell line. Interestingly, we found that MEK inhibition had greater effect on 22RV1 cells compared to LNCaP, V16D or ENZ-resistant cells MR49C and MR49F cells. In vitro, combination AKT and MEK blockade had evidence of synergy observed in some cell lines and assays, but this was not consistent across all results. In vivo, the combination of AKT and MEK inhibition resulted in more consistent tumor growth inhibition of MR49F xenografts and longer disease specific survival compared to AKT inhibitor monotherapy. As in our in vitro study, 22RV1 xenografts were more resistant to AKT inhibition while they were more sensitive to MEK inhibition. Our results suggest that targeting AKT and MEK in combination may be a valuable strategy in prostate cancer when both pathways are activated and further support the importance of characterizing the dominant oncogenic pathway in each patient’s tumor in order to select optimal therapy. PMID:27046225

  9. Combined AKT and MEK Pathway Blockade in Pre-Clinical Models of Enzalutamide-Resistant Prostate Cancer.

    PubMed

    Toren, Paul; Kim, Soojin; Johnson, Fraser; Zoubeidi, Amina

    2016-01-01

    Despite recent improvements in patient outcomes using newer androgen receptor (AR) pathway inhibitors, treatment resistance in castrate resistant prostate cancer (CRPC) continues to remain a clinical problem. Co-targeting alternate resistance pathways are of significant interest to treat CRPC and delay the onset of resistance. Both the AKT and MEK signaling pathways become activated as prostate cancer develops resistance to AR-targeted therapies. This pre-clinical study explores co-targeting these pathways in AR-positive prostate cancer models. Using various in vitro models of prostate cancer disease states including androgen dependent (LNCaP), CRPC (V16D and 22RV1) and ENZ-resistant prostate cancer (MR49C and MR49F), we evaluate the relevance of targeting both AKT and MEK pathways. Our data reveal that AKT inhibition induces apoptosis and inhibits cell growth in PTEN null cell lines independently of their sensitivity to hormone therapy; however, AKT inhibition had no effect on the PTEN positive 22RV1 cell line. Interestingly, we found that MEK inhibition had greater effect on 22RV1 cells compared to LNCaP, V16D or ENZ-resistant cells MR49C and MR49F cells. In vitro, combination AKT and MEK blockade had evidence of synergy observed in some cell lines and assays, but this was not consistent across all results. In vivo, the combination of AKT and MEK inhibition resulted in more consistent tumor growth inhibition of MR49F xenografts and longer disease specific survival compared to AKT inhibitor monotherapy. As in our in vitro study, 22RV1 xenografts were more resistant to AKT inhibition while they were more sensitive to MEK inhibition. Our results suggest that targeting AKT and MEK in combination may be a valuable strategy in prostate cancer when both pathways are activated and further support the importance of characterizing the dominant oncogenic pathway in each patient's tumor in order to select optimal therapy.

  10. Allosteric regulation of rhomboid intramembrane proteolysis

    PubMed Central

    Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

    2014-01-01

    Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

  11. Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression.

    PubMed

    Yoon, Sang-Oh; Shin, Sejeong; Lee, Ho-Jae; Chun, Hyo-Kon; Chung, An-Sik

    2006-11-01

    Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.

  12. Site-directed substitution of Ser1406 of hamster CAD with glutamic acid alters allosteric regulation of carbamyl phosphate synthetase II.

    PubMed

    Banerjei, L C; Davidson, J N

    1997-01-01

    Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylated in vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylated in vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation. PMID:9218000

  13. Allosteric Modulators for the Treatment of Schizophrenia: Targeting Glutamatergic Networks

    PubMed Central

    Menniti, Frank S.; Lindsley, Craig W.; Conn, P. Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A.

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved. PMID:23409764

  14. Entropic mechanism of large fluctuation in allosteric transition.

    PubMed

    Itoh, Kazuhito; Sasai, Masaki

    2010-04-27

    A statistical mechanical model of allosteric transitions in proteins is developed by extending the structure-based model of protein folding to cases of multiple native conformations. The partition function is calculated exactly within the model and the free-energy surface reflecting allostery is derived. This approach is applied to an example protein, the receiver domain of the bacterial enhancer-binding protein NtrC. The model predicts the large entropy associated with a combinatorial number of preexisting transition routes. This large entropy lowers the free-energy barrier of the allosteric transition, which explains the large structural fluctuation observed in the NMR data of NtrC. The global allosteric transformation of NtrC is explained by the shift of preexisting distribution of conformations upon phosphorylation, but the local structural adjustment around the phosphorylation site is explained by the complementary induced-fit mechanism. Structural disordering accompanied by fluctuating interactions specific to two allosteric conformations underlies a large number of routes of allosteric transition. PMID:20385843

  15. Allosteric modulators for the treatment of schizophrenia: targeting glutamatergic networks.

    PubMed

    Menniti, Frank S; Lindsley, Craig W; Conn, P Jeffrey; Pandit, Jayvardhan; Zagouras, Panayiotis; Volkmann, Robert A

    2013-01-01

    Schizophrenia is a highly debilitating mental disorder which afflicts approximately 1% of the global population. Cognitive and negative deficits account for the lifelong disability associated with schizophrenia, whose symptoms are not effectively addressed by current treatments. New medicines are needed to treat these aspects of the disease. Neurodevelopmental, neuropathological, genetic, and behavioral pharmacological data indicate that schizophrenia stems from a dysfunction of glutamate synaptic transmission, particularly in frontal cortical networks. A number of novel pre- and postsynaptic mechanisms affecting glutamatergic synaptic transmission have emerged as viable targets for schizophrenia. While developing orthosteric glutamatergic agents for these targets has proven extremely difficult, targeting allosteric sites of these targets has emerged as a promising alternative. From a medicinal chemistry perspective, allosteric sites provide an opportunity of finding agents with better drug-like properties and greater target specificity. Furthermore, allosteric modulators are better suited to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission. Herein, we review neuropathological and genomic/genetic evidence underscoring the importance of glutamate synaptic dysfunction in the etiology of schizophrenia and make a case for allosteric targets for therapeutic intervention. We review progress in identifying allosteric modulators of AMPA receptors, NMDA receptors, and metabotropic glutamate receptors, all with the aim of restoring physiological glutamatergic synaptic transmission. Challenges remain given the complexity of schizophrenia and the difficulty in studying cognition in animals and humans. Nonetheless, important compounds have emerged from these efforts and promising preclinical and variable clinical validation has been achieved.

  16. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

  17. Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new opportunities.

