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1.
Nature ; 629(8011): 435-442, 2024 May.
Article En | MEDLINE | ID: mdl-38658751

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.


Allosteric Regulation , Drug Discovery , Enzyme Inhibitors , Proteomics , Werner Syndrome Helicase , Animals , Female , Humans , Male , Mice , Allosteric Regulation/drug effects , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Cysteine/drug effects , Cysteine/metabolism , DNA Breaks, Double-Stranded/drug effects , Drug Discovery/methods , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Microsatellite Instability , Models, Molecular , Werner Syndrome Helicase/antagonists & inhibitors , Werner Syndrome Helicase/chemistry , Werner Syndrome Helicase/metabolism , Xenograft Model Antitumor Assays , Cell Death/drug effects , Adenosine Triphosphate/metabolism
3.
Nat Chem Biol ; 18(12): 1388-1398, 2022 12.
Article En | MEDLINE | ID: mdl-36097295

The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.


Cysteine , Proteomics , Signal Transduction , Cytokines , Protein Isoforms
4.
RSC Chem Biol ; 2(2): 354-367, 2021 Apr 01.
Article En | MEDLINE | ID: mdl-34458789

Targeted covalent inhibitors have regained widespread attention in drug discovery and have emerged as powerful tools for basic biomedical research. Fueled by considerable improvements in mass spectrometry sensitivity and sample processing, chemoproteomic strategies have revealed thousands of proteins that can be covalently modified by reactive small molecules. Fragment-based drug discovery, which has traditionally been used in a target-centric fashion, is now being deployed on a proteome-wide scale thereby expanding its utility to both the discovery of novel covalent ligands and their cognate protein targets. This powerful approach is allowing 'high-throughput' serendipitous discovery of cryptic pockets leading to the identification of pharmacological modulators of proteins previously viewed as "undruggable". The reactive fragment toolkit has been enabled by recent advances in the development of new chemistries that target residues other than cysteine including lysine and tyrosine. Here, we review the emerging area of covalent fragment-based ligand discovery, which integrates the benefits of covalent targeting and fragment-based medicinal chemistry. We discuss how the two strategies synergize to facilitate the efficient discovery of new pharmacological modulators of established and new therapeutic target proteins.

5.
RSC Chem Biol ; 2(2): 670-671, 2021 Apr 01.
Article En | MEDLINE | ID: mdl-34459829

[This corrects the article DOI: 10.1039/D0CB00222D.].

6.
Cell ; 172(3): 578-589.e17, 2018 01 25.
Article En | MEDLINE | ID: mdl-29373830

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.


Antineoplastic Agents/pharmacology , Neoplasms, Experimental/drug therapy , Piperazines/pharmacology , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Quinazolines/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Proliferation/drug effects , Cells, Cultured , Female , HCT116 Cells , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Docking Simulation , Mutation , Piperazines/chemistry , Piperazines/therapeutic use , Protein Binding , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Quinazolines/chemistry , Quinazolines/therapeutic use
8.
Blood ; 128(2): 239-48, 2016 07 14.
Article En | MEDLINE | ID: mdl-27151888

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, yet 40% to 50% of patients will eventually succumb to their disease, demonstrating a pressing need for novel therapeutic options. Gene expression profiling has identified messenger RNAs that lead to transformation, but critical events transforming cells are normally executed by kinases. Therefore, we hypothesized that previously unrecognized kinases may contribute to DLBCL pathogenesis. We performed the first comprehensive analysis of global kinase activity in DLBCL, to identify novel therapeutic targets, and discovered that germinal center kinase (GCK) was extensively activated. GCK RNA interference and small molecule inhibition induced cell-cycle arrest and apoptosis in DLBCL cell lines and primary tumors in vitro and decreased the tumor growth rate in vivo, resulting in a significantly extended lifespan of mice bearing DLBCL xenografts. GCK expression was also linked to adverse clinical outcome in a cohort of 151 primary DLBCL patients. These studies demonstrate, for the first time, that GCK is a molecular therapeutic target in DLBCL tumors and that inhibiting GCK may significantly extend DLBCL patient survival. Because the majority of DLBCL tumors (∼80%) exhibit activation of GCK, this therapy may be applicable to most patients.


