RESUMEN
Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Preparaciones Farmacéuticas/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Alanina/análogos & derivados , Alanina/farmacología , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/ultraestructura , Azepinas/farmacología , Sitios de Unión , Microscopía por Crioelectrón , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Oxadiazoles/química , Oxadiazoles/farmacología , Presenilina-1/química , Presenilina-1/metabolismo , Unión Proteica/efectos de los fármacos , Conformación Proteica , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteoma/análisis , Proteómica/métodos , Azepinas/química , Azepinas/metabolismo , Azepinas/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , Estradiol/farmacología , Humanos , Marcaje Isotópico , Células Jurkat , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Proteolisis/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Espectrometría de Masas en Tándem , Triazoles/química , Triazoles/metabolismo , Triazoles/farmacologíaRESUMEN
The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.
Asunto(s)
Apoptosis , Cromatina/metabolismo , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Apoptosis/efectos de los fármacos , Azepinas/farmacología , Azepinas/uso terapéutico , Bencimidazoles/farmacología , Bencimidazoles/uso terapéutico , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Inversión Cromosómica/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , ADN/química , ADN/metabolismo , ADN Helicasas/metabolismo , Modelos Animales de Enfermedad , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription.
Asunto(s)
Elementos de Facilitación Genéticos , MicroARNs/metabolismo , Animales , Azepinas/farmacología , Regulación de la Expresión Génica , Código de Histonas , Humanos , Ratones , Neoplasias/genética , Especificidad de Órganos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Factores de Transcripción/metabolismo , Transcripción Genética , Triazoles/farmacologíaRESUMEN
Fragile X syndrome (FXS) is a leading genetic cause of intellectual disability and autism. FXS results from the loss of function of fragile X mental retardation protein (FMRP), which represses translation of target transcripts. Most of the well-characterized target transcripts of FMRP are synaptic proteins, yet targeting these proteins has not provided effective treatments. We examined a group of FMRP targets that encode transcriptional regulators, particularly chromatin-associated proteins. Loss of FMRP in mice results in widespread changes in chromatin regulation and aberrant gene expression. To determine if targeting epigenetic factors could reverse phenotypes associated with the disorder, we focused on Brd4, a BET protein and chromatin reader targeted by FMRP. Inhibition of Brd4 function alleviated many of the phenotypes associated with FXS. We conclude that loss of FMRP results in significant epigenetic misregulation and that targeting transcription via epigenetic regulators like Brd4 may provide new treatments for FXS.
Asunto(s)
Azepinas/farmacología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/tratamiento farmacológico , Síndrome del Cromosoma X Frágil/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/farmacología , Animales , Células Cultivadas , Epigénesis Genética , Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Ratones , Ratones Noqueados , Naftiridinas/farmacología , Neuronas/metabolismo , Fenazinas , Transcripción GenéticaRESUMEN
γ-Secretase proteases have been associated with pathology in Alzheimer disease (AD), but we are just beginning to understand their basic mechanisms and physiological roles. A negative drug trial with a broad spectrum γ-secretase inhibitor in AD patients has severely dampened enthusiasm for the potential of pursuing γ-secretase research therapeutically. This pessimism is unwarranted: analysis of available information presented here demonstrates significant confounds for interpreting the outcome of the trial and argues that the major lessons pertain to broad knowledge gaps that are imperative to fill.
Asunto(s)
Alanina/análogos & derivados , Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Azepinas/efectos adversos , Ensayos Clínicos como Asunto , Alanina/efectos adversos , Alanina/farmacocinética , Alanina/uso terapéutico , Péptidos beta-Amiloides/metabolismo , Azepinas/farmacocinética , Azepinas/uso terapéutico , Encéfalo/efectos de los fármacos , Cognición/efectos de los fármacos , HumanosRESUMEN
Chromatin regulators have become attractive targets for cancer therapy, but it is unclear why inhibition of these ubiquitous regulators should have gene-specific effects in tumor cells. Here, we investigate how inhibition of the widely expressed transcriptional coactivator BRD4 leads to selective inhibition of the MYC oncogene in multiple myeloma (MM). BRD4 and Mediator were found to co-occupy thousands of enhancers associated with active genes. They also co-occupied a small set of exceptionally large super-enhancers associated with genes that feature prominently in MM biology, including the MYC oncogene. Treatment of MM tumor cells with the BET-bromodomain inhibitor JQ1 led to preferential loss of BRD4 at super-enhancers and consequent transcription elongation defects that preferentially impacted genes with super-enhancers, including MYC. Super-enhancers were found at key oncogenic drivers in many other tumor cells. These observations have implications for the discovery of cancer therapeutics directed at components of super-enhancers in diverse tumor types.
Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Elementos de Facilitación Genéticos , Complejo Mediador/metabolismo , Neoplasias/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Triazoles/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Cromatina , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Humanos , Complejo Mediador/antagonistas & inhibidores , Mieloma Múltiple/genética , Proteínas Nucleares/antagonistas & inhibidores , Elongación de la Transcripción Genética , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.
Asunto(s)
Resistencia a Antineoplásicos/genética , Proteínas/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Azepinas/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Triazoles/farmacología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
Asunto(s)
Azepinas/farmