36 results on '"Dar AC"'
Search Results
2. The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.
- Author
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Ryan MB, Quade B, Schenk N, Fang Z, Zingg M, Cohen SE, Swalm BM, Li C, Özen A, Ye C, Ritorto MS, Huang X, Dar AC, Han Y, Hoeflich KP, Hale M, and Hagel M
- Subjects
- Humans, Animals, Mice, raf Kinases metabolism, raf Kinases antagonists & inhibitors, Cell Line, Tumor, ras Proteins metabolism, Xenograft Model Antitumor Assays, Brain metabolism, Brain drug effects, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, MAP Kinase Signaling System drug effects, Neoplasms drug therapy, Neoplasms metabolism
- Abstract
Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor., (©2024 The Authors; Published by the American Association for Cancer Research.)
- Published
- 2024
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3. Live-cell target engagement of allosteric MEKi on MEK-RAF/KSR-14-3-3 complexes.
- Author
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Marsiglia WM, Chow A, Khan ZM, He L, and Dar AC
- Subjects
- MAP Kinase Signaling System, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins B-raf genetics, Biological Assay
- Abstract
The RAS-mitogen-activated protein kinase (MAPK) pathway includes KSR, RAF, MEK and the phospho-regulatory sensor 14-3-3. Specific assemblies among these components drive various diseases and likely dictate efficacy for numerous targeted therapies, including allosteric MEK inhibitors (MEKi). However, directly measuring drug interactions on physiological RAS-MAPK complexes in live cells has been inherently challenging to query and therefore remains poorly understood. Here we present a series of NanoBRET-based assays to quantify direct target engagement of MEKi on MEK1 and higher-order MEK1-bound complexes with ARAF, BRAF, CRAF, KSR1 and KSR2 in the presence and absence of 14-3-3 in living cells. We find distinct MEKi preferences among these complexes that can be compiled to generate inhibitor binding profiles. Further, these assays can report on the influence of the pathogenic BRAF-V600E mutant on MEKi binding. Taken together, these approaches can be used as a platform to screen for compounds intended to target specific complexes in the RAS-MAPK cascade., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2024
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4. WNTinib is a multi-kinase inhibitor with specificity against β-catenin mutant hepatocellular carcinoma.
- Author
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Rialdi A, Duffy M, Scopton AP, Fonseca F, Zhao JN, Schwarz M, Molina-Sanchez P, Mzoughi S, Arceci E, Abril-Fornaguera J, Meadows A, Ruiz de Galarreta M, Torre D, Reyes K, Lim YT, Rosemann F, Khan ZM, Mohammed K, Wang X, Yu X, Lakshmanan M, Rajarethinam R, Tan SY, Jin J, Villanueva A, Michailidis E, De Jong YP, Rice CM, Marazzi I, Hasson D, Llovet JM, Sobota RM, Lujambio A, Guccione E, and Dar AC
- Subjects
- Humans, Mice, Animals, beta Catenin genetics, beta Catenin metabolism, Transcription Factors metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Carcinoma, Hepatocellular drug therapy, Carcinoma, Hepatocellular genetics, Liver Neoplasms drug therapy, Liver Neoplasms genetics, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use
- Abstract
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. β-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant β-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2023
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5. RASopathy mutations open new insights into the mechanism of BRAF activation.
- Author
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Dar AC and Brady DC
- Subjects
- Mutation, Protein Domains, Proto-Oncogene Proteins B-raf genetics, Proto-Oncogene Proteins B-raf metabolism
- Abstract
Spencer-Smith et al. (2022)
1 investigate multiple functions of the BRAF cysteine-rich domain (CRD), finding distinct classes of RASopathy-associated BRAF mutations and unique features among RAF paralogs that may contribute to the spectrum of mutations observed in disease., Competing Interests: Declaration of interests D.C.B. holds ownership in Merlon Inc. and Elaeis Therapeutics, is an inventor on the patent application 20,150,017,261 (entitled “Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway”), and is a member of the Molecular Cell Advisory Board. A.C.D. is a founder, shareholder, advisory board member, and consultant to Nested Therapeutics., (Copyright © 2022. Published by Elsevier Inc.)- Published
- 2022
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6. Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR.
- Author
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Schwarz M, Torre D, Lozano-Ojalvo D, Tan AT, Tabaglio T, Mzoughi S, Sanchez-Tarjuelo R, Le Bert N, Lim JME, Hatem S, Tuballes K, Camara C, Lopez-Granados E, Paz-Artal E, Correa-Rocha R, Ortiz A, Lopez-Hoyos M, Portoles J, Cervera I, Gonzalez-Perez M, Bodega-Mayor I, Conde P, Oteo-Iglesias J, Borobia AM, Carcas AJ, Frías J, Belda-Iniesta C, Ho JSY, Nunez K, Hekmaty S, Mohammed K, Marsiglia WM, Carreño JM, Dar AC, Berin C, Nicoletti G, Della Noce I, Colombo L, Lapucci C, Santoro G, Ferrari M, Nie K, Patel M, Barcessat V, Gnjatic S, Harris J, Sebra R, Merad M, Krammer F, Kim-Schulze S, Marazzi I, Bertoletti A, Ochando J, and Guccione E
- Subjects
- Humans, Immunity, Cellular, Polymerase Chain Reaction, T-Lymphocytes, SARS-CoV-2 genetics, COVID-19
- Abstract
Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2022
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7. Targeting drug-resistant mutations in ALK.