Apoptosis , Cell Cycle Checkpoints , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Lymphoma, Large B-Cell, Diffuse/ethnology , Neoplasm Proteins/biosynthesis , Protein Serine-Threonine Kinases/biosynthesis , Animals , Cell Line, Tumor , Germinal Center Kinases , Heterografts , Humans , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Mice , Neoplasm Transplantation
9.
PLoS One ; 11(3): e0152934, 2016.
Article En | MEDLINE | ID: mdl-27031502

We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.


Adenocarcinoma/genetics , Casein Kinase Ialpha/genetics , Colonic Neoplasms/genetics , Mutation, Missense , Adenocarcinoma/pathology , Base Sequence , Casein Kinase Ialpha/chemistry , Catalytic Domain , Colon/pathology , Colonic Neoplasms/pathology , HEK293 Cells , Humans , Molecular Sequence Data , Proteomics/methods
10.
Cancer Discov ; 6(3): 316-29, 2016 Mar.
Article En | MEDLINE | ID: mdl-26739882

UNLABELLED: KRAS gain-of-function mutations occur in approximately 30% of all human cancers. Despite more than 30 years of KRAS-focused research and development efforts, no targeted therapy has been discovered for cancers with KRAS mutations. Here, we describe ARS-853, a selective, covalent inhibitor of KRAS(G12C) that inhibits mutant KRAS-driven signaling by binding to the GDP-bound oncoprotein and preventing activation. Based on the rates of engagement and inhibition observed for ARS-853, along with a mutant-specific mass spectrometry-based assay for assessing KRAS activation status, we show that the nucleotide state of KRAS(G12C) is in a state of dynamic flux that can be modulated by upstream signaling factors. These studies provide convincing evidence that the KRAS(G12C) mutation generates a "hyperexcitable" rather than a "statically active" state and that targeting the inactive, GDP-bound form is a promising approach for generating novel anti-RAS therapeutics. SIGNIFICANCE: A cell-active, mutant-specific, covalent inhibitor of KRAS(G12C) is described that targets the GDP-bound, inactive state and prevents subsequent activation. Using this novel compound, we demonstrate that KRAS(G12C) oncoprotein rapidly cycles bound nucleotide and responds to upstream signaling inputs to maintain a highly active state.


Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , ras Proteins/antagonists & inhibitors , ras Proteins/chemistry , Biomarkers , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Ligands , Models, Biological , Models, Molecular , Molecular Conformation , Recombinant Proteins , Signal Transduction/drug effects , Structure-Activity Relationship , ras Proteins/genetics , ras Proteins/metabolism
11.
Blood ; 125(20): 3133-43, 2015 May 14.
Article En | MEDLINE | ID: mdl-25833960

Oncogenic forms of NRAS are frequently associated with hematologic malignancies and other cancers, making them important therapeutic targets. Inhibition of individual downstream effector molecules (eg, RAF kinase) have been complicated by the rapid development of resistance or activation of bypass pathways. For the purpose of identifying novel targets in NRAS-transformed cells, we performed a chemical screen using mutant NRAS transformed Ba/F3 cells to identify compounds with selective cytotoxicity. One of the compounds identified, GNF-7, potently and selectively inhibited NRAS-dependent cells in preclinical models of acute myelogenous leukemia and acute lymphoblastic leukemia. Mechanistic analysis revealed that its effects were mediated in part through combined inhibition of ACK1/AKT and of mitogen-activated protein kinase kinase kinase kinase 2 (germinal center kinase). Similar to genetic synthetic lethal approaches, these results suggest that small molecule screens can be used to identity novel therapeutic targets in cells addicted to RAS oncogenes.