    PubMed

    Moaddel, Ruin; Jozwiak, Krzysztof; Wainer, Irving W

    2007-09-01

    Allosteric, non-competitive inhibitors (NCIs) of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to produce a wide variety of clinically relevant responses. Many of the observed effects are desired as the nAChR is the therapeutic target, while others are undesired consequences due to off-target binding at the nAChR. Thus, the determination of whether or not a lead drug candidate is an NCI should play an important role in drug discovery programs. However, the current experimental techniques used to identify NCIs are challenging, expensive, and time consuming. This review focuses on an alternative approach to the investigation of interactions between test compounds and nAChRs based upon liquid chromatographic stationary phases containing cellular fragments from cell lines expressing nAChRs. The development and validation of these phases as well as their use in drug discovery and pharmacophore modeling are discussed. PMID:17238157

  18. Blocking the PI3K/AKT pathway enhances mammalian reovirus replication by repressing IFN-stimulated genes

    PubMed Central

    Tian, Jin; Zhang, Xiaozhan; Wu, Hongxia; Liu, Chunguo; Li, Zhijie; Hu, Xiaoliang; Su, Shuo; Wang, Lin-Fa; Qu, Liandong

    2015-01-01

    Many host cellular signaling pathways were activated and exploited by virus infection for more efficient replication. The PI3K/Akt pathway has recently attracted considerable interest due to its role in regulating virus replication. This study demonstrated for the first time that the mammalian reovirus strains Masked Palm Civet/China/2004 (MPC/04) and Bat/China/2003 (B/03) can induce transient activation of the PI3K/Akt pathway early in infection in vitro. When UV-treated, both viruses activated PI3K/Akt signaling, indicating that the virus/receptor interaction was sufficient to activate PI3K/Akt. Reovirus virions can use both clathrin- and caveolae-mediated endocytosis, but only chlorpromazine, a specific inhibitor of clathrin-mediated endocytosis, or siRNA targeting clathrin suppressed Akt phosphorylation. We also identified the upstream molecules of the PI3K pathway. Virus infection induced phosphorylation of focal adhesion kinase (FAK) but not Gab1, and blockage of FAK phosphorylation suppressed Akt phosphorylation. Blockage of PI3K/Akt activation increased virus RNA synthesis and viral yield. We also found that reovirus infection activated the IFN-stimulated response element (ISRE) in an interferon-independent manner and up-regulated IFN-stimulated genes (ISGs) via the PI3K/Akt/EMSY pathway. Suppression of PI3K/Akt activation impaired the induction of ISRE and down-regulated the expression of ISGs. Overexpression of ISG15 and Viperin inhibited virus replication, and knockdown of either enhanced virus replication. Collectively, these results demonstrate that PI3K/Akt activated by mammalian reovirus serves as a pathway for sensing and then inhibiting virus replication/infection. PMID:26388843

  19. The role of the PI3K-Akt signal transduction pathway in Autographa californica multiple nucleopolyhedrovirus infection of Spodoptera frugiperda cells

    SciTech Connect

    Xiao Wei; Yang Yi; Weng Qingbei; Lin Tiehao; Yuan Meijin; Yang Kai; Pang Yi

    2009-08-15

    Many viruses activate the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, thereby modulating diverse downstream signaling pathways associated with antiapoptosis, proliferation, cell cycling, protein synthesis and glucose metabolism, in order to augment their replication. To date, the role of the PI3K-Akt pathway in Baculovirus replication has not been defined. In the present study, we demonstrate that infection of Sf9 cells with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) elevated cellular Akt phosphorylation at 1 h post-infection. The maximum Akt phosphorylation occurred at 6 h post-infection and remained unchanged until 18 h post-infection. The PI3K-specific inhibitor, LY294002, suppressed Akt phosphorylation in a dose-dependent manner, suggesting that AcMNPV-induced Akt phosphorylation is PI3K-dependent. The inhibition of PI3K-Akt activation by LY294002 significantly reduced the viral yield, including a reduction in budded viruses and occlusion bodies. The virus production was reduced only when the inhibitor was added within 24 h of infection, implying that activation of PI3K occurred early in infection. Correspondingly, both viral DNA replication and late (VP39) and very late (POLH) viral protein expression were impaired by LY294002 treatment; LY294002 had no effect on immediate-early (IE1) and early-late (GP64) protein expression. These results demonstrate that the PI3K-Akt pathway is required for efficient Baculovirus replication.

  20. Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

    PubMed

    Bertacchini, J; Guida, M; Accordi, B; Mediani, L; Martelli, A M; Barozzi, P; Petricoin, E; Liotta, L; Milani, G; Giordan, M; Luppi, M; Forghieri, F; De Pol, A; Cocco, L; Basso, G; Marmiroli, S

    2014-11-01

    Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

  1. Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms.

    PubMed

    Wang, Qi; Yu, Wan-Ni; Chen, Xinyu; Peng, Xiao-Ding; Jeon, Sang-Min; Birnbaum, Morris J; Guzman, Grace; Hay, Nissim

    2016-04-11

    Akt is frequently hyperactivated in human cancers and is targeted for cancer therapy. However, the physiological consequences of systemic Akt isoform inhibition were not fully explored. We showed that while combined Akt1 and Akt3 deletion in adult mice is tolerated, combined Akt1 and Akt2 deletion induced rapid mortality. Akt2(-/-) mice survived hepatic Akt1 deletion but all developed spontaneous hepatocellular carcinoma (HCC), which is associated with FoxO-dependent liver injury and inflammation. The gene expression signature of HCC-bearing livers is similar to aggressive human HCC. Consistently, neither Akt1(-/-) nor Akt2(-/-) mice are resistant to diethylnitrosamine-induced hepatocarcinogenesis, and Akt2(-/-) mice display a high incidence of lung metastasis. Thus, in contrast to other cancers, hepatic Akt inhibition induces liver injury that could promote HCC. PMID:26996309

  2. PI3K/AKT Signaling Pathway Is Essential for Survival of Induced Pluripotent Stem Cells

    PubMed Central

    Hossini, Amir M.; Quast, Annika S.; Plötz, Michael; Grauel, Katharina; Exner, Tarik; Küchler, Judit; Stachelscheid, Harald; Eberle, Jürgen; Rabien, Anja

    2016-01-01

    Apoptosis is a highly conserved biochemical mechanism which is tightly controlled in cells. It contributes to maintenance of tissue homeostasis and normally eliminates highly proliferative cells with malignant properties. Induced pluripotent stem cells (iPSCs) have recently been described with significant functional and morphological similarities to embryonic stem cells. Human iPSCs are of great hope for regenerative medicine due to their broad potential to differentiate into specialized cell types in culture. They may be useful for exploring disease mechanisms and may provide the basis for future cell-based replacement therapies. However, there is only poor insight into iPSCs cell signaling as the regulation of apoptosis. In this study, we focused our attention on the apoptotic response of Alzheimer fibroblast-derived iPSCs and two other Alzheimer free iPSCs to five biologically relevant kinase inhibitors as well as to the death ligand TRAIL. To our knowledge, we are the first to report that the relatively high basal apoptotic rate of iPSCs is strongly suppressed by the pancaspase inhibitor QVD-Oph, thus underlining the dependency on proapoptotic caspase cascades. Furthermore, wortmannin, an inhibitor of phosphoinositid-3 kinase / Akt signaling (PI3K-AKT), dramatically and rapidly induced apoptosis in iPSCs. In contrast, parental fibroblasts as well as iPSC-derived neuronal cells were not responsive. The resulting condensation and fragmentation of DNA and decrease of the membrane potential are typical features of apoptosis. Comparable effects were observed with an AKT inhibitor (MK-2206). Wortmannin resulted in disappearance of phosphorylated AKT and activation of the main effector caspase-3 in iPSCs. These results clearly demonstrate for the first time that PI3K-AKT represents a highly essential survival signaling pathway in iPSCs. The findings provide improved understanding on the underlying mechanisms of apoptosis regulation in iPSCs. PMID:27138223

  3. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias.