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He L and Dar AC
- Subjects
- Anaplastic Lymphoma Kinase genetics, Humans, Mutation, Carcinoma, Non-Small-Cell Lung genetics, Lung Neoplasms genetics
- Published
- 2022
- Full Text
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8. Understanding and drugging RAS: 40 years to break the tip of the iceberg.
- Author
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Brady DC, Hmeljak J, and Dar AC
- Subjects
- Antineoplastic Agents pharmacology, Humans, Mutation genetics, Protein Processing, Post-Translational, Signal Transduction genetics, Neoplasms drug therapy, Neoplasms genetics, Neoplasms metabolism, ras Proteins drug effects, ras Proteins genetics, ras Proteins metabolism
- Abstract
Several cancers and rare genetic diseases are caused by dysregulation in the RAS signaling pathway. RAS proteins serve as molecular switches that regulate pathways involved in cellular growth, differentiation and survival. These pathways have been an intense area of investigation for four decades, since the initial identification of somatic RAS mutations linked to human cancers. In the past few years, inhibitors against several RAS effectors, as well as direct inhibitors of the K-RAS mutant G12C, have been developed. This Special Issue in DMM includes original Research articles on RAS-driven cancers and RASopathies. The articles provide insights into mechanisms and biomarkers, and evaluate therapeutic targets. Several articles also present new disease models, whereas others describe technologies or approaches to evaluate the function of RAS in vivo. The collection also includes a series of Review articles on RAS biology and translational aspects of defining and treating RAS-driven diseases. In this Editorial, we summarize this collection and discuss the potential impact of the articles within this evolving area of research. We also identify areas of growth and possible future developments., (© 2022. Published by The Company of Biologists Ltd.)
- Published
- 2022
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9. Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.
- Author
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Chow A, Khan ZM, Marsiglia WM, and Dar AC
- Subjects
- Binding Sites, Molecular Conformation, Phosphorylation, Proto-Oncogene Proteins c-raf metabolism, Signal Transduction, Protein Kinases chemistry, Protein Serine-Threonine Kinases
- Abstract
Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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10. Type II Binders Targeting the "GLR-Out" Conformation of the Pseudokinase STRADα.
- Author
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Smith RHB, Khan ZM, Ung PM, Scopton AP, Silber L, Mack SM, Real AM, Schlessinger A, and Dar AC
- Subjects
- Humans, Protein Domains, Protein Stability, Adaptor Proteins, Vesicular Transport chemistry, Adenosine Triphosphate chemistry
- Abstract
Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.
- Published
- 2021
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11. Structural basis for the action of the drug trametinib at KSR-bound MEK.
- Author
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Khan ZM, Real AM, Marsiglia WM, Chow A, Duffy ME, Yerabolu JR, Scopton AP, and Dar AC
- Subjects
- Amino Acid Sequence, Animals, Binding Sites drug effects, Humans, Mice, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Models, Molecular, Protein Binding drug effects, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology, Substrate Specificity, raf Kinases chemistry, raf Kinases metabolism, Mitogen-Activated Protein Kinase Kinases chemistry, Mitogen-Activated Protein Kinase Kinases metabolism, Protein Kinases chemistry, Protein Kinases metabolism, Pyridones chemistry, Pyridones pharmacology, Pyrimidinones chemistry, Pyrimidinones pharmacology
- Abstract
The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology
1 , and more recently in immunotherapy2,3 and ageing4 . However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10 . Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.- Published
- 2020
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12. Tubular transcriptional co-activator with PDZ-binding motif protects against ischemic acute kidney injury.
- Author
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Wu CL, Chang CC, Yang TH, Tsai AC, Wang JL, Chang CH, and Tarng DC
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- Acute Kidney Injury etiology, Acute Kidney Injury physiopathology, Adaptor Proteins, Signal Transducing, Animals, Apoptosis, Epithelial Cells cytology, Epithelial Cells metabolism, Female, Humans, Kidney Tubules cytology, Kidney Tubules metabolism, Male, Mice, Mice, Inbred C57BL, NF-kappa B genetics, NF-kappa B metabolism, Reperfusion Injury metabolism, Signal Transduction, Trans-Activators genetics, Acute Kidney Injury metabolism, Acute Kidney Injury prevention & control, Reperfusion Injury complications, Trans-Activators metabolism
- Abstract
Transcriptional co-activator with PDZ-binding motif (TAZ) is a key downstream effector of the Hippo tumor-suppressor pathway. The functions of TAZ in the kidney, especially in tubular epithelial cells, are not well-known. To elucidate the adaptive expression, protective effects on kidney injury, and signaling pathways of TAZ in response to acute kidney injury (AKI), we used in vitro (hypoxia-treated human renal proximal tubular epithelial cells [RPTECs]) and in vivo (mouse ischemia-reperfusion injury [IRI]) models of ischemic AKI. After ischemic AKI, TAZ was up-regulated in RPTECs and the renal cortex or tubules. Up-regulation of TAZ in RPTECs subjected to hypoxia was controlled by IκB kinase (IKK)/nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) signaling. TAZ overexpression attenuated hypoxic and oxidative injury, inhibited apoptosis and activation of p38 and c-Jun N-terminal kinase (JNK) proteins, and promoted wound healing in an RPTEC monolayer. However, TAZ knockdown aggravated hypoxic injury, apoptosis, and activation of p38 and JNK signaling, delayed wound closure of an RPTEC monolayer, and promoted G0/G1 phase cell-cycle arrest. Chloroquine and verteporfin treatment produced similar results to TAZ overexpression and knockdown in RPTECs, respectively. Compared with vehicle-treated mice, chloroquine treatment increased TAZ in the renal cortex and tubules, improved renal function, and attenuated tubular injury and tubular apoptosis after renal IRI, whereas TAZ siRNA and verteporfin decreased TAZ in the renal cortex and tubules, deteriorated renal failure and tubular injury, and aggravated tubular apoptosis. Our findings indicate the renoprotective role of tubular TAZ in ischemic AKI. Drugs augmenting (e.g., chloroquine) or suppressing (e.g., verteporfin) TAZ in the kidney might be beneficial or deleterious to patients with AKI., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2020
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13. Ploidy Leads a Molecular Motor to Walk Different Paths to Drug Resistance.
