RESUMO
T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells. Mice with DUBA-deficient T cells developed exacerbated inflammation in the small intestine after challenge with anti-CD3 antibodies. DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated RORγt in response to TGF-ß signalling. Our data identify DUBA as a cell-intrinsic suppressor of IL-17 production.
Assuntos
Interleucina-17/biossíntese , Biossíntese de Proteínas , Células Th17/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Estabilidade Enzimática , Feminino , Inflamação/genética , Inflamação/patologia , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transdução de Sinais , Especificidade por Substrato , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/deficiência , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
ISWI chromatin remodelers mobilize nucleosomes to control DNA accessibility. Complexes isolated to date pair one of six regulatory subunits with one of two highly similar ATPases. However, we find that each endogenously expressed ATPase co-purifies with every regulatory subunit, substantially increasing the diversity of ISWI complexes, and we additionally identify BAZ2B as a novel, seventh regulatory subunit. Through reconstitution of catalytically active human ISWI complexes, we demonstrate that the new interactions described here are stable and direct. Finally, we profile the nucleosome remodeling functions of the now expanded family of ISWI chromatin remodelers. By revealing the combinatorial nature of ISWI complexes, we provide a basis for better understanding ISWI function in normal settings and disease.
Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas Cromossômicas não Histona , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Nucleossomos/genética , Ligação Proteica , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteína 2 de Ligação ao Retinoblastoma/antagonistas & inibidores , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.
Assuntos
Proteína de Ligação a CREB/antagonistas & inibidores , Proteína p300 Associada a E1A/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Bromodomain-containing protein 9 (BRD9), an epigenetic "reader" of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.
Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Linhagem Celular Tumoral , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Piridonas/química , Piridonas/farmacologia , Retinal Desidrogenase , Fatores de Transcrição/metabolismoRESUMO
The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Cloreto de Sódio/farmacologia , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Bromodomains are acetyl-lysine binding modules that are found in different classes of chromatin-interacting proteins. Among these are large chromatin remodeling complexes such as BAF and PBAF (variants of human SWI/SNF). Previous work has identified chemical probes targeting a subset of the bromodomains present in the BAF and PBAF complexes. Selective inhibitors of the individual bromodomains have proven challenging to discover, as the domains are highly similar. Here, elaboration of an aminopyridazine scaffold used previously to develop probes for the bromodomains of SMARCA2, SMARCA4, and the fifth bromodomain of PBRM1 yielded compounds with both potency and unusual selectivity for the second bromodomain of PBRM1. One of these, GNE-235, and its enantiomer control GNE-234 are suggested for initial cellular investigations of the function of the second bromodomain of PBRM1.
Assuntos
Proteínas que Contêm Bromodomínio , DNA Helicases , Proteínas Nucleares , Humanos , Proteínas de Ligação a DNA , Domínios Proteicos , Fatores de Transcrição , Proteínas que Contêm Bromodomínio/antagonistas & inibidoresRESUMO
Genetic code expansion has proven invaluable to the elucidation of functions of defined protein modifications through the site-specific incorporation of noncanonical amino acids. The use of nonhydrolyzable derivatives of post-translational modifications can greatly increase site stoichiometry and half-life. Investigating acetyllysine reader domain (bromodomain) interactions with acetylated nonhistone proteins is challenging due to the limited tools available and dynamic nature of this post-translational modification. Here, we demonstrate that bromodomains bind acetyllysine peptides and those substituted with an acetyllysine derivative, trifluoroacetyllysine, with similar affinity and selectivity. Importantly, both trifluoroacetyllysine and acetyllysine can be site-specifically incorporated into proteins expressed in bacterial and mammalian cells, and the strong electron-withdrawing trifluoro substituent makes the latter resistant to deacetylation by sirtuins (SIRTs). The controlled expression of SIRT-resistant, site-specifically acetylated transcription factors expands the set of available tools for determining the function of acetylation, and it serves as a template for investigating bromodomain interactions with acetylated transcription factors.
Assuntos
Lisina , Sirtuínas , Acetilação , Animais , Lisina/química , Mamíferos/metabolismo , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.