    PubMed

    Bolognini, Daniele; Moss, Catherine E; Nilsson, Karolina; Petersson, Annika U; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B; Milligan, Graeme

    2016-09-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11 Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  4. A Novel Allosteric Activator of Free Fatty Acid 2 Receptor Displays Unique Gi-functional Bias*

    PubMed Central

    Bolognini, Daniele; Moss, Catherine E.; Nilsson, Karolina; Petersson, Annika U.; Donnelly, Iona; Sergeev, Eugenia; König, Gabriele M.; Kostenis, Evi; Kurowska-Stolarska, Mariola; Miller, Ashley; Dekker, Niek; Tobin, Andrew B.

    2016-01-01

    The short chain fatty acid receptor FFA2 is able to stimulate signaling via both Gi- and Gq/G11-promoted pathways. These pathways are believed to control distinct physiological end points but FFA2 receptor ligands appropriate to test this hypothesis have been lacking. Herein, we characterize AZ1729, a novel FFA2 regulator that acts as a direct allosteric agonist and as a positive allosteric modulator, increasing the activity of the endogenously produced short chain fatty acid propionate in Gi-mediated pathways, but not at those transduced by Gq/G11. Using AZ1729 in combination with direct inhibitors of Gi and Gq/G11 family G proteins demonstrated that although both arms contribute to propionate-mediated regulation of phospho-ERK1/2 MAP kinase signaling in FFA2-expressing 293 cells, the Gq/G11-mediated pathway is predominant. We extend these studies by employing AZ1729 to dissect physiological FFA2 signaling pathways. The capacity of AZ1729 to act at FFA2 receptors to inhibit β-adrenoreceptor agonist-promoted lipolysis in primary mouse adipocytes and to promote chemotaxis of isolated human neutrophils confirmed these as FFA2 processes mediated by Gi signaling, whereas, in concert with blockade by the Gq/G11 inhibitor FR900359, the inability of AZ1729 to mimic or regulate propionate-mediated release of GLP-1 from mouse colonic preparations defined this physiological response as an end point transduced via activation of Gq/G11. PMID:27385588

  5. Anisotropic energy flow and allosteric ligand binding in albumin

    NASA Astrophysics Data System (ADS)

    Li, Guifeng; Magana, Donny; Dyer, R. Brian

    2014-01-01

    Allosteric interactions in proteins generally involve propagation of local structural changes through the protein to a remote site. Anisotropic energy transport is thought to couple the remote sites, but the nature of this process is poorly understood. Here, we report the relationship between energy flow through the structure of bovine serum albumin and allosteric interactions between remote ligand binding sites of the protein. Ultrafast infrared spectroscopy is used to probe the flow of energy through the protein backbone following excitation of a heater dye, a metalloporphyrin or malachite green, bound to different binding sites in the protein. We observe ballistic and anisotropic energy flow through the protein structure following input of thermal energy into the flexible ligand binding sites, without local heating of the rigid helix bundles that connect these sites. This efficient energy transport mechanism enables the allosteric propagation of binding energy through the connecting helix structures.

  6. Studying the allosteric energy cycle by isothermal titration calorimetry.

    PubMed

    Martinez-Julvez, Marta; Abian, Olga; Vega, Sonia; Medina, Milagros; Velazquez-Campoy, Adrian

    2012-01-01

    Isothermal titration calorimetry (ITC) is a powerful biophysical technique which allows a complete thermodynamic characterization of protein interactions with other molecules. The possibility of dissecting the Gibbs energy of interaction into its enthalpic and entropic contributions, as well as the detailed additional information experimentally accessible on the intermolecular interactions (stoichiometry, cooperativity, heat capacity changes, and coupled equilibria), make ITC a suitable technique for studying allosteric interactions in proteins. Two experimental methodologies for the characterization of allosteric heterotropic ligand interactions by ITC are described in this chapter, illustrated with two proteins with markedly different structural and functional features: a photosynthetic electron transfer protein and a drug target viral protease.

  7. Human recombinant H2 relaxin induces AKT and GSK3β phosphorylation and HTR-8/SVneo cell proliferation.

    PubMed

    Astuti, Yoni; Nakabayashi, Koji; Deguchi, Masashi; Ebina, Yasuhiko; Yamada, Hideto

    2015-01-01

    Relaxin is essential for trophoblast development during pregnancy. Evidence shows that relaxin increases trophoblast cell migration capacity. Here, we show the effect of relaxin on protein kinase B (AKT) activation and glycogen synthase kinase 3-beta (GSK3β) inactivation as well as on the proliferation of HTR-8/SVneo cells, a model of human extravillous trophoblast (EVT). HTR-8/SVneo cells were treated with different doses of human recombinant (rH2) relaxin in serum-deprived conditions and treated for increasing time with 1 ng/mL of rH2 relaxin. Western blot analysis was performed to detect pAKT, AKT, pGSK3β, GSK3β, and actin expression. Proliferation of HTR-8/SVneo cells was analyzed by MTS assay. rH2 relaxin treatment increased the ratio of pAKT/AKT, pGSK3β/GSK3β, and proliferation in HTR-8/SVneo cells. Furthermore, AKT and GSK3β activation by rH2 relaxin was inhibited by a phosphoinositide 3-kinase (PI3K) inhibitor. This study suggests that rH2 relaxin induces AKT and GSK3β phosphorylation as well as proliferation in HTR-8/SVneo cells. PMID:25868609

  8. Activation of PI3K/Akt pathway limits JNK-mediated apoptosis during EV71 infection.

    PubMed

    Zhang, Hua; Li, Fengqi; Pan, Ziye; Wu, Zhijun; Wang, Yanhong; Cui, Yudong

    2014-11-01

    Apoptosis is frequently induced to inhibit virus replication during infection of Enterovirus 71 (EV71). On the contrary, anti-apoptotic pathway, such as PI3K/Akt pathway, is simultaneously exploited by EV71 to accomplish the viral life cycle. The relationship by which EV71-induced apoptosis and PI3K/Akt signaling pathway remains to be elucidated. In this study, we demonstrated that EV71 infection altered Bax conformation and triggered its redistribution from the cytosol to mitochondria in RD cells. Subsequently, cytochrome c was released from mitochondria to cytosol. We also found that c-Jun NH2-terminal kinase (JNK) was activated during EV71 infection. The JNK specific inhibitor significantly inhibited Bax activation and cytochrome c release, suggesting that EV71-induced apoptosis was involved into a JNK-dependent manner. Meanwhile, EV71-induced Akt phosphorylation involved a PI3K-dependent mechanism. Inhibition of the PI3K/Akt pathway enhanced JNK phosphorylation and the JNK-mediated apoptosis upon EV71 infection. Moreover, PI3K/Akt pathway phosphorylated apoptosis signal-regulating kinase 1 (ASK1) and negatively regulated the ASK1 activity. Knockdown of ASK1 significantly decreased JNK phosphorylation, which implied that ASK1 phosphorylation by Akt inhibited ASK1-mediated JNK activation. Collectively, these data reveal that activation of the PI3K/Akt pathway limits JNK-mediated apoptosis by phosphorylating and inactivating ASK1 during EV71 infection.