GTP Phosphohydrolases/genetics , Leukemia/genetics , Membrane Proteins/genetics , Mutation , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Disease Models, Animal , Drug Screening Assays, Antitumor , GTP Phosphohydrolases/metabolism , Germinal Center Kinases , Humans , Leukemia/drug therapy , Leukemia/metabolism , Leukemia/mortality , Leukemia/pathology , Membrane Proteins/metabolism , Mice , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidinones/pharmacology , Signal Transduction/drug effects , Small Molecule Libraries , Xenograft Model Antitumor Assays
12.
J Biomol Screen ; 19(6): 936-46, 2014 Jul.
Article En | MEDLINE | ID: mdl-24464431

During viral infection of human cells, host kinases mediate signaling activities that are used by all viruses for replication; therefore, targeting of host kinases is of broad therapeutic interest. Here, host kinases were globally screened during human influenza virus (H1N1) infection to determine the time-dependent effects of virus infection and replication on kinase function. Desthiobiotin-labeled analogs of adenosine triphosphate and adenosine diphosphate were used to probe and covalently label host kinases in infected cell lysates, and probe affinity was determined. Using infected human A549 cells, we screened for time-dependent signal changes and identified host kinases whose probe affinities differed significantly when compared to uninfected cells. Our screen identified 10 novel host kinases that have not been previously shown to be involved with influenza virus replication, and we validated the functional importance of these novel kinases during infection using targeted small interfering RNAs (siRNAs). The effects of kinase-targeted siRNA knockdowns on replicating virus levels were measured by quantitative reverse-transcription PCR and cytoprotection assays. We identified several novel host kinases that, when knocked down, enhanced or reduced the viral load in cell culture. This preliminary work represents the first screen of the changing host kinome in influenza virus-infected human cells.


Influenza A Virus, H1N1 Subtype/physiology , Influenza, Human/enzymology , Protein Serine-Threonine Kinases/chemistry , RNA, Small Interfering/genetics , Virus Replication , A549 Cells , Adenosine Diphosphate/chemistry , Adenosine Triphosphate/chemistry , Apoptosis , Biotin/analogs & derivatives , Biotin/chemistry , Cell Survival , Chromatography, Liquid , Drug Discovery , Humans , Mass Spectrometry , NIMA-Related Kinase 1/chemistry , Peptides/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Serine-Threonine Kinase 3 , Viral Load
13.
Mol Cell Proteomics ; 12(9): 2481-96, 2013 Sep.
Article En | MEDLINE | ID: mdl-23722185

Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)(1) probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188.


Adenosine Triphosphate/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Molecular Probes/metabolism , Protein Kinases/metabolism , Acylation , Amino Acid Sequence , Arabidopsis Proteins/chemistry , Binding Sites , Biotin/analogs & derivatives , Biotin/chemistry , Biotin/metabolism , Chaperonin 60/metabolism , Mass Spectrometry , Molecular Sequence Data , Oxidation-Reduction , Peptides/chemistry , Peptides/metabolism , Protein Binding , Proteome/metabolism , Receptors, Cell Surface/metabolism , Staining and Labeling
14.
Cancer Res ; 73(8): 2574-86, 2013 Apr 15.
Article En | MEDLINE | ID: mdl-23436801

mTOR is a highly conserved serine/threonine protein kinase that serves as a central regulator of cell growth, survival, and autophagy. Deregulation of the PI3K/Akt/mTOR signaling pathway occurs commonly in cancer and numerous inhibitors targeting the ATP-binding site of these kinases are currently undergoing clinical evaluation. Here, we report the characterization of Torin2, a second-generation ATP-competitive inhibitor that is potent and selective for mTOR with a superior pharmacokinetic profile to previous inhibitors. Torin2 inhibited mTORC1-dependent T389 phosphorylation on S6K (RPS6KB1) with an EC(50) of 250 pmol/L with approximately 800-fold selectivity for cellular mTOR versus phosphoinositide 3-kinase (PI3K). Torin2 also exhibited potent biochemical and cellular activity against phosphatidylinositol-3 kinase-like kinase (PIKK) family kinases including ATM (EC(50), 28 nmol/L), ATR (EC(50), 35 nmol/L), and DNA-PK (EC(50), 118 nmol/L; PRKDC), the inhibition of which sensitized cells to Irradiation. Similar to the earlier generation compound Torin1 and in contrast to other reported mTOR inhibitors, Torin2 inhibited mTOR kinase and mTORC1 signaling activities in a sustained manner suggestive of a slow dissociation from the kinase. Cancer cell treatment with Torin2 for 24 hours resulted in a prolonged block in negative feedback and consequent T308 phosphorylation on Akt. These effects were associated with strong growth inhibition in vitro. Single-agent treatment with Torin2 in vivo did not yield significant efficacy against KRAS-driven lung tumors, but the combination of Torin2 with mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor AZD6244 yielded a significant growth inhibition. Taken together, our findings establish Torin2 as a strong candidate for clinical evaluation in a broad number of oncologic settings where mTOR signaling has a pathogenic role.