Assuntos
DNA Helicases , Fatores de Transcrição , Proteínas de Ligação a DNA , Humanos , Proteínas Nucleares , Domínios ProteicosRESUMO
Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Sítios de Ligação , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Insetos , Cinética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMO
The chromosomal passenger complex (CPC) has been identified as a master regulator of mitosis. In particular, proper chromosome segregation and cytokinesis depend on the correct localization and function of the CPC. Within the complex, the kinase Aurora B associates with Incenp, Survivin, and Borealin. The stoichiometry of the complex as well as a complete understanding of how these four components interact with each other remains to be elucidated. Here, we identify a new domain of Borealin. We determined its structure using NMR spectroscopy and discovered a novel dimerization motif. Interestingly, we found that substitutions at Borealin T230, recently identified as an Mps1 phosphorylation site, can modulate the dimerization state of Borealin. Mutation of this single residue to alanine or valine impairs Aurora B activity during mitosis and causes chromosome segregation defects. This study reveals that Mps1 regulates the CPC through a novel Borealin domain.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromossomos Humanos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Dimerização , Humanos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
Aurora kinases are important mitotic regulators, and Aurora kinase inhibitors are under investigation as treatments for cancer. An ongoing debate in the field is which Aurora kinase is the better drug target. A new study (Girdler et al., 2008, in this issue of Chemistry & Biology) pushes the case forward for Aurora B.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinase B , Aurora Quinases , Humanos , Mitose/efeitos dos fármacos , Mitose/fisiologia , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-molecule targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chemical probe discovery to understand the biological potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compounds have since advanced to human clinical trials. Here, we review the current state of BET inhibitor biology in relation to clinical development, and we discuss the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications. The lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.
Assuntos
Desenvolvimento de Medicamentos/métodos , Fatores de Transcrição/antagonistas & inibidores , Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Ensaios Clínicos como Assunto , Epigênese Genética , Humanos , Terapia de Alvo Molecular , Fatores de Transcrição/genéticaRESUMO
The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC50 of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.
Assuntos
Benzimidazóis/metabolismo , Desenho de Fármacos , Sondas Moleculares/metabolismo , Fator de Transcrição TFIID/química , Fator de Transcrição TFIID/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Domínios ProteicosRESUMO
The cell-extrinsic apoptotic pathway triggers programmed cell death in response to certain ligands that bind to cell-surface death receptors. Apoptosis is essential for normal development and homeostasis in metazoans, and furthermore, selective activation of the cell-extrinsic pathway in tumor cells holds considerable promise for cancer therapy. We used phage display to identify peptides and synthetic antibodies that specifically bind to the human proapoptotic death receptor DR5. Despite great differences in overall size and structure, the DR5-binding peptides and antibodies shared a tripeptide motif, which was conserved within a disulfide-constrained loop of the peptides and the third complementarity determining region of the antibody heavy chains. The X-ray crystal structure of an antibody in complex with DR5 revealed that the tripeptide motif is buried at the core of the interface, confirming its central role in antigen recognition. We found that certain peptides and antibodies exhibited potent proapoptotic activity against DR5-expressing SK-MES-1 lung carcinoma cells. These phage-derived ligands may be useful for elucidating DR5 activation at the molecular level and for creating synthetic agonists of proapoptotic death receptors.
Assuntos
Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas/química , Oligopeptídeos/química , Receptores do Fator de Necrose Tumoral/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Biblioteca de Peptídeos , Conformação Proteica , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/agonistas , Receptores do Fator de Necrose Tumoral/imunologiaRESUMO
Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival.ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Fatores de Transcrição/fisiologia , Sistemas CRISPR-Cas , Linhagem Celular , Cromatina/metabolismo , DNA/metabolismo , Edição de Genes , Humanos , Estrutura Molecular , Fatores de Transcrição/químicaRESUMO
The biological function of bromodomains, epigenetic readers of acetylated lysine residues, remains largely unknown. Herein we report our efforts to discover a potent and selective inhibitor of the bromodomain of cat eye syndrome chromosome region candidate 2 (CECR2). Screening of our internal medicinal chemistry collection led to the identification of a pyrrolopyridone chemical lead, and subsequent structure-based drug design led to a potent and selective CECR2 bromodomain inhibitor (GNE-886) suitable for use as an in vitro tool compound.
RESUMO
The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.
Assuntos
Histona Acetiltransferases/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Piridonas/farmacologia , Pirróis/farmacologia , Fatores Associados à Proteína de Ligação a TATA/antagonistas & inibidores , Fator de Transcrição TFIID/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Água/química , Sítios de Ligação/efeitos dos fármacos , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Fluorometria , Histona Acetiltransferases/metabolismo , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Proteínas Nucleares/metabolismo , Piridonas/síntese química , Piridonas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/metabolismoRESUMO
CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.
RESUMO
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.