  9. Crosstalk Between MAPK/ERK and PI3K/AKT Signal Pathways During Brain Ischemia/Reperfusion

    PubMed Central

    Zhou, Jing; Du, Ting; Li, Baoman; Rong, Yan; Verkhratsky, Alexei

    2015-01-01

    The epidermal growth factor receptor (EGFR) is linked to the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Raf/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2) signaling pathways. During brain ischemia/reperfusion, EGFR could be transactivated, which stimulates these intracellular signaling cascades that either protect cells or potentiate cell injury. In the present study, we investigated the activation of EGFR, PI3K/AKT, and Raf/MAPK/ERK1/2 during ischemia or reperfusion of the brain using the middle cerebral artery occlusion model. We found that EGFR was phosphorylated and transactivated during both ischemia and reperfusion periods. During ischemia, the activity of PI3K/AKT pathway was significantly increased, as judged from the strong phosphorylation of AKT; this activation was suppressed by the inhibitors of EGFR and Zn-dependent metalloproteinase. Ischemia, however, did not induce ERK1/2 phosphorylation, which was dependent on reperfusion. Coimmunoprecipitation of Son of sevenless 1 (SOS1) with EGFR showed increased association between the receptor and SOS1 in ischemia, indicating the inhibitory node downstream of SOS1. The inhibitory phosphorylation site of Raf-1 at Ser259, but not its stimulatory phosphorylation site at Ser338, was phosphorylated during ischemia. Furthermore, ischemia prompted the interaction between Raf-1 and AKT, while both the inhibitors of PI3K and AKT not only abolished AKT phosphorylation but also restored ERK1/2 phosphorylation. All these findings suggest that Raf/MAPK/ERK1/2 signal pathway is inhibited by AKT via direct phosphorylation and inhibition at Raf-1 node during ischemia. During reperfusion, we observed a significant increase of ERK1/2 phosphorylation but no change in AKT phosphorylation. Inhibitors of reactive oxygen species and phosphatase and tensin homolog restored AKT phosphorylation but abolished ERK1/2 phosphorylation, suggesting that the reactive oxygen species

  10. TGF-β1 induces human aortic vascular smooth muscle cell phenotype switch through PI3K/AKT/ID2 signaling

    PubMed Central

    Zhu, Shui-Bo; Zhu, Jian; Zhou, Zi-Zi; Xi, Er-Ping; Wang, Rong-Ping; Zhang, Yu

    2015-01-01

    The vascular smooth muscle cell (VSMC) phenotypic switch is considered to be the key pathophysiological change in various cardiovascular diseases, such as aortic dissection, atherosclerosis, and hypertension. The results in this study showed that TGF-β1 promotes the proliferation, migration and morphological changes of VSMC.TGF-β1 promoted the expressions of PI3K, P-PI3K, AKT, P-AKT, ID2, and OPN protein and suppressed the expressions of α-SMA and SM22α protein; the opposite results were observed for TGF-β1 inhibitor group, AKT inhibitor group and Combined inhibitors group. After the stimulation of TGF-β1 signaling, the mRNA levels of PI3K, AKT, ID2, and OPN were the highest, while the mRNA levels of α-SMA and SM22α were the lowest; the opposite results were found in the same groups above. These results suggested the PI3K/AKT/ID2 signaling pathway is involved in TGF-β1-mediated human aortic VSMC phenotypic switching, that is from a contractile to synthetic phenotype, and Combined inhibitors was more effective in inhibiting the phenotypic switch than a single inhibitor. The Combined inhibitors experiments may provide new avenues for the prevention and treatment of thoracic aortic dissection (TAD) that are based on the pathological effects of phenotypic switching. PMID:26885273

  11. Seven transmembrane receptors as nature's prototype allosteric protein: de-emphasizing the geography of binding.

    PubMed

    Kenakin, Terry P

    2008-09-01

    The article in this issue by Redka et al. (p. 834) illustrates some interesting interactions between classified orthosteric (bind to the same recognition site as endogenous agonist) and allosteric (bind to a different site) ligands. Of particular interest are the methods used to deal with an obfuscating factor in these kinds of studies, namely the propensity of seven transmembrane receptors to form dimers and thus demonstrate allosteric effects through binding at the orthosteric site. The judicious use of kinetics to detect and quantify allosteric action also is demonstrated. The various unique properties of allosteric modulators are discussed in the context of the increasing prevalence of allosteric ligands as investigational drugs.

  12. Identification and Characterization of an Allosteric Inhibitory Site on Dihydropteroate Synthase

    PubMed Central

    2015-01-01

    The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development. Here, we report the discovery of the first known allosteric inhibitor of DHPS. NMR and crystallographic studies reveal that it engages a previously unknown binding site at the dimer interface. Kinetic data show that this inhibitor does not prevent substrate binding but rather exerts its effect at a later step in the catalytic cycle. Molecular dynamics simulations and quasi-harmonic analyses suggest that the effect of inhibitor binding is transmitted from the dimer interface to the active-site loops that are known to assume an obligatory ordered substructure during catalysis. Together with the kinetics results, these structural and dynamics data suggest an inhibitory mechanism in which binding at the dimer interface impacts loop movements that are required for product release. Our results potentially provide a novel target site for the development of new antibiotics. PMID:24650357

  13. Restoration of Akt activity by the bisperoxovanadium compound bpV(pic) attenuates hippocampal apoptosis in experimental neonatal pneumococcal meningitis.

