ABSTRACT
An in-depth multi-omic molecular characterisation of poly(adenosine 5'-diphosphate [ADP]-ribose) polymerase (PARP) inhibitors revealed a distinct poly-pharmacology of niraparib (Zejula®) mediated by its interaction with lanosterol synthase (LSS), which is not observed with other PARP inhibitors. Niraparib, in a similar way to the LSS inhibitor Ro-48-8071, induced activation of the 24,25-epoxysterol shunt pathway, which is a regulatory signalling branch of the cholesterol biosynthesis pathway. Interestingly, the combination of a LSS inhibitor with a PARP inhibitor that does not bind to LSS, such as olaparib, had an additive effect on killing of cancer cells to levels comparable to Niraparib as single agent. In addition, the combination of PARP inhibitors and statins, inhibitors of HMGCR, an enzyme catalysing the rate-limiting step in the mevalonate pathway, had a synergistic effect on tumor cell killing in cell lines and patient-derived ovarian tumor organoids. These observations suggest that concomitant inhibition of cholesterol biosynthesis pathway and PARP activity might result in stronger efficacy of these inhibitors against tumor types highly dependent on cholesterol metabolism.
ABSTRACT
Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.
Subject(s)
Werner Syndrome Helicase , Humans , Werner Syndrome Helicase/genetics , Mice , Animals , Microsatellite Instability , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/pathology , Cell Line, Tumor , Xenograft Model Antitumor Assays , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic useABSTRACT
Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes.
Subject(s)
Poly(ADP-ribose) Polymerase Inhibitors , Proteome , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Multiomics , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proteomics/methodsABSTRACT
DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.
Subject(s)
Azacitidine , Leukemia, Myeloid, Acute , Animals , Azacitidine/pharmacology , DNA/metabolism , DNA Methylation , DNA Modification Methylases/genetics , Decitabine/pharmacology , Leukemia, Myeloid, Acute/drug therapy , MiceABSTRACT
In order to understand the full mechanism of action of candidate drug molecules, it is critical to thoroughly characterize their interactions with endogenously expressed pharmacological targets and potentially undesired off-targets. Here we describe a chemoproteomics approach that is based on functionalized analogs of the compound of interest to affinity enrich target proteins from cell or tissue extracts. Experiments are designed as competition binding assays where free parental compound is spiked at a range of concentrations into the extracts to compete specific binders off the immobilized compound matrix. Quantification of matrix-bound proteins enables generation of dose-response curves and half-binding concentrations. In addition, the influence of the affinity matrix on the equilibrium is determined in rebinding experiments. TMT10 isobaric mass tags enable analyzing repeat binding and dose-dependent competition samples in a single mass spectrometry analysis run, thus enabling the efficient identification of targets, apparent dissociation constants, and selectivity of small molecules in a single experiment. The workflow is exemplified with the kinase inhibitor sunitinib.
Subject(s)
Protein Kinase Inhibitors/metabolism , Proteins/analysis , Proteomics , Sunitinib/metabolism , Tandem Mass Spectrometry , Animals , Binding, Competitive , Female , Humans , Placenta/metabolism , Pregnancy , Protein Binding , Protein Kinase Inhibitors/pharmacology , Research Design , Sunitinib/pharmacologyABSTRACT
The transcriptional coactivator BRD4 has a fundamental role in transcription regulation and thus became a promising epigenetic therapeutic candidate to target diverse pathologies. However, the regulation of BRD4 by posttranslational modifications has been largely unexplored. Here, we show that BRD4 is methylated on chromatin at lysine-99 by the protein lysine methyltransferase SETD6. BRD4 methylation negatively regulates the expression of genes that are involved in translation and inhibits total mRNA translation in cells. Mechanistically, we provide evidence that supports a model where BRD4 methylation by SETD6 does not have a direct role in the association with acetylated histone H4 at chromatin. However, this methylation specifically determines the recruitment of the transcription factor E2F1 to selected target genes that are involved in mRNA translation. Together, our findings reveal a previously unknown molecular mechanism for BRD4 methylation-dependent gene-specific targeting, which may serve as a new direction for the development of therapeutic applications.
Subject(s)
Cell Cycle Proteins , Nuclear Proteins , Protein Methyltransferases , Transcription Factors , Cell Cycle Proteins/genetics , Chromatin , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Methylation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Biosynthesis , Protein Methyltransferases/genetics , Protein Processing, Post-Translational , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Multiplexed quantitative proteomics enabled complex workflows to study the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The isobaric mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity by which such experiments could be performed. Here we evaluate a recent generation of 16-plex isobaric mass tags and demonstrate the sensitive and time efficient identification of Staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over seven concentrations per temperature enabled comprehensive selectivity profiling of Staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.