- Author
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Real AM, Marsiglia WM, and Dar AC
- Subjects
- Bridged Bicyclo Compounds, Heterocyclic, Drug Resistance, Haploidy, Humans, Diploidy, Neoplasms
- Abstract
In this issue of Cell Chemical Biology, Pisa et al. (2020) find that haploid and diploid cells differentially develop resistance to the CENP-E inhibitor GSK923295. The results highlight the power of tumor cells to evade growth inhibition and potentially inform the design of next-generation CENP-E drugs to overcome resistance., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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14. The Alteration of CTNNBIP1 in Lung Cancer.
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Chang JM, Tsai AC, Huang WR, and Tseng RC
- Subjects
- Cell Line, Tumor, Cell Movement, Female, Humans, Lung Neoplasms metabolism, Lung Neoplasms pathology, Male, Wnt Signaling Pathway, beta Catenin metabolism, Gene Expression Regulation, Neoplastic, Lung Neoplasms genetics, beta Catenin genetics
- Abstract
β-catenin is a major component of the Wnt/β-catenin signaling pathway, and is known to play a role in lung tumorigenesis. β-catenin-interacting protein 1 (CTNNBIP1) is a known repressor of β-catenin transactivation. However, little is known about the role of CTNNBIP1 in lung cancer. The aim of this study was to carry out a molecular analysis of CTNNBIP1 and its effect on β-catenin signaling, using samples from lung cancer patients and various lung cancer cell lines. Our results indicate a significant inverse correlation between the CTNNBIP1 mRNA expression levels and the CTNNBIP1 promoter hypermethylation, which suggests that the promoter hypermethylation is responsible for the low levels of CTNNBIP1 present in many lung cancer patient samples. The ectopic expression of CTNNBIP1 is able to reduce the β-catenin transactivation; this then brings about a decrease in the expression of β-catenin-targeted genes, such as matrix metalloproteinase 7 (MMP7). Conversely, CTNNBIP1 knockdown is able to increase β-catenin transactivation and the expression of MMP7. In agreement with these findings, a low level of CTNNBIP1 was found to be correlated with a high level of MMP7 when a publicly available microarray dataset for lung cancer was analyzed. Also, in agreement with the above, the ectopic expression of CTNNBIP1 inhibits the migration of lung cancer cells, whereas the CTNNBIP1 knockdown increases cancer cell migration. Our findings suggest that CTNNBIP1 is a suppressor of cancer migration, thus making it a potential prognostic predictor for lung cancer.
- Published
- 2019
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15. Phenotype-Based Screens with Conformation-Specific Inhibitors Reveal p38 Gamma and Delta as Targets for HCC Polypharmacology.
- Author
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Yu JX, Craig AJ, Duffy ME, Villacorta-Martin C, Miguela V, Ruiz de Galarreta M, Scopton AP, Silber L, Maldonado AY, Rialdi A, Guccione E, Lujambio A, Villanueva A, and Dar AC
- Subjects
- Animals, Carcinoma, Hepatocellular pathology, Cell Line, Tumor, Drug Screening Assays, Antitumor methods, Female, Heterocyclic Compounds, 4 or More Rings chemistry, Heterocyclic Compounds, 4 or More Rings pharmacokinetics, Humans, Kaplan-Meier Estimate, Liver Neoplasms pathology, Male, Mice, Inbred C57BL, Mice, Nude, Mitogen-Activated Protein Kinase 12 chemistry, Mitogen-Activated Protein Kinase 13 chemistry, Phenotype, Polypharmacology, Carcinoma, Hepatocellular drug therapy, Heterocyclic Compounds, 4 or More Rings pharmacology, Liver Neoplasms drug therapy, Mitogen-Activated Protein Kinase 12 antagonists & inhibitors, Mitogen-Activated Protein Kinase 13 antagonists & inhibitors, Xenograft Model Antitumor Assays methods
- Abstract
The approved kinase inhibitors for hepatocellular carcinoma (HCC) are not matched to specific mutations within tumors. This has presented a daunting challenge; without a clear target or mechanism, no straightforward path has existed to guide the development of improved therapies for HCC. Here, we combine phenotypic screens with a class of conformation-specific kinase inhibitors termed type II to identify a multikinase inhibitor, AD80, with antitumoral activity across a variety of HCC preclinical models, including mouse xenografts. Mass spectrometry profiling found a number of kinases as putative targets for AD80, including several receptor and cytoplasmic protein kinases. Among these, we found p38 gamma and delta as direct targets of AD80. Notably, a closely related analog of AD80 lacking p38δ/γ activity, but retaining several other off-target kinases, lost significant activity in several HCC models. Moreover, forced and sustained MKK6 → p38→ATF2 signaling led to a significant reduction of AD80 activity within HCC cell lines. Together with HCC survival data in The Cancer Genome Atlas and RNA-seq analysis, we suggest p38 delta and gamma as therapeutic targets in HCC and an "AD80 inhibition signature" as identifying those patients with best clinical outcomes., (©2019 American Association for Cancer Research.)