    PubMed

    Sury, Matthias D; Vorlet-Fawer, Lorianne; Agarinis, Claudia; Yousefi, Shida; Grandgirard, Denis; Leib, Stephen L; Christen, Stephan

    2011-01-01

    Pneumococcal meningitis causes apoptosis of developing neurons in the dentate gyrus of the hippocampus. The death of these cells is accompanied with long-term learning and memory deficits in meningitis survivors. Here, we studied the role of the PI3K/Akt (protein kinase B) survival pathway in hippocampal apoptosis in a well-characterized infant rat model of pneumococcal meningitis. Meningitis was accompanied by a significant decrease of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) and of phosphorylated (i.e., activated) Akt in the hippocampus. At the cellular level, phosphorylated Akt was decreased in both the granular layer and the subgranular zone of the dentate gyrus, the region where the developing neurons undergo apoptosis. Protein levels and activity of PTEN, the major antagonist of PI3K, were unaltered by infection, suggesting that the observed decrease in PIP(3) and Akt phosphorylation is a result of decreased PI3K signaling. Treatment with the PTEN inhibitor bpV(pic) restored Akt activity and significantly attenuated hippocampal apoptosis. Co-treatment with the specific PI3K inhibitor LY294002 reversed the restoration of Akt activity and attenuation of hippocampal apoptosis, while it had no significant effect on these parameters on its own. These results indicate that the inhibitory effect of bpV(pic) on apoptosis was mediated by PI3K-dependent activation of Akt, strongly suggesting that bpV(pic) acted on PTEN. Treatment with bpV(pic) also partially inhibited the concentration of bacteria and cytokines in the CSF, but this effect was not reversed by LY294002, indicating that the effect of bpV(pic) on apoptosis was independent of its effect on CSF bacterial burden and cytokine levels. These results indicate that the PI3K/Akt pathway plays an important role in the death and survival of developing hippocampal neurons during the acute phase of pneumococcal meningitis.

  14. RICTOR involvement in the PI3K/AKT pathway regulation in melanocytes and melanoma

    PubMed Central

    Laugier, Florence; Finet-Benyair, Adeline; André, Jocelyne; Rachakonda, P. Sivaramakrishna; Kumar, Rajiv; Bensussan, Armand; Dumaz, Nicolas

    2015-01-01

    Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development. PMID:26356562

  15. RICTOR involvement in the PI3K/AKT pathway regulation in melanocytes and melanoma.

    PubMed

    Laugier, Florence; Finet-Benyair, Adeline; André, Jocelyne; Rachakonda, P Sivaramakrishna; Kumar, Rajiv; Bensussan, Armand; Dumaz, Nicolas

    2015-09-29

    Several studies have highlighted the importance of the PI3K pathway in melanocytes and its frequent over-activation in melanoma. However, little is known about regulation of the PI3K pathway in melanocytic cells. We showed that normal human melanocytes are less sensitive to selective PI3K or mTOR inhibitors than to dual PI3K/mTOR inhibitors. The resistance to PI3K inhibitor was due to a rapid AKT reactivation limiting the inhibitor effect on proliferation. Reactivation of AKT was linked to a feedback mechanism involving the mTORC2 complex and in particular its scaffold protein RICTOR. RICTOR overexpression in melanocytes disrupted the negative feedback, activated the AKT pathway and stimulated clonogenicity highlighting the importance of this feedback to restrict melanocyte proliferation. We found that the RICTOR locus is frequently amplified and overexpressed in melanoma and that RICTOR over-expression in NRAS-transformed melanocytes stimulates their clonogenicity, demonstrating that RICTOR amplification can cooperate with NRAS mutation to stimulate melanoma proliferation. These results show that RICTOR plays a central role in PI3K pathway negative feedback in melanocytes and that its deregulation could be involved in melanoma development.

  16. Efficacy of Selective PDE4D Negative Allosteric Modulators in the Object Retrieval Task in Female Cynomolgus Monkeys (Macaca fascicularis)

    PubMed Central

    Sutcliffe, Jane S.; Beaumont, Vahri; Watson, James M.; Chew, Chang Sing; Beconi, Maria; Hutcheson, Daniel M.; Dominguez, Celia; Munoz-Sanjuan, Ignacio

    2014-01-01

    Cyclic adenosine monophosphate (cAMP) signalling plays an important role in synaptic plasticity and information processing in the hippocampal and basal ganglia systems. The augmentation of cAMP signalling through the selective inhibition of phosphodiesterases represents a viable strategy to treat disorders associated with dysfunction of these circuits. The phosphodiesterase (PDE) type 4 inhibitor rolipram has shown significant pro-cognitive effects in neurological disease models, both in rodents and primates. However, competitive non-isoform selective PDE4 inhibitors have a low therapeutic index which has stalled their clinical development. Here, we demonstrate the pro-cognitive effects of selective negative allosteric modulators (NAMs) of PDE4D, D159687 and D159797 in female Cynomolgous macaques, in the object retrieval detour task. The efficacy displayed by these NAMs in a primate cognitive task which engages the corticostriatal circuitry, together with their suitable pharmacokinetic properties and safety profiles, suggests that clinical development of these allosteric modulators should be considered for the treatment of a variety of brain disorders associated with cognitive decline. PMID:25050979

  17. The Role of PI3K/Akt/mTOR Signaling in Gastric Carcinoma

    PubMed Central

    Matsuoka, Tasuku; Yashiro, Masakazu

    2014-01-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is one of the key signaling pathways induced by various receptor-tyrosine kinases. Accumulating evidence shows that this pathway is an important promoter of cell growth, metabolism, survival, metastasis, and resistance to chemotherapy. Genetic alterations in the PI3K/Akt/mTOR pathway in gastric carcinoma have often been demonstrated. Many kinds of molecular targeting therapies are currently undergoing clinical testing in patients with solid tumors. However, with the exception of the ErbB2-targeting antibody, targeting agents, including PI3K/Akt/mTOR inhibitors, have not been approved for treatment of patients with gastric carcinoma. This review summarizes the current knowledge on PI3K/Akt/mTOR signaling in the pathogenesis of gastric carcinoma and the possible therapeutic targets for gastric carcinoma. Improved knowledge of the PI3K/Akt/mTOR pathway in gastric carcinoma will be useful in understanding the mechanisms of tumor development and for identifying ideal targets of anticancer therapy for gastric carcinoma. PMID:25003395

  18. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling

    PubMed Central

    Han, Yong-seok; Lee, Jun Hee; Lee, Sang Hun

    2015-01-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer. PMID:25995820

  19. Antitumor Effects of Fucoidan on Human Colon Cancer Cells via Activation of Akt Signaling.

    PubMed

    Han, Yong-Seok; Lee, Jun Hee; Lee, Sang Hun

    2015-05-01

    We identified a novel Akt signaling mechanism that mediates fucoidan-induced suppression of human colon cancer cell (HT29) proliferation and anticancer effects. Fucoidan treatment significantly inhibited growth, induced G1-phase-associated upregulation of p21WAF1 expression, and suppressed cyclin and cyclin-dependent kinase expression in HT29 colon cancer cells. Additionally, fucoidan treatment activated the Akt signaling pathway, which was inhibited by treatment with an Akt inhibitor. The inhibition of Akt activation reversed the fucoidan-induced decrease in cell proliferation, the induction of G1-phase-associated p21WAF1 expression, and the reduction in cell cycle regulatory protein expression. Intraperitoneal injection of fucoidan reduced tumor volume; this enhanced antitumor efficacy was associated with induction of apoptosis and decreased angiogenesis. These data suggest that the activation of Akt signaling is involved in the growth inhibition of colon cancer cells treated with fucoidan. Thus, fucoidan may serve as a potential therapeutic agent for colon cancer.