Subject(s)
Pharmaceutical Preparations , Proteomics , Mass Spectrometry , Protein Stability , ProteomeABSTRACT
Pan-bromodomain and extra terminal (BET) inhibitors interact equipotently with all eight bromodomains of the BET family of proteins. They have shown profound efficacy in vitro and in vivo in oncology and immunomodulatory models, and a number of them are currently in clinical trials where significant safety signals have been reported. It is therefore important to understand the functional contribution of each bromodomain to assess the opportunity to tease apart efficacy and toxicity. This article discloses the in vitro and cellular activity profiles of GSK789, a potent, cell-permeable, and highly selective inhibitor of the first bromodomains of the BET family.
Subject(s)
Naphthyridines/chemistry , Transcription Factors/antagonists & inhibitors , ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , ATPases Associated with Diverse Cellular Activities/metabolism , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Binding Sites , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Half-Life , Humans , Molecular Dynamics Simulation , Naphthyridines/metabolism , Naphthyridines/pharmacology , Protein Domains , Quinolones/chemistry , Quinolones/metabolism , Quinolones/pharmacology , Transcription Factors/metabolismABSTRACT
Non-BET bromodomain-containing proteins have become attractive targets for the development of novel therapeutics targeting epigenetic pathways. To help facilitate the target validation of this class of proteins, structurally diverse small-molecule ligands and methodologies to produce selective inhibitors in a predictable fashion are in high demand. Herein, we report the development and application of atypical acetyl-lysine (KAc) methyl mimetics to take advantage of the differential stability of conserved water molecules in the bromodomain binding site. Discovery of the n-butyl group as an atypical KAc methyl mimetic allowed generation of 31 (GSK6776) as a soluble, permeable, and selective BRD7/9 inhibitor from a pyridazinone template. The n-butyl group was then used to enhance the bromodomain selectivity of an existing BRD9 inhibitor and to transform pan-bromodomain inhibitors into BRD7/9 selective compounds. Finally, a solvent-exposed vector was defined from the pyridazinone template to enable bifunctional molecule synthesis, and affinity enrichment chemoproteomic experiments were used to confirm several of the endogenous protein partners of BRD7 and BRD9, which form part of the chromatin remodeling PBAF and BAF complexes, respectively.
Subject(s)
Chromosomal Proteins, Non-Histone/antagonists & inhibitors , Lysine/chemistry , Pyridazines/chemistry , Transcription Factors/antagonists & inhibitors , Binding Sites , Chromosomal Proteins, Non-Histone/metabolism , Crystallography, X-Ray , Humans , Ligands , Molecular Dynamics Simulation , Protein Structure, Tertiary , Pyridazines/metabolism , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.
Subject(s)
Gene Expression Regulation , Prokaryotic Cells/metabolism , Proteins/chemistry , Proteins/metabolism , Proteome/analysis , Transition Temperature , Animals , Electron Transport Chain Complex Proteins/metabolism , Humans , Mitochondria/metabolism , Protein Stability , Software , Species SpecificityABSTRACT
The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Histone Acetyltransferases/antagonists & inhibitors , Immunologic Factors/pharmacology , Molecular Targeted Therapy , Transcription Factors/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Agents/therapeutic use , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Drug Discovery , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Histone Acetyltransferases/chemistry , Histone Acetyltransferases/genetics , Humans , Immune System Diseases/drug therapy , Immunologic Factors/chemistry , Immunologic Factors/therapeutic use , Inflammation/drug therapy , Neoplasms/drug therapy , Protein Domains/drug effects , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.
Subject(s)
Blood/metabolism , Proteome/chemistry , Proteome/metabolism , Small Molecule Libraries/administration & dosage , Animals , Azepines/administration & dosage , Azepines/pharmacology , Hep G2 Cells , Humans , Kidney/chemistry , Kidney/metabolism , Liver/chemistry , Liver/metabolism , Lung/chemistry , Lung/metabolism , Male , Mass Spectrometry , Mice , Organ Specificity , Panobinostat/administration & dosage , Panobinostat/pharmacology , Protein Stability , Rats , Small Molecule Libraries/pharmacology , Spleen/chemistry , Spleen/metabolism , Testis/chemistry , Testis/metabolism , Thermodynamics , Triazoles/administration & dosage , Triazoles/pharmacology , Vemurafenib/administration & dosage , Vemurafenib/pharmacologyABSTRACT
Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.