- Published
- 2019
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16. Integrated computational and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network.
- Author
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Ung PMU, Sonoshita M, Scopton AP, Dar AC, Cagan RL, and Schlessinger A
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- Animals, Computational Biology methods, Drosophila, Models, Biological, Neoplasms, Experimental enzymology, Neoplasms, Experimental metabolism, Antineoplastic Agents pharmacology, Carcinoma, Neuroendocrine enzymology, Carcinoma, Neuroendocrine metabolism, Drug Evaluation, Preclinical methods, Protein Kinase Inhibitors pharmacology, Protein Kinases drug effects, Protein Kinases metabolism, Thyroid Neoplasms enzymology, Thyroid Neoplasms metabolism
- Abstract
Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2019
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17. A whole-animal platform to advance a clinical kinase inhibitor into new disease space.
- Author
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Sonoshita M, Scopton AP, Ung PMU, Murray MA, Silber L, Maldonado AY, Real A, Schlessinger A, Cagan RL, and Dar AC
- Subjects
- Animals, Animals, Genetically Modified, Cell Line, Tumor, Cell Movement, Disease Models, Animal, Drug Design, Female, HCT116 Cells, Humans, Male, Mice, Mice, Inbred ICR, Molecular Docking Simulation, Neoplasm Transplantation, Protein Isoforms, Proto-Oncogene Proteins c-raf metabolism, Signal Transduction, Sorafenib pharmacology, Carcinoma metabolism, Carcinoma, Neuroendocrine metabolism, Drosophila metabolism, Protein Kinase Inhibitors pharmacology, Thyroid Neoplasms metabolism
- Abstract
Synthetic tailoring of approved drugs for new indications is often difficult, as the most appropriate targets may not be readily apparent, and therefore few roadmaps exist to guide chemistry. Here, we report a multidisciplinary approach for accessing novel target and chemical space starting from an FDA-approved kinase inhibitor. By combining chemical and genetic modifier screening with computational modeling, we identify distinct kinases that strongly enhance ('pro-targets') or limit ('anti-targets') whole-animal activity of the clinical kinase inhibitor sorafenib in a Drosophila medullary thyroid carcinoma (MTC) model. We demonstrate that RAF-the original intended sorafenib target-and MKNK kinases function as pharmacological liabilities because of inhibitor-induced transactivation and negative feedback, respectively. Through progressive synthetic refinement, we report a new class of 'tumor calibrated inhibitors' with unique polypharmacology and strongly improved therapeutic index in fly and human MTC xenograft models. This platform provides a rational approach to creating new high-efficacy and low-toxicity drugs.
- Published
- 2018
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18. CNS Anticancer Drug Discovery and Development: 2016 conference insights.
- Author
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Levin VA, Abrey LE, Heffron TP, Tonge PJ, Dar AC, Weiss WA, and Gallo JM
- Abstract
CNS Anticancer Drug Discovery and Development, 16-17 November 2016, Scottsdale, AZ, USA The 2016 second CNS Anticancer Drug Discovery and Development Conference addressed diverse viewpoints about why new drug discovery/development focused on CNS cancers has been sorely lacking. Despite more than 70,000 individuals in the USA being diagnosed with a primary brain malignancy and 151,669-286,486 suffering from metastatic CNS cancer, in 1999, temozolomide was the last drug approved by the US FDA as an anticancer agent for high-grade gliomas. Among the topics discussed were economic factors and pharmaceutical risk assessments, regulatory constraints and perceptions and the need for improved imaging surrogates of drug activity. Included were modeling tumor growth and drug effects in a medical environment in which direct tumor sampling for biological effects can be problematic, potential new drugs under investigation and targets for drug discovery and development. The long trajectory and diverse impediments to novel drug discovery, and expectation that more than one drug will be needed to adequately inhibit critical intracellular tumor pathways were viewed as major disincentives for most pharmaceutical/biotechnology companies. While there were a few unanimities, one consensus is the need for continued and focused discussion among academic and industry scientists and clinicians to address tumor targets, new drug chemistry, and more time- and cost-efficient clinical trials based on surrogate end points.
- Published
- 2017
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19. Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.
- Author
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Dhawan NS, Scopton AP, and Dar AC
- Subjects
- Alleles, Allosteric Regulation drug effects, Cell Line, Enzyme Stability drug effects, Humans, Mitogen-Activated Protein Kinase Kinases antagonists & inhibitors, Mitogen-Activated Protein Kinase Kinases chemistry, Mitogen-Activated Protein Kinase Kinases metabolism, Models, Molecular, Mutation, Neoplasms drug therapy, Neoplasms enzymology, Neoplasms genetics, Neoplasms metabolism, Oncogenes genetics, Phosphorylation drug effects, Protein Binding, Protein Conformation drug effects, Protein Multimerization drug effects, Protein Serine-Threonine Kinases genetics, Pyridones pharmacology, Pyrimidinones pharmacology, raf Kinases chemistry, raf Kinases metabolism, ras Proteins genetics, ras Proteins metabolism, MAP Kinase Signaling System drug effects, Oncogenes drug effects, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism, Quinazolines pharmacology, ras Proteins antagonists & inhibitors
- Abstract
Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.