  20. TBK1 Directly Engages Akt/PKB Survival Signaling to Support Oncogenic Transformation

    PubMed Central

    Ou, Yi-Hung; Torres, Michael; Ram, Rosalyn; Formstecher, Etienne; Roland, Christina; Cheng, Tzuling; Brekken, Rolf; Wurz, Ryan; Tasker, Andrew; Polverino, Tony; Tan, Seng-Lai; White, Michael A.

    2011-01-01

    The innate immune signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling. PMID:21329883

  1. Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer

    PubMed Central

    van der Mijn, Johannes C.; Sol, Nik; Mellema, Wouter; Jimenez, Connie R.; Piersma, Sander R.; Dekker, Henk; Schutte, Lisette M.; Smit, Egbert F.; Broxterman, Henk J.; Skog, Johan; Tannous, Bakhos A.; Wurdinger, Thomas; Verheul, Henk M. W.

    2014-01-01

    Background Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). Conclusion Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer. PMID:25491250

  2. Allosteric Interactions between the Myristate- and ATP-Site of the Abl Kinase

    PubMed Central

    Iacob, Roxana E.; Zhang, Jianming; Gray, Nathanael S.; Engen, John R.

    2011-01-01

    Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors. PMID:21264348

  3. Metal ion coupled protein folding and allosteric motions

    NASA Astrophysics Data System (ADS)

    Wang, Wei

    2014-03-01

    Many proteins need the help of cofactors for their successful folding and functioning. Metal ions, i.e., Zn2+, Ca2+, and Mg2+ etc., are typical biological cofactors. Binding of metal ions can reshape the energy landscapes of proteins, thereby modifying the folding and allosteric motions. For example, such binding may make the intrinsically disordered proteins have funneled energy landscapes, consequently, ensures their spontaneous folding. In addition, the binding may activate certain biological processes by inducing related conformational changes of regulation proteins. However, how the local interactions involving the metal ion binding can induce the global conformational motions of proteins remains elusive. Investigating such question requires multiple models with different details, including quantum mechanics, atomistic models, and coarse grained models. In our recent work, we have been developing such multiscale methods which can reasonably model the metal ion binding induced charge transfer, protonation/deprotonation, and large conformational motions of proteins. With such multiscale model, we elucidated the zinc-binding induced folding mechanism of classical zinc finger and the calcium-binding induced dynamic symmetry breaking in the allosteric motions of calmodulin. In addition, we studied the coupling of folding, calcium binding and allosteric motions of calmodulin domains. In this talk, I will introduce the above progresses on the metal ion coupled protein folding and allosteric motions. We thank the finacial support from NSFC and the 973 project.

  4. The structure and allosteric regulation of glutamate dehydrogenase.

    PubMed

    Li, Ming; Li, Changhong; Allen, Aron; Stanley, Charles A; Smith, Thomas J

    2011-09-01

    Glutamate dehydrogenase (GDH) has been extensively studied for more than 50 years. Of particular interest is the fact that, while considered by most to be a 'housekeeping' enzyme, the animal form of GDH is heavily regulated by a wide array of allosteric effectors and exhibits extensive inter-subunit communication. While the chemical mechanism for GDH has remained unchanged through epochs of evolution, it was not clear how or why animals needed to evolve such a finely tuned form of this enzyme. As reviewed here, recent studies have begun to elucidate these issues. Allosteric regulation first appears in the Ciliates and may have arisen to accommodate evolutionary changes in organelle function. The occurrence of allosteric regulation appears to be coincident with the formation of an 'antenna' like feature rising off the tops of the subunits that may be necessary to facilitate regulation. In animals, this regulation further evolved as GDH became integrated into a number of other regulatory pathways. In particular, mutations in GDH that abrogate GTP inhibition result in dangerously high serum levels of insulin and ammonium. Therefore, allosteric regulation of GDH plays an important role in insulin homeostasis. Finally, several compounds have been identified that block GDH-mediated insulin secretion that may be to not only find use in treating these insulin disorders but to kill tumors that require glutamine metabolism for cellular energy.

  5. Activation of muscarinic acetylcholine receptors via their allosteric binding sites.

    PubMed Central

    Jakubík, J; Bacáková, L; Lisá, V; el-Fakahany, E E; Tucek, S

    1996-01-01

    Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation. PMID:8710935

  6. Deeper Insights into the Allosteric Modulation of Ionotropic Glutamate Receptors.

    PubMed

    Regan, Michael C; Furukawa, Hiro

    2016-09-21

    Two articles in this issue of Neuron (Yelshanskaya et al., 2016; Yi et al., 2016) explore the structural basis of allosteric inhibition in ionotropic glutamate receptors, providing key insights into how iGluRs function in the brain as well as how they might be pharmacologically modulated in neurological disorders and disease. PMID:27657445

  7. Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

    PubMed

    Rodríguez-Peña, Ana B; Grande, Maria T; Eleno, Nélida; Arévalo, Miguel; Guerrero, Carmen; Santos, Eugerio; López-Novoa, José M

    2008-07-01

    Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

  8. Molecular Characterization of Synovial Sarcoma in Children and Adolescents: Evidence of Akt Activation1

    PubMed Central

    Bozzi, Fabio; Ferrari, Andrea; Negri, Tiziana; Conca, Elena; Luca, Da Riva; Losa, Marco; Casieri, Paola; Orsenigo, Marta; Lampis, Andrea; Meazza, Cristina; Casanova, Michela; Pierotti, Marco A; Tamborini, Elena; Pilotti, Silvana

    2008-01-01

    Synovial sarcoma (SS) is the most frequent nonrhabdomyosarcomatous soft tissue sarcoma encountered in adolescents and young adults, and despite advances in the treatment of local disease, metastases remain the main cause of death. The aim of this study was to characterize a single-center series of pediatric SS molecularly to seek any biomarkers or pathways that might make suitable targets for new agents. Seventeen cases of pediatric SS showing the SYT-SSX fusion transcript were screened immunohistochemically, biochemically, molecularly, and cytogenetically (depending on the available material) to investigate any expression/activation of epidermal growth factor receptor, platelet-derived growth factor receptor alpha (PDGFRα), PDGFRβ, Akt, and deregulated Wnt pathway. The most relevant outcome was the finding of activated epidermal growth factor receptor, PDGFRα, and PDGFRβ, which activated Akt in both the monophasic and biphasic histologic subtypes. Consistently, Akt activation was completely abolished in an SS cell line assay when stimulated by PDGF-AA and treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Our results also showed the nuclear localization of β-catenin and cyclin D1 gene products in monophasic SS and the movement of β-catenin into the cytoplasm in the glandular component of the biphasic subtype. Although they need to be confirmed in larger series, these preliminary data suggest that therapeutic strategies including specific inhibitors of the phosphatidylinositol 3-kinase/Akt pathway might be exploited in SS. PMID:18633459