Subject(s)
Dasatinib/pharmacology , Janus Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proteome/metabolism , Proteomics/methods , Animals , Chromatography, Affinity/methods , Dogs , HEK293 Cells , Humans , Janus Kinases/metabolism , K562 Cells , Mice , Microspheres , Monocytes/metabolism , Protein Binding , Proteome/chemistry , Sepharose/analogs & derivatives , Substrate SpecificityABSTRACT
Adenosine triphosphate (ATP) plays fundamental roles in cellular biochemistry and was recently discovered to function as a biological hydrotrope. Here, we use mass spectrometry to interrogate ATP-mediated regulation of protein thermal stability and protein solubility on a proteome-wide scale. Thermal proteome profiling reveals high affinity interactions of ATP as a substrate and as an allosteric modulator that has widespread influence on protein complexes and their stability. Further, we develop a strategy for proteome-wide solubility profiling, and discover ATP-dependent solubilization of at least 25% of the insoluble proteome. ATP increases the solubility of positively charged, intrinsically disordered proteins, and their susceptibility for solubilization varies depending on their localization to different membrane-less organelles. Moreover, a few proteins, exhibit an ATP-dependent decrease in solubility, likely reflecting polymer formation. Our data provides a proteome-wide, quantitative insight into how ATP influences protein structure and solubility across the spectrum of physiologically relevant concentrations.
Subject(s)
Adenosine Triphosphate/metabolism , Proteome/metabolism , Adenosine Triphosphate/chemistry , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Jurkat Cells , Mass Spectrometry , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding , Protein Stability , Proteome/chemistry , Proteomics/methods , SolubilityABSTRACT
ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.
Subject(s)
ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Drug Discovery , Nuclear Proteins/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription Factors/antagonists & inhibitors , Cell Cycle Proteins , Humans , Models, Molecular , Molecular Structure , Protein Conformation , Protein Domains , Structure-Activity RelationshipABSTRACT
Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed "multiplexed proteome dynamics profiling" (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.
Subject(s)
HSP90 Heat-Shock Proteins/metabolism , Proteome/analysis , Proteomics/methods , Azepines/chemistry , Azepines/metabolism , Azepines/pharmacology , Cell Line , Chromatography, High Pressure Liquid , Cluster Analysis , Estradiol/pharmacology , Humans , Isotope Labeling , Jurkat Cells , MCF-7 Cells , Neoplasm Proteins/metabolism , Proteins/antagonists & inhibitors , Proteins/metabolism , Proteolysis/drug effects , Receptors, Estrogen/metabolism , Tandem Mass Spectrometry , Triazoles/chemistry , Triazoles/metabolism , Triazoles/pharmacologyABSTRACT
Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to â¼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.
Subject(s)
Lysine/chemistry , Phosphatidylinositol 3-Kinases/chemistry , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Catalytic Domain/drug effects , Cell Line , Class I Phosphatidylinositol 3-Kinases , Drug Discovery , Humans , Lysine/metabolism , Mice , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/metabolism , ProteomicsABSTRACT
[This corrects the article on p. 474 in vol. 10, PMID: 27857680.].
ABSTRACT
Chemoproteomics profiling of kinase inhibitors with kinobeads enables the assessment of inhibitor potency and selectivity for endogenously expressed protein kinases in cell lines and tissues. Using a small panel of targeted covalent inhibitors, we demonstrate the importance of measuring covalent target binding in live cells. We present a differential kinobeads profiling strategy for covalent kinase inhibitors where a compound is added either to live cells or to a cell extract that enables the comprehensive assessment of inhibitor selectivity for covalent and noncovalent targets. We found that Acalabrutinib, CC-292, and Ibrutinib potently and covalently bind TEC family kinases, but only Ibrutinib also potently binds to BLK. ZAK was identified as a submicromolar affinity Ibrutinib off-target due to covalent modification of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150 next to the DFG loop, demonstrating an alternative route to covalent inactivation of this kinase, e.g., to inhibit canonical TGF-ß dependent processes.
Subject(s)
Acrylamides/pharmacology , Benzamides/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Acrylamides/chemistry , Adenine/analogs & derivatives , B-Lymphocytes , Benzamides/chemistry , Cell Line , Humans , Piperidines , Protein Binding , Protein-Tyrosine Kinases/metabolism , Proteomics/methods , Pyrazines/chemistry , Pyrazoles/chemistry , Pyrimidines/chemistryABSTRACT
The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.