RESUMO
Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.
Assuntos
Proteômica , Fatores de Transcrição , Humanos , Proteômica/métodos , Cisteína/metabolismo , LigantesRESUMO
Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers.
Assuntos
Membrana Celular/metabolismo , Hidrolases/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas ras/metabolismo , Proliferação de Células , Células Cultivadas , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/patologia , Lipoilação , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura MolecularRESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.7b00126.].
RESUMO
Histone deacetylase (HDAC) activity is modulated inâ vivo by post-translational modifications and formation of multiprotein complexes. Novel chemical tools to study how these factors affect engagement of HDAC isoforms by HDAC inhibitors (HDACi) in cells and tissues are needed. In this study, a synthetic strategy to access chemically diverse photoreactive probes (PRPs) was developed and used to prepare seven novel HDAC PRPs 9-15. The classâ I HDAC isoform engagement by PRPs was determined in biochemical assays and photolabeling experiments in live SET-2, HepG2, HuH7, and HEK293T cell lines and in mouse liver tissue. Unlike the HDAC protein abundance and biochemical activity against recombinant HDACs, the chemotype of the PRPs and the type of cells were key in defining the engagement of HDAC isoforms in live cells. Our findings suggest that engagement of HDAC isoforms by HDACi inâ vivo may be substantially modulated in a cell- and tissue-type-dependent manner.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Imagem Óptica , Marcadores de Fotoafinidade/farmacologia , Animais , Células Cultivadas , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Fígado/diagnóstico por imagem , Camundongos , Camundongos da Linhagem 129 , Marcadores de Fotoafinidade/síntese química , Marcadores de Fotoafinidade/químicaRESUMO
Histone deacetylase (HDAC) catalyzes the removal of acetyl marks from histones, effectively regulating gene expression. Genome wide chromatin immunoprecipitation (ChIP) studies have shown HDACs are present on numerous active and repressed genes. However, HDAC inhibitors (HDACi) only regulate a small subset of this population in a cell type dependent fashion. To determine genomic locations directly targeted by HDACi, we developed a chromatin precipitation method using a photoreactive HDAC inhibitor probe (photomate). We validate this method by analyzing several canonical HDACi regulated genes, CDKN1A and FOSL1, and compare it to traditional ChIP using HDAC1 antibodies. We show that HDACi target HDACs bound at the promoter regions but not gene bodies, differing from HDAC1 antibody-based ChIP in the case of CDKN1A. This approach is anticipated to be useful for genome wide studies to identify the subset of genes directly regulated by an HDACi in a given cell type.
RESUMO
Histone deacetylase (HDAC) catalytic activity is regulated by formation of co-regulator complexes and post-translational modification. Whether these mechanisms are transformed in cancer and how this affects the binding and selectivity of HDAC inhibitors (HDACis) is unclear. In this study, we developed a method that identified a 3- to 16-fold increase in HDACi selectivity for HDAC3 in triple-negative breast cancer (TNBC) cells in comparison with luminal subtypes that was not predicted by current practice measurements with recombinant proteins. We found this increase was caused by c-Jun N-terminal kinase (JNK) phosphorylation of HDAC3, was independent of HDAC3 complex composition or subcellular localization, and was associated with a 5-fold increase in HDAC3 enzymatic activity. This study points to HDAC3 and the JNK axes as targets in TNBC, highlights how HDAC phosphorylation affects HDACi binding and selectivity, and outlines a method to identify changes in individual HDAC isoforms catalytic activity, applicable to any disease state.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Células MCF-7 , Fosforilação , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The mechanism of action of histone deacetylase inhibitors (HDACi) is mainly attributed to the inhibition of the deacetylase catalytic activity for their histone substrates. In this study, we analyzed the abundance of class I HDACs in the cytosolic, nuclear soluble and chromatin bound cellular fractions in breast cancer cells after HDACi treatment. We found that potent N-hydroxy propenamide-based HDACi induced a concentration dependent decrease in the HDAC1 associated with chromatin and a lasting concomitant increase in cytoplasmic HDAC1 while maintaining total protein expression. No such change occurred with HDAC2 or 8, however, an increase in cytoplasmic non-phosphorylated HDAC3 was also observed. The subcellular re-equilibration of HDAC1 was subsequent to the accumulation of acetylated histones and might be cell cycle dependent. This study suggests that the biological activity of a subset of N-hydroxy propenamide-based HDACi may stem from direct competition with histone substrates of HDACs as well as from spatial separation from their substrates in the nucleus and/or change in post-translational modification status of HDACs.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Inibidores de Histona Desacetilases/química , Histonas/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Mitógenos/farmacologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
Histone deacetylase 8 (HDAC8) is a promising drug target for multiple therapeutic applications. Here, we describe the modeling, design, synthesis, and biological evaluation of a novel series of C1-substituted tetrahydroisoquinoline (TIQ)-based HDAC8 inhibitors. Minimization of entropic loss upon ligand binding and use of the unique HDAC8 "open" conformation of the binding site yielded a successful strategy for improvement of both HDAC8 potency and selectivity. The TIQ-based 3g and 3n exhibited the highest 82 and 55 nM HDAC8 potency and 330- and 135-fold selectivity over HDAC1, respectively. Selectivity over other class I isoforms was comparable or better, whereas inhibition of HDAC6, a class II HDAC isoform, was below 50% at 10 µM. The cytotoxicity of 3g and 3n was evaluated in neuroblastoma cell lines, and 3n displayed concentration-dependent cytotoxicity similar to or better than that of PCI-34051. The selectivity of 3g and 3n was confirmed in SH-SY5Y cells as both did not increase the acetylation of histone H3 and α-tubulin. Discovery of the novel TIQ chemotype paves the way for the development of HDAC8 selective inhibitors for therapeutic applications.