  9. Heat stress activates AKT via focal adhesion kinase-mediated pathway in neonatal rat ventricular myocytes.

    PubMed

    Wei, Hongguang; Vander Heide, Richard S

    2008-08-01

    Heat stress (HS)-induced cardioprotection is associated with increased paxillin localization to the membrane fraction of neonatal rat ventricular myocytes (NRVM). The purpose of this study was 1) to examine the subcellular signaling pathways activated by HS; 2) to determine whether myocardial stress organizes and activates an integrated survival pathway; and 3) to investigate potential downstream cytoprotective proteins activated by HS. After HS, NRVM were subjected to chemical inhibitors (CI) designed to simulate ischemia by inhibiting both glycolysis and mitochondrial respiration. Protein kinase B (AKT) expression (wild type) was increased selectively with an adenoviral vector. Cell signaling was analyzed with Western blot analysis, while oncosis/apoptosis was assayed by measuring Trypan blue exclusion and/or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, respectively. HS increased phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 but did not adversely affect the viability of NRVM before CI. HS increased association between FAK and phosphatidylinositol 3-kinase as well as causing a significant increase in AKT activity. Increased expression of wild-type AKT protected myocytes from both oncotic and apoptotic cell death. Increased expression of a FAK inhibitor, FRNK, reduced AKT phosphorylation in response to HS both at time 0 and after 10 min of CI compared with myocytes expressing empty virus. We conclude that myocardial stress activates cytoskeleton-based signaling pathways that are associated with protection from lethal cell injury.

  10. Akt signaling dynamics in individual cells

    PubMed Central

    Gross, Sean M.; Rotwein, Peter

    2015-01-01

    ABSTRACT The protein kinase Akt (for which there are three isoforms) is a key intracellular mediator of many biological processes, yet knowledge of Akt signaling dynamics is limited. Here, we have constructed a fluorescent reporter molecule in a lentiviral delivery system to assess Akt kinase activity at the single cell level. The reporter, a fusion between a modified FoxO1 transcription factor and clover, a green fluorescent protein, rapidly translocates from the nucleus to the cytoplasm in response to Akt stimulation. Because of its long half-life and the intensity of clover fluorescence, the sensor provides a robust readout that can be tracked for days under a range of biological conditions. Using this reporter, we find that stimulation of Akt activity by IGF-I is encoded into stable and reproducible analog responses at the population level, but that single cell signaling outcomes are variable. This reporter, which provides a simple and dynamic measure of Akt activity, should be compatible with many cell types and experimental platforms, and thus opens the door to new insights into how Akt regulates its biological responses. PMID:26040286

  11. Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells

    SciTech Connect

    Horiuchi, Rie; Akimoto, Takayuki; Hong, Zhang; Ushida, Takashi

    2012-08-15

    Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17 Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling. -- Highlights: Black-Right-Pointing-Pointer The expression of Nanog, which is an essential regulator of 'stemness' was reduced during embryonic stem (ES) cell differentiation. Black-Right-Pointing-Pointer Cyclic mechanical strain attenuated the reduction of Nanog expression. Black-Right-Pointing-Pointer Cyclic mechanical strain promoted PI3K-Akt signaling and mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor and an inhibitor of actin polymerization.

  12. Osteocalcin attenuates high fat diet-induced impairment of endothelium-dependent relaxation through Akt/eNOS-dependent pathway

    PubMed Central

    2014-01-01

    Background Recent studies have demonstrated a protective effect of osteocalcin (OCN) on glucose homeostasis and metabolic syndrome. However, its role in vascular function remains unknown. This study investigated the contribution of OCN to the pathogenesis of endothelial dysfunction in the thoracic aorta of apolipoprotein E-deficient (ApoE-KO) mice. Methods Eight-week-old ApoE–KO mice were given chow or high fat diet (HFD) for 12 weeks with or without daily intraperitoneal injection of OCN. Intraperitoneal glucose tolerance test (IPGTT), insulin tolerance test (ITT),measurement of serum lipid profiles and blood pressure were carried out. Endothelium-dependent relaxation (EDR) was measured by wire myography. Human umbilical vein endothelial cells (HUVECs) were used to study the role of OCN on eNOS levels in vitro. PI3K inhibitor (LY294002) and Akt inhibitor V were used ex-vivo to determine whether PI3K/Akt/eNOS contributes to the beneficial effect of OCN for the vascular or not. Results Daily injections of OCN can significantly improve lipid metabolism, glucose tolerance and insulin sensitivity in ApoE-KO mice. In ApoE-KO mice fed with HFD, the OCN-treated mice displayed an improved acetylcholine-stimulated EDR compared to the vehicle-treated group. In addition, compared to vehicle-treated HUVECs, OCN-treated HUVECs displayed increased activation of the Akt-eNOS signaling pathway, as evidenced by significantly higher levels of phosphorylated Akt and eNOS. Furthermore, a similar beneficial effect of OCN on thoracic aorta was observed using ex vivo organ culture of isolated mouse aortic segment. However, this effect was attenuated upon co-incubation with PI3K inhibitor or Akt inhibitor V. Conclusions Our study demonstrates that OCN has an endothelial-protective effect in atherosclerosis through mediating the PI3K/Akt/eNOS signaling pathway. PMID:24708830

  13. Cofactor binding triggers a molecular switch to allosterically activate human UDP-α-D-glucose 6-dehydrogenase.

    PubMed

    Sennett, Nicholas C; Kadirvelraj, Renuka; Wood, Zachary A

    2012-11-20

    Human UDP-α-D-glucose dehydrogenase (hUGDH) catalyzes the NAD(+)-dependent oxidation of UDP-α-D-glucose (UDG) to produce UDP-α-D-glucuronic acid. The oligomeric structure of hUGDH is dynamic and can form two distinct hexameric complexes in solution. The active form of hUGDH consists of dimers that undergo a concentration-dependent association to form a hexamer with 32 symmetry. In the presence of the allosteric feedback inhibitor UDP-α-D-xylose (UDX), hUGDH changes shape to form an inactive, horseshoe-shaped complex. Previous studies have identified the UDX-induced allosteric mechanism that changes the hexameric structure to inhibit the enzyme. Here, we investigate the role of the 32 symmetry hexamer in the catalytic cycle. We engineered a stable hUGDH dimer by introducing a charge-switch substitution (K94E) in the hexamer-building interface (hUGDH(K94E)). The k(cat) of hUGDH(K94E) is ~160-fold lower than that of the wild-type enzyme, suggesting that the hexamer is the catalytically relevant state. We also show that cofactor binding triggers the formation of the 32 symmetry hexamer, but UDG is needed for the stability of the complex. The hUGDH(K94E) crystal structure at 2.08 Å resolution identifies loop(88-110) as the cofactor-responsive allosteric switch that drives hexamer formation; loop(88-110) directly links cofactor binding to the stability of the hexamer-building interface. In the interface, loop(88-110) packs against the Thr131-loop/α6 helix, the allosteric switch that responds to the feedback inhibitor UDX. We also identify a structural element (the S-loop) that explains the indirect stabilization of the hexamer by substrate and supports a sequential, ordered binding of the substrate and cofactor. These observations support a model in which (i) UDG binds to the dimer and stabilizes the S-loop to promote cofactor binding and (ii) cofactor binding orders loop(88-110) to induce formation of the catalytically active hexamer.