- Published
- 2016
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20. CNS Anticancer Drug Discovery and Development Conference White Paper.
- Author
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Levin VA, Tonge PJ, Gallo JM, Birtwistle MR, Dar AC, Iavarone A, Paddison PJ, Heffron TP, Elmquist WF, Lachowicz JE, Johnson TW, White FM, Sul J, Smith QR, Shen W, Sarkaria JN, Samala R, Wen PY, Berry DA, and Petter RC
- Subjects
- Animals, Clinical Trials as Topic, Disease Models, Animal, Disease-Free Survival, Endpoint Determination, Humans, Treatment Outcome, Antineoplastic Agents therapeutic use, Central Nervous System Neoplasms drug therapy, Drug Discovery, Glioma drug therapy, Medulloblastoma drug therapy
- Abstract
Following the first CNS Anticancer Drug Discovery and Development Conference, the speakers from the first 4 sessions and organizers of the conference created this White Paper hoping to stimulate more and better CNS anticancer drug discovery and development. The first part of the White Paper reviews, comments, and, in some cases, expands on the 4 session areas critical to new drug development: pharmacological challenges, recent drug approaches, drug targets and discovery, and clinical paths. Following this concise review of the science and clinical aspects of new CNS anticancer drug discovery and development, we discuss, under the rubric "Accelerating Drug Discovery and Development for Brain Tumors," further reasons why the pharmaceutical industry and academia have failed to develop new anticancer drugs for CNS malignancies and what it will take to change the current status quo and develop the drugs so desperately needed by our patients with malignant CNS tumors. While this White Paper is not a formal roadmap to that end, it should be an educational guide to clinicians and scientists to help move a stagnant field forward., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2015
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21. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
- Author
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Mendez AS, Alfaro J, Morales-Soto MA, Dar AC, McCullagh E, Gotthardt K, Li H, Acosta-Alvear D, Sidrauski C, Korennykh AV, Bernales S, Shokat KM, and Walter P
- Subjects
- Adenosine Triphosphate analogs & derivatives, Adenosine Triphosphate chemical synthesis, Animals, Biological Assay, Cell Line, Cell Survival drug effects, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Endoplasmic Reticulum Stress, Endoribonucleases genetics, Endoribonucleases metabolism, Enzyme Activation, Escherichia coli genetics, Escherichia coli metabolism, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts enzymology, Gene Expression, Genes, Reporter, HEK293 Cells, Humans, Mice, Molecular Mimicry, Protein Kinase Inhibitors chemical synthesis, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Regulatory Factor X Transcription Factors, Sulfur Radioisotopes, Transcription Factors genetics, Transcription Factors metabolism, Unfolded Protein Response genetics, eIF-2 Kinase genetics, eIF-2 Kinase metabolism, Adenosine Triphosphate pharmacology, Endoribonucleases antagonists & inhibitors, Protein Kinase Inhibitors pharmacology, Protein Serine-Threonine Kinases antagonists & inhibitors, Unfolded Protein Response drug effects, eIF-2 Kinase antagonists & inhibitors
- Abstract
Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors.
- Published
- 2015
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22. Hepatitis C virus genetics affects miR-122 requirements and response to miR-122 inhibitors.
- Author
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Israelow B, Mullokandov G, Agudo J, Sourisseau M, Bashir A, Maldonado AY, Dar AC, Brown BD, and Evans MJ
- Subjects
- Base Sequence, Hepacivirus physiology, Hepatitis C genetics, Hepatitis C virology, Humans, Liver metabolism, Liver virology, MicroRNAs genetics, Molecular Sequence Data, Virus Replication, Hepacivirus genetics, Hepatitis C metabolism, MicroRNAs metabolism, Point Mutation
- Abstract
Hepatitis C virus (HCV) replication is dependent on a liver-specific microRNA (miRNA), miR-122. A recent clinical trial reported that transient inhibition of miR-122 reduced viral titres in HCV-infected patients. Here we set out to better understand how miR-122 inhibition influences HCV replication over time. Unexpectedly, we observed the emergence of an HCV variant that is resistant to miR-122 knockdown. Next-generation sequencing revealed that this was due to a single nucleotide change at position 28 (G28A) of the HCV genome, which falls between the two miR-122 seed-binding sites. Naturally occurring HCV isolates encoding G28A are similarly resistant to miR-122 inhibition, indicating that subtle differences in viral sequence, even outside the seed-binding site, greatly influence HCV's miR-122 concentration requirement. In addition, we found that HCV itself reduces miR-122's activity in the cell, possibly through binding and sequestering miR-122. Our study provides insight into the interaction between miR-122 and HCV, including viral adaptation to reduced miR-122 bioavailability, and has implications for the development of anti-miR-122-based HCV drugs.
- Published
- 2014
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23. Structure-guided inhibitor design expands the scope of analog-sensitive kinase technology.
