RESUMO
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.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Proteoma/análise , Proteômica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacologia , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Estradiol/farmacologia , Humanos , Marcação por Isótopo , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Espectrometria de Massas em Tandem , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
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.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Proteoma , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Multiômica , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteômica/métodosRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Células Procarióticas/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteoma/análise , Temperatura de Transição , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/metabolismo , Estabilidade Proteica , Software , Especificidade da EspécieRESUMO
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.
Assuntos
Preparações Farmacêuticas , Proteômica , Espectrometria de Massas , Estabilidade Proteica , ProteomaRESUMO
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.
Assuntos
Lisina/química , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Classe I de Fosfatidilinositol 3-Quinases , Descoberta de Drogas , Humanos , Lisina/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , ProteômicaRESUMO
We extended thermal proteome profiling to detect transmembrane protein-small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell-receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.
Assuntos
Proteínas de Membrana/metabolismo , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Células CACO-2 , Temperatura Alta , Humanos , Células Jurkat , Células K562 , Ligantes , Ligação Proteica , Estabilidade Proteica , Proteoma/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Vanadatos/farmacologiaRESUMO
We describe a two-dimensional thermal proteome profiling strategy that can be combined with an orthogonal chemoproteomics approach to enable comprehensive target profiling of the marketed histone deacetylase inhibitor panobinostat. The N-hydroxycinnamide moiety is identified as critical for potent and tetrahydrobiopterin-competitive inhibition of phenylalanine hydroxylase leading to increases in phenylalanine and decreases in tyrosine levels. These findings provide a rationale for adverse clinical observations and suggest repurposing of the drug for treatment of tyrosinemia.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Fenilalanina Hidroxilase/antagonistas & inibidores , Temperatura , Relação Dose-Resposta a Droga , Células Hep G2 , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Indóis/química , Estrutura Molecular , Panobinostat , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/metabolismo , Relação Estrutura-AtividadeRESUMO
Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low mass range of tandem MS spectra for relative quantification. The recent extension of TMT multiplexing to 10 conditions has been enabled by utilizing neutron encoded tags with reporter ion m/z differences of 6 mDa. The baseline resolution of these closely spaced tags is possible due to the high resolving power of current day mass spectrometers. In this work we evaluated the performance of the TMT10 isobaric mass tags on the Q Exactive Orbitrap mass spectrometers for the first time and demonstrated comparable quantification accuracy and precision to what can be achieved on the Orbitrap Elite mass spectrometers. However, we discovered, upon analysis of complex proteomics samples on the Q Exactive Orbitrap mass spectrometers, that the proximate TMT10 reporter ion pairs become prone to coalescence. The fusion of the different reporter ion signals into a single measurable entity has a detrimental effect on peptide and protein quantification. We established that the main reason for coalescence is the commonly accepted maximum ion target for MS2 spectra of 1e6 on the Q Exactive instruments. The coalescence artifact was completely removed by lowering the maximum ion target for MS2 spectra from 1e6 to 2e5 without any losses in identification depth or quantification quality of proteins.
Assuntos
Espectrometria de Massas em Tandem/métodos , Íons , NêutronsRESUMO
We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Interleucina-17/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Ligação Competitiva , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Estrutura Molecular , Ratos , Ratos Wistar , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacocinética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
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.
RESUMO
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.