  14. Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins

    PubMed Central

    Jain, Mayur V.; Shareef, Ahmad; Likus, Wirginia; Cieślar-Pobuda, Artur; Ghavami, Saeid; Łos, Marek J.

    2016-01-01

    Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway. PMID:26967567

  15. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

    Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

  16. Drug design from the cryptic inhibitor envelope

    PubMed Central

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  17. Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1.

    PubMed

    Dowal, Louisa; Sim, Derek S; Dilks, James R; Blair, Price; Beaudry, Sarah; Denker, Bradley M; Koukos, Georgios; Kuliopulos, Athan; Flaumenhaft, Robert

    2011-02-15

    G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α(2A)-adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G(αq) but not G(α12). The compound inhibited thrombus formation in vivo following vascular injury with an IC(50) of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation. PMID:21282664

  18. Identification of drugs competing with d-tubocurarine for an allosteric site on cardiac muscarinic receptors.

    PubMed

    Waelbroeck, M

    1994-10-01

    d-Tubocurarine behaved as a weak allosteric inhibitor of N-[3H] methylscopolamine binding to cardiac M2 muscarinic receptors. In a low ionic strength buffer devoid of bivalent ions, d-tubocurarine recognized cardiac M2 receptors in the micromolar concentration range and decreased their affinity for N-[3H]methylscopolamine by at most 4-fold. To identify the compounds that preferentially recognize this accessory site (as opposed to the classical muscarinic binding site), we measured the inhibition by different drugs of N-[3H]methylscopolamine binding, in the absence or presence of d-tubocurarine. The effect of gallamine was competitively inhibited by d-tubocurarine; both drugs compete for the same accessory site on muscarinic receptors. The effects of dexetimide, levetimide, 4-diphenylacetoxy-N-ethylpiperidine ethobromide, AF-DX 116, and telenzepine on N-[3H]methylscopolamine binding were not affected or were barely affected by d-tubocurarine; these compounds preferentially recognize another binding site (probably the muscarinic binding site). The dose-effect curves for pentamethylene-bis(4-diphenylacetoxymethylpiperidine) bromide and methoctramine were shifted, but at most 10-fold, by d-tubocurarine. It is likely that (in this low ionic strength incubation buffer) methoctramine and pentamethylene-bis(4-diphenylacetoxymethylpiperidine)bromide had comparable affinities for the muscarinic site and the accessory site. d-Tubocurarine competitively inhibited their binding to the accessory site and allosterically inhibited their binding to the muscarinic site. This resulted in a large decrease (40-60-fold) of their overall affinity for muscarinic receptors. PMID:7969047

  19. Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

    PubMed Central

    2012-01-01

    Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux

  20. PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

    SciTech Connect

    Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki; Watanabe, Takumi; Odaka, Yoshiaki; Ozaki, Kakeru; Yoneda, Yukio

    2014-07-18

    Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylation of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.

  1. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  2. Inhibition of akt enhances the chemopreventive effects of topical rapamycin in mouse skin

    USGS Publications Warehouse

    Dickinson, Sally E; Janda, Jaroslav; Criswell, Jane; Blohm-Mangone, Karen; Olson, Erik R.; Liu, Zhonglin; Barber, Christie; Rusche, Jadrian J.; Petricoin, Emmanuel; Calvert, Valerie; Einspahr, Janine G.; Dickinson, Jesse; Stratton, Steven P.; Curiel-Lewandrowski, Clara; Saboda, Kathylynn; Hu, Chengcheng; Bode, Ann M.; Dong, Zigang; Alberts, David S.; Bowden, G. Timothy

    2016-01-01

    The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced non-melanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin, (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared to those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here we explored the use of topical rapamycin as a chemopreventive agent in the context of solar simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared to controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared to vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC.

  3. A novel form of constitutively active farnesylated Akt1 prevents mammary epithelial cells from anoikis and suppresses chemotherapy-induced apoptosis.

    PubMed

    Schmidt, M; Hövelmann, S; Beckers, T L

    2002-10-01

    Protein kinase B/Akt has been described as a central mediator of anti-apoptotic signals transduced by the PI3 kinase. Although the role of Akt in the suppression of apoptosis is well elucidated, a potential function of Akt in tumorigenesis and chemoresistance is less intensively documented. In this study, we describe the construction of a novel form of constitutively active Akt1, which relies on the deletion of its pleckstrin homology domain and the insertion of a C-terminal farnesylation sequence. Stable cell lines were generated with MCF10A mammary epithelial cells and A549 human NSCLC cells expressing constitutively active Akt1. Enigneered MCF10A cells were rendered resistant towards apoptosis resulting from loss of cellular substrate attachment (anoikis). We investigated the chemosensitivity of A549 cells expressing farnesylated Akt vs control cells. A profoundly decreased sensitivity towards Mitoxantrone and cisplatin was observed in cells expressing farnesylated Akt. No significant difference in sensitivity however was observed upon treatment with cell cycle specific chemotherapeutic agents like paclitaxel. Our data suggest, that Akt is a central mediator in the suppression of anoikis and modulation of chemotherapy-induced apoptosis. Therefore it represents a promising target for small molecule inhibitors to shift the apoptotic threshold in cancer cells after treatment with standard chemotherapy.

  4. The Regulation of Lipid Deposition by Insulin in Goose Liver Cells Is Mediated by the PI3K-AKT-mTOR Signaling Pathway

    PubMed Central

    Han, Chunchun; Wei, Shouhai; He, Fang; Liu, Dandan; Wan, Huofu; Liu, Hehe; Li, Liang; Xu, Hongyong; Du, Xiaohui; Xu, Feng

    2015-01-01

    Background We previously showed that the fatty liver formations observed in overfed geese are accompanied by the activation of the PI3K-Akt-mTOR pathway and an increase in plasma insulin concentrations. Recent studies have suggested a crucial role for the PI3K-Akt-mTOR pathway in regulating lipid metabolism; therefore, we hypothesized that insulin affects goose hepatocellular lipid metabolism through the PI3K-Akt-mTOR signaling pathway. Methods Goose primary hepatocytes were isolated and treated with serum-free media supplemented with PI3K-Akt-mTOR pathway inhibitors (LY294002, rapamycin, and NVP-BEZ235, respectively) and 50 or 150 nmol/L insulin. Results Insulin induced strong effects on lipid accumulation as well as the mRNA and protein levels of genes involved in lipogenesis, fatty acid oxidation, and VLDL-TG assembly and secretion in primary goose hepatocytes. The stimulatory effect of insulin on lipogenesis was significantly decreased by treatment with PI3K-Akt-mTOR inhibitors. These inhibitors also rescued the insulin-induced down-regulation of fatty acid oxidation and VLDL-TG assembly and secretion. Conclusion These findings suggest that the stimulatory effect of insulin on lipid deposition is mediated by PI3K-Akt-mTOR regulation of lipogenesis, fatty acid oxidation, and VLDL-TG assembly and secretion in goose hepatocytes. PMID:25945932