Adenosine Triphosphate/metabolism , Cell Cycle Proteins/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Naphthyridines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tumor Suppressor Proteins/antagonists & inhibitors , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Ataxia Telangiectasia Mutated Proteins , Autophagy/drug effects , Benzimidazoles/pharmacology , Binding, Competitive , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Synergism , Humans , Kinetics , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Naphthyridines/administration & dosage , Naphthyridines/chemistry , Protein Binding , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor Assays , ras Proteins/genetics
15.
ACS Med Chem Lett ; 3(8): 658-662, 2012 Aug 09.
Article En | MEDLINE | ID: mdl-23066449

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) are present in a subset of Parkinson's disease (PD) patients and may represent an attractive therapeutic target. Here we report a 2-anilino-4-methylamino-5-chloropyrimidine, HG-10-102-01(4) is a potent and selective inhibitor of wild-type LRRK2 and the G2019S mutant. Compound 4 substantially inhibits Ser910 and Ser935 phosphorylation of both wild-type LRRK2 and G2019S mutant at a concentration of 0.1-0.3 µM in cells and is the first compound reported to be capable of inhibiting Ser910 and Ser935 phosphorylation in mouse brain following intraperitoneal delivery of doses as low as 50 mg/kg.

16.
ACS Chem Biol ; 7(12): 2019-26, 2012 Dec 21.
Article En | MEDLINE | ID: mdl-22999307

Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5' untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales.


Biomarkers/metabolism , DNA-Activated Protein Kinase/metabolism , Dengue/enzymology , Proteomics , 5' Untranslated Regions , Blotting, Western , Cell Line , Humans , Signal Transduction
17.
Cell ; 150(2): 426-40, 2012 Jul 20.
Article En | MEDLINE | ID: mdl-22817901

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.


Apoptosis , Proteome/analysis , Proteome/metabolism , Caspases/metabolism , Humans , Models, Molecular , Phosphorylation , Proteolysis , Signal Transduction
18.
J Biol Chem ; 287(13): 9742-9752, 2012 Mar 23.
Article En | MEDLINE | ID: mdl-22223645

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 µM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 µM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 µM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.


Enzyme Inhibitors/pharmacokinetics , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Adenosine Triphosphate , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Humans , Kinetics , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Proteins/antagonists & inhibitors , Proteins/metabolism , Proteomics/methods , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism
19.
Chem Biol ; 18(6): 699-710, 2011 Jun 24.
Article En | MEDLINE | ID: mdl-21700206

Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here, we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against >200 kinases in native cell proteomes and reveal biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected on live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.


Protein Kinases/chemistry , Adenosine Triphosphate/chemistry , Cell Line, Tumor , Dasatinib , Humans , MAP Kinase Kinase 5/antagonists & inhibitors , MAP Kinase Kinase 5/metabolism , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Thiazoles/chemistry , Thiazoles/pharmacology , raf Kinases/antagonists & inhibitors , raf Kinases/genetics , raf Kinases/metabolism
20.
Proc Natl Acad Sci U S A ; 108(12): 4834-9, 2011 Mar 22.
Article En | MEDLINE | ID: mdl-21383145

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.


Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/metabolism , Autophagy/drug effects , Endoplasmic Reticulum/enzymology , Enzyme Inhibitors/pharmacology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/metabolism , Quinazolines/pharmacology , Ubiquitin/metabolism , Adenosine Triphosphatases/genetics , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Caspase 3/genetics , Caspase 3/metabolism , Caspase 7/genetics , Caspase 7/metabolism , Cell Line , Endoplasmic Reticulum/genetics , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Mice , Neoplasms/drug therapy , Neoplasms/enzymology , Nuclear Proteins/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Quinazolines/chemical synthesis , Quinazolines/chemistry , Ubiquitin/genetics
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