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Zhang C, Lopez MS, Dar AC, Ladow E, Finkbeiner S, Yun CH, Eck MJ, and Shokat KM
- Subjects
- Crystallography, X-Ray, Humans, Models, Molecular, Protein Kinases chemistry, Protein Kinases metabolism, Drug Design, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology
- Abstract
Engineered analog-sensitive (AS) protein kinases have emerged as powerful tools for dissecting phospho-signaling pathways, for elucidating the cellular function of individual kinases, and for deciphering unanticipated effects of clinical therapeutics. A crucial and necessary feature of this technology is a bioorthogonal small molecule that is innocuous toward native cellular systems but potently inhibits the engineered kinase. In order to generalize this method, we sought a molecule capable of targeting divergent AS-kinases. Here we employ X-ray crystallography and medicinal chemistry to unravel the mechanism of current inhibitors and use these insights to design the most potent, selective, and general AS-kinase inhibitors reported to date. We use large-scale kinase inhibitor profiling to characterize the selectivity of these molecules as well as examine the consequences of potential off-target effects in chemical genetic experiments. The molecules reported here will serve as powerful tools in efforts to extend AS-kinase technology to the entire kinome and the principles discovered may help in the design of other engineered enzyme/ligand pairs.
- Published
- 2013
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- View/download PDF
24. A pickup in pseudokinase activity.
- Author
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Dar AC
- Subjects
- Biocatalysis, Catalytic Domain, Models, Molecular, Phosphorylation, Protein Conformation, Protein Kinases chemistry, Protein Kinases metabolism
- Abstract
Kinases catalyse the phosphorylation of target substrates on hydroxy group-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Through this biochemical activity, kinases play critical roles in many signal transduction and disease pathways. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive on the basis of mutations of key conserved active-site residues. However, recent biochemical and structural analyses have revealed several enzymatically active pseudokinases, suggesting either that novel mechanisms of phosphorylation are at play or that the constraints for highly conserved active-site residues are looser than originally anticipated. The purpose of the present review is to summarize several of the active pseudokinases, and one in particular termed KSR (kinase suppressor of Ras), which was recently found to possess a kinase activity that can become accelerated through an allosteric mechanism. Utilization of catalytic activity or structural features of the kinase fold may be key to the function of many pseudokinases.
- Published
- 2013
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- View/download PDF
25. Chemical genetic discovery of targets and anti-targets for cancer polypharmacology.
- Author
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Dar AC, Das TK, Shokat KM, and Cagan RL
- Subjects
- Animals, Benzenesulfonates pharmacology, Cell Transformation, Neoplastic drug effects, Cell Transformation, Neoplastic pathology, Disease Models, Animal, Drosophila Proteins antagonists & inhibitors, Drosophila Proteins genetics, Drosophila Proteins metabolism, Drosophila melanogaster drug effects, Drosophila melanogaster genetics, Drug Evaluation, Preclinical, Drug-Related Side Effects and Adverse Reactions, Extracellular Signal-Regulated MAP Kinases antagonists & inhibitors, Extracellular Signal-Regulated MAP Kinases metabolism, Heterocyclic Compounds, 4 or More Rings adverse effects, Heterocyclic Compounds, 4 or More Rings chemistry, Heterocyclic Compounds, 4 or More Rings pharmacology, Heterocyclic Compounds, 4 or More Rings therapeutic use, Humans, Male, Multiple Endocrine Neoplasia Type 2b enzymology, Niacinamide analogs & derivatives, Phenylurea Compounds, Protein Kinase Inhibitors adverse effects, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors pharmacology, Protein Kinase Inhibitors therapeutic use, Proto-Oncogene Proteins c-ret genetics, Proto-Oncogene Proteins c-ret metabolism, Pyridines pharmacology, Receptor Protein-Tyrosine Kinases antagonists & inhibitors, Signal Transduction drug effects, Sorafenib, Survival Rate, Xenograft Model Antitumor Assays, src-Family Kinases antagonists & inhibitors, src-Family Kinases metabolism, Molecular Targeted Therapy, Multiple Endocrine Neoplasia Type 2b drug therapy, Multiple Endocrine Neoplasia Type 2b genetics, Polypharmacy
- Abstract
The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the 'anti-target' Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.
- Published
- 2012
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26. A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK.
- Author
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Brennan DF, Dar AC, Hertz NT, Chao WC, Burlingame AL, Shokat KM, and Barford D
- Subjects
- Adenosine Triphosphate metabolism, Allosteric Regulation physiology, Animals, Biocatalysis, Catalytic Domain, Crystallography, X-Ray, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases metabolism, Humans, Models, Molecular, Phosphorylation, Protein Multimerization, Protein Structure, Quaternary, Proto-Oncogene Proteins B-raf chemistry, Proto-Oncogene Proteins B-raf genetics, Rabbits, Signal Transduction, MAP Kinase Kinase 1 chemistry, MAP Kinase Kinase 1 metabolism, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism, Proto-Oncogene Proteins B-raf metabolism
- Abstract
In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.
