RESUMEN
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.
Asunto(s)
ADN Helicasas , Factores de Transcripción , Proteínas de Unión al ADN , Humanos , Proteínas Nucleares , Dominios ProteicosRESUMEN
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.
Asunto(s)
Bencimidazoles/metabolismo , Diseño de Fármacos , Sondas Moleculares/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Resistencia a Medicamentos/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Línea Celular Tumoral , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Piridonas/química , Piridonas/farmacología , Retinal-Deshidrogenasa , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Histona Acetiltransferasas/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Piridonas/farmacología , Pirroles/farmacología , Factores Asociados con la Proteína de Unión a TATA/antagonistas & inhibidores , Factor de Transcripción TFIID/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Agua/química , Sitios de Unión/efectos de los fármacos , Proteínas de Ciclo Celular , Relación Dosis-Respuesta a Droga , Transferencia Resonante de Energía de Fluorescencia , Fluorometría , Histona Acetiltransferasas/metabolismo , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Proteínas Nucleares/metabolismo , Piridonas/síntesis química , Piridonas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-Actividad , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Acetilación/efectos de los fármacos , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/metabolismo , Factores de Transcripción Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Interleukin (IL) 17-producing T helper (T(H)17) cells have been selected through evolution for their ability to control fungal and bacterial infections. It is also firmly established that their aberrant generation and activation results in autoimmune conditions. Using a characterized potent and selective small molecule inhibitor, we show that the bromodomain and extra-terminal domain (BET) family of chromatin adaptors plays fundamental and selective roles in human and murine T(H)17 differentiation from naive CD4(+) T cells, as well as in the activation of previously differentiated T(H)17 cells. We provide evidence that BET controls T(H)17 differentiation in a bromodomain-dependent manner through a mechanism that includes the direct regulation of multiple effector T(H)17-associated cytokines, including IL17, IL21, and GMCSF. We also demonstrate that BET family members Brd2 and Brd4 associate with the Il17 locus in T(H)17 cells, and that this association requires bromodomains. We recapitulate the critical role of BET bromodomains in T(H)17 differentiation in vivo and show that therapeutic dosing of the BET inhibitor is efficacious in mouse models of autoimmunity. Our results identify the BET family of proteins as a fundamental link between chromatin signaling and T(H)17 biology, and support the notion of BET inhibition as a point of therapeutic intervention in autoimmune conditions.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Células Th17/inmunología , Células Th17/patología , Animales , Comunicación Autocrina/genética , Autoinmunidad/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Citocinas/genética , Citocinas/metabolismo , Sitios Genéticos/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Unión Proteica/genética , Transcripción GenéticaRESUMEN
The Toll signaling pathway is required for the innate immune response against fungi and Gram-positive bacteria in Drosophila. Here we show that the endosomal proteins Myopic (Mop) and Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) are required for the activation of the Toll signaling pathway. This requirement is observed in cultured cells and in flies, and epistasis experiments show that the Mop protein functions upstream of the MyD88 adaptor and the Pelle kinase. Mop and Hrs, which are critical components of the ESCRT-0 endocytosis complex, colocalize with the Toll receptor in endosomes. We conclude that endocytosis is required for the activation of the Toll signaling pathway.
Asunto(s)
Proteínas de Drosophila/inmunología , Drosophila melanogaster/inmunología , Endocitosis , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Inmunidad Innata , Fosfoproteínas/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Transducción de Señal , Receptores Toll-Like/inmunología , Animales , Línea Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Fosfoproteínas/genética , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , Receptores Toll-Like/metabolismoRESUMEN
Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.
Asunto(s)
Intrones/genética , Mitocondrias/genética , Chaperonas Moleculares/metabolismo , ARN Helicasas/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN/biosíntesis , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/fisiología , ARN Helicasas DEAD-box , Intrones/fisiología , Mitocondrias/metabolismo , Mutación , Biosíntesis de Proteínas/fisiología , ARN Helicasas/genética , Procesamiento Postranscripcional del ARN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
Group II intron homing in yeast mitochondria is initiated at active target sites by activities of intron-encoded ribonucleoprotein (RNP) particles, but is completed by competing recombination and repair mechanisms. Intron aI1 transposes in haploid cells at low frequency to target sites in mtDNA that resemble the exon 1-exon 2 (E1/E2) homing site. This study investigates a system in which aI1 can transpose in crosses (i.e., in trans). Surprisingly, replacing an inefficient transposition site with an active E1/E2 site supports <1% transposition of aI1. Instead, the ectopic site was mainly converted to the related sequence in donor mtDNA in a process we call "abortive transposition." Efficient abortive events depend on sequences in both E1 and E2, suggesting that most events result from cleavage of the target site by the intron RNP particles, gapping, and recombinational repair using homologous sequences in donor mtDNA. A donor strain that lacks RT activity carries out little abortive transposition, indicating that cDNA synthesis actually promotes abortive events. We also infer that some intermediates abort by ejecting the intron RNA from the DNA target by forward splicing. These experiments provide new insights to group II intron transposition and homing mechanisms in yeast mitochondria.
Asunto(s)
Elementos Transponibles de ADN/genética , Reordenamiento Génico/genética , Mitocondrias/genética , Modelos Genéticos , Ribonucleoproteínas/metabolismo , Levaduras/genética , Cruzamientos Genéticos , Ciclooxigenasa 1 , ADN Complementario/genética , Intrones/genética , Polimorfismo de Longitud del Fragmento de Restricción , Prostaglandina-Endoperóxido Sintasas/genética , Ribonucleoproteínas/genéticaRESUMEN
Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.
Asunto(s)
Intrones , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Sitios de Unión/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Mitocondrial/química , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN/química , ARN/genética , ARN/metabolismo , Empalme del ARN , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN MitocondrialRESUMEN
Group II introns are catalytic RNAs and mobile retrotransposable elements known to be present in the genomes of some nonmarine bacteria and eukaryotic organelles. Here we report the discovery of group II introns in a bacterial mat sample collected from a deep-sea hydrothermal vent near 9 degrees N on the East Pacific Rise. One of the introns was shown to self-splice in vitro. This is the first example of marine bacterial introns from molecular population structure studies of microorganisms that live in the proximity of hydrothermal vents. These types of mobile genetic elements may prove useful in improving our understanding of bacterial genome evolution and may serve as valuable markers in comparative studies of bacterial communities.