Assuntos
Helicase da Síndrome de Werner , Humanos , Helicase da Síndrome de Werner/genética , Camundongos , Animais , Instabilidade de Microssatélites , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêuticoRESUMO
Isobaric mass tag-based quantitative proteomics strategies such as iTRAQ and TMT utilize reporter ions in the low-mass range of tandem MS spectra for relative quantification. The number of samples that can be compared in a single experiment (multiplexing) is limited by the number of different reporter ions that can be generated by differential stable isotope incorporation ((15)N, (13)C) across the reporter and the mass balancing parts of the reagents. Here, we demonstrate that a higher multiplexing rate can be achieved by utilizing the 6 mDa mass difference between (15)N- and (13)C-containing reporter fragments, in combination with high-resolution mass spectrometry. Two variants of the TMT127 and TMT129 reagents are available; these are distinguished by the position and the nature of the incorporated stable isotope in the reporter portions of the labels (TMT127L, (12)C(8)H(16)(15)N(1)(+); TMT127H, (12)C(7)(13)C(1)H(16)(14)N(1)(+); TMT129L, (12)C(6)(13)C(2)H(16)(15)N(1)(+); and TMT129H, (12)C(5)(13)C(3)H(16)(14)N(1)(+)). We demonstrate that these variants can be baseline-resolved in Orbitrap Elite higher-energy collision-induced dissociation spectra recorded with a 96 ms transient enabling comparable dynamic range, precision, and accuracy of quantification as 1 Da spaced reporter ions. The increased multiplexing rate enabled determination of inhibitor potencies in chemoproteomic kinase assays covering a wider range of compound concentrations in a single experiment, compared to conventional 6-plex TMT-based assays.
Assuntos
Espectrometria de Massas em Tandem/métodos , Dasatinibe , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Indicadores e Reagentes/química , Células K562 , Gravidez , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologiaRESUMO
Herein, we disclose the discovery of a series of 7-substituted triazolopyridines which culminated in the identification of 14 (CZC24758), a potent, orally bioavailable small-molecule inhibitor of PI3Kγ, an attractive drug target for inflammatory and autoimmune disorders. Compound 14 has excellent selectivity across the kinome, demonstrates good potency in cell based assays and furthermore exhibits in vivo efficacy in a collagen induced arthritis model in mouse after oral dosing.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Animais , Descoberta de Drogas , Granulócitos/efeitos dos fármacos , Ligação de Hidrogênio , Inflamação/tratamento farmacológico , Masculino , Camundongos , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Piridinas/uso terapêutico , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/uso terapêutico , Triazóis/química , Triazóis/uso terapêuticoRESUMO
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.
Assuntos
Inibidores de Proteínas Quinases/metabolismo , Proteínas/análise , Proteômica , Sunitinibe/metabolismo , Espectrometria de Massas em Tandem , Animais , Ligação Competitiva , Feminino , Humanos , Placenta/metabolismo , Gravidez , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Projetos de Pesquisa , Sunitinibe/farmacologiaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular , Proteínas Nucleares , Proteínas Metiltransferases , Fatores de Transcrição , Proteínas de Ciclo Celular/genética , Cromatina , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Proteínas Metiltransferases/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Animais , Azacitidina/farmacologia , DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/genética , Decitabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , CamundongosRESUMO
Mass spectrometry-based quantitative proteomics has become an important component of biological and clinical research. Although such analyses typically assume that a protein's peptide fragments are observed with equal likelihood, only a few so-called 'proteotypic' peptides are repeatedly and consistently identified for any given protein present in a mixture. Using >600,000 peptide identifications generated by four proteomic platforms, we empirically identified >16,000 proteotypic peptides for 4,030 distinct yeast proteins. Characteristic physicochemical properties of these peptides were used to develop a computational tool that can predict proteotypic peptides for any protein from any organism, for a given platform, with >85% cumulative accuracy. Possible applications of proteotypic peptides include validation of protein identifications, absolute quantification of proteins, annotation of coding sequences in genomes, and characterization of the physical principles governing key elements of mass spectrometric workflows (e.g., digestion, chromatography, ionization and fragmentation).
Assuntos
Algoritmos , Perfilação da Expressão Gênica/métodos , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodos , Peptídeos/química , Proteoma/química , Análise de Sequência de Proteína/métodos , Peptídeos/análise , Proteoma/análiseRESUMO
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.
Assuntos
Naftiridinas/química , Fatores de Transcrição/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Sítios de Ligação , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Meia-Vida , Humanos , Simulação de Dinâmica Molecular , Naftiridinas/metabolismo , Naftiridinas/farmacologia , Domínios Proteicos , Quinolonas/química , Quinolonas/metabolismo , Quinolonas/farmacologia , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Lisina/química , Piridazinas/química , Fatores de Transcrição/antagonistas & inibidores , Sítios de Ligação , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Piridazinas/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
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.