- Published
- 2011
- Full Text
- View/download PDF
27. The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling.
- Author
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Dar AC and Shokat KM
- Subjects
- Adenosine Triphosphate metabolism, Amino Acid Sequence, Animals, Binding Sites, Computational Biology, Drug Discovery, Humans, Models, Molecular, Molecular Sequence Data, Molecular Structure, Mutation, Neoplasms enzymology, Neoplasms genetics, Protein Conformation, Protein Kinases genetics, Sequence Alignment, Protein Kinase Inhibitors chemistry, Protein Kinase Inhibitors metabolism, Protein Kinase Inhibitors pharmacology, Protein Kinases chemistry, Protein Kinases metabolism, Signal Transduction drug effects
- Abstract
Kinases are highly regulated enzymes with diverse mechanisms controlling their catalytic output. Over time, chemical discovery efforts for kinases have produced ATP-competitive compounds, allosteric regulators, irreversible binders, and highly specific inhibitors. These distinct classes of small molecules have revealed many novel aspects about kinase-mediated signaling, and some have progressed from simple tool compounds into clinically validated therapeutics. This review explores several small-molecule inhibitors for kinases highlighting elaborate mechanisms by which kinase function is modulated. A complete surprise of targeted kinase drug discovery has been the finding of ATP-competitive inhibitors that behave as agonists, rather than antagonists, of their direct kinase target. These studies hint at a connection between ATP-binding site occupancy and networks of communication that are independent of kinase catalysis. Indeed, kinase inhibitors that induce changes in protein localization, protein-protein interactions, and even enhancement of catalytic activity of the target kinase have been found. The relevance of these findings to the therapeutic efficacy of kinase inhibitors and to the future identification of new classes of drug targets is discussed.
- Published
- 2011
- Full Text
- View/download PDF
28. Chemical genetic approach for kinase-substrate mapping by covalent capture of thiophosphopeptides and analysis by mass spectrometry.
- Author
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Hertz NT, Wang BT, Allen JJ, Zhang C, Dar AC, Burlingame AL, and Shokat KM
- Abstract
Mapping kinase-substrate interactions demands robust methods to rapidly and unequivocally identify substrates from complex protein mixtures. Toward this goal, we present a method in which a kinase, engineered to utilize synthetic ATPγS analogs, specifically thiophosphorylates its substrates in a complex lysate. The thiophosphate label provides a bio-orthogonal tag that can be used to affinity purify and identify labeled proteins. Following the labeling reaction, proteins are digested with trypsin; thiol-containing peptides are then covalently captured and non-thiol-containing peptides are washed from the resin. Oxidation-promoted hydrolysis, at sites of thiophosphorylation, releases phosphopeptides for analysis by tandem mass spectrometry. By incorporating two specificity gates-kinase engineering and peptide affinity purification-this method yields high-confidence substrate identifications. This method gives both the identity of the substrates and phosphorylation-site localization. With this information, investigators can analyze the biological significance of the phosphorylation mark immediately following confirmation of the kinase-substrate relationship. Here, we provide an optimized version of this technique to further enable widespread utilization of this technology. Curr. Protoc. Chem Biol. 2:15-36. © 2010 by John Wiley & Sons, Inc.
- Published
- 2010
- Full Text
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29. Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.
- Author
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Seo EJ, Liu F, Kawagishi-Kobayashi M, Ung TL, Cao C, Dar AC, Sicheri F, and Dever TE
- Subjects
- Binding Sites, Eukaryotic Initiation Factor-2 chemistry, Eukaryotic Initiation Factor-2 metabolism, Models, Molecular, Mutation, Phosphorylation, Poxviridae metabolism, Protein Conformation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Substrate Specificity, Viral Proteins chemistry, eIF-2 Kinase chemistry, eIF-2 Kinase genetics, Viral Proteins metabolism, eIF-2 Kinase antagonists & inhibitors
- Abstract
As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.
- Published
- 2008
- Full Text
- View/download PDF
30. Small molecule recognition of c-Src via the Imatinib-binding conformation.
- Author
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Dar AC, Lopez MS, and Shokat KM
- Subjects
- Benzamides, Crystallography, X-Ray, Imatinib Mesylate, Models, Molecular, Molecular Structure, Protein Binding, Proto-Oncogene Proteins pp60(c-src) chemistry, Proto-Oncogene Proteins pp60(c-src) genetics, Pharmaceutical Preparations chemistry, Piperazines chemistry, Proto-Oncogene Proteins pp60(c-src) metabolism, Pyrimidines chemistry
- Abstract
The cancer drug, Imatinib, is a selective Abl kinase inhibitor that does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last 15 years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation, effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the DFG-out conformation of c-Src with high affinity. We report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavorable DFG-out conformation in c-Src. Structural comparison between c-Src in complex with these inhibitors allows us to speculate on the differential selectivity of Imatinib for c-Src and Abl.
- Published
- 2008
- Full Text
- View/download PDF
31. Mechanistic link between PKR dimerization, autophosphorylation, and eIF2alpha substrate recognition.
- Author
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Dey M, Cao C, Dar AC, Tamura T, Ozato K, Sicheri F, and Dever TE
- Subjects
- Catalysis, Dimerization, Eukaryotic Initiation Factor-2 chemistry, Eukaryotic Initiation Factor-2 genetics, Mutagenesis, Site-Directed, Phosphorylation, Protein Binding, Protein Conformation, Protein Structure, Secondary, RNA, Double-Stranded chemistry, RNA, Double-Stranded metabolism, Saccharomyces cerevisiae metabolism, eIF-2 Kinase chemistry, eIF-2 Kinase genetics, Eukaryotic Initiation Factor-2 metabolism, eIF-2 Kinase metabolism
- Abstract
The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.
- Published
- 2005
- Full Text
- View/download PDF
32. Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR.
- Author
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Dar AC, Dever TE, and Sicheri F
- Subjects
- Animals, Crystallography, X-Ray, Dimerization, Eukaryotic Initiation Factor-2 chemistry, Humans, Mice, Models, Molecular, Molecular Sequence Data, Phosphorylation, Protein Binding, Protein Conformation, RNA, Double-Stranded chemistry, RNA, Double-Stranded metabolism, Saccharomyces cerevisiae chemistry, Sequence Homology, Amino Acid, eIF-2 Kinase chemistry, Eukaryotic Initiation Factor-2 metabolism, Protein Structure, Secondary, eIF-2 Kinase metabolism
- Abstract
In response to binding viral double-stranded RNA byproducts within a cell, the RNA-dependent protein kinase PKR phosphorylates the alpha subunit of the translation initiation factor eIF2 on a regulatory site, Ser51. This triggers the general shutdown of protein synthesis and inhibition of viral propagation. To understand the basis for substrate recognition by and the regulation of PKR, we determined X-ray crystal structures of the catalytic domain of PKR in complex with eIF2alpha. The structures reveal that eIF2alpha binds to the C-terminal catalytic lobe while catalytic-domain dimerization is mediated by the N-terminal lobe. In addition to inducing a local unfolding of the Ser51 acceptor site in eIF2alpha, its mode of binding to PKR affords the Ser51 site full access to the catalytic cleft of PKR. The generality and implications of the structural mechanisms uncovered for PKR to the larger family of four human eIF2alpha protein kinases are discussed.
- Published
- 2005
- Full Text
- View/download PDF
33. PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha.
- Author
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Dey M, Trieselmann B, Locke EG, Lu J, Cao C, Dar AC, Krishnamoorthy T, Dong J, Sicheri F, and Dever TE
- Subjects
- Amino Acid Sequence, Amino Acid Substitution, Conserved Sequence, DNA Mutational Analysis, Eukaryotic Initiation Factor-2 chemistry, Eukaryotic Initiation Factor-2 metabolism, Eukaryotic Initiation Factor-2B genetics, Eukaryotic Initiation Factor-2B physiology, Molecular Sequence Data, Mutation genetics, Peptide Chain Initiation, Translational physiology, Phosphorylation, Protein Conformation, Protein Kinases genetics, Protein Kinases metabolism, Protein Serine-Threonine Kinases, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Serine genetics, Serine metabolism, eIF-2 Kinase genetics, eIF-2 Kinase metabolism, Eukaryotic Initiation Factor-2 genetics, Eukaryotic Initiation Factor-2B metabolism, Peptide Chain Initiation, Translational genetics, Protein Kinases physiology, Saccharomyces cerevisiae physiology, Saccharomyces cerevisiae Proteins physiology, eIF-2 Kinase physiology
- Abstract
Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.
- Published
- 2005
- Full Text
- View/download PDF
34. Formin leaky cap allows elongation in the presence of tight capping proteins.
- Author
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Zigmond SH, Evangelista M, Boone C, Yang C, Dar AC, Sicheri F, Forkey J, and Pring M
- Subjects
- Chromatography, Chromatography, Gel, Dimerization, Saccharomyces cerevisiae Proteins metabolism, Actin Cytoskeleton metabolism, Microfilament Proteins genetics, Microfilament Proteins metabolism, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae Proteins genetics
- Abstract
Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.
- Published
- 2003
- Full Text
- View/download PDF
35. Slipped-strand DNAs formed by long (CAG)*(CTG) repeats: slipped-out repeats and slip-out junctions.
- Author
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Pearson CE, Tam M, Wang YH, Montgomery SE, Dar AC, Cleary JD, and Nichol K
- Subjects
- DNA metabolism, DNA, Single-Stranded metabolism, DNA-Binding Proteins metabolism, Deoxyribonuclease I metabolism, Deoxyribonucleases, Type II Site-Specific metabolism, Humans, Nucleic Acid Conformation, Nucleic Acid Heteroduplexes chemistry, Nucleic Acid Heteroduplexes metabolism, Nucleic Acid Heteroduplexes ultrastructure, Single-Strand Specific DNA and RNA Endonucleases metabolism, DNA chemistry, Repetitive Sequences, Nucleic Acid
- Abstract
The disease-associated expansion of (CTG)*(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)*(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson-Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients-suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors.
- Published
- 2002
- Full Text
- View/download PDF
36. X-ray crystal structure and functional analysis of vaccinia virus K3L reveals molecular determinants for PKR subversion and substrate recognition.
- Author
-
Dar AC and Sicheri F
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Binding Sites, Crystallography, X-Ray, Dimerization, Fluorescence Polarization, Humans, Kinetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Binding, Protein Conformation, Sequence Homology, Amino Acid, Structure-Activity Relationship, Substrate Specificity, Viral Proteins genetics, eIF-2 Kinase metabolism, Vaccinia virus chemistry, Viral Proteins chemistry, Viral Proteins metabolism, eIF-2 Kinase antagonists & inhibitors
- Abstract
The vaccinia virus protein K3L subverts the mammalian antiviral defense mechanism by inhibiting the RNA-dependent protein kinase PKR. K3L is a structural mimic of PKR's natural substrate, the translation initiation factor eIF2alpha. To further our understanding of K3L inhibitory function and PKR substrate recognition, we have solved the 1.8 A X-ray crystal structure of K3L. The structure consists of a five-strand beta barrel with an intervening helix insert region similar in topology to the functionally divergent S1 domain. Mutational analysis identifies two proximal regions of the K3L structure as possessing specialized PKR binding and inhibitory function. Further analysis reveals that PKR dimerization composes a key switch that regulates both its catalytic activation and its molecular recognition of K3L and eIF2alpha.
- Published
- 2002
- Full Text
- View/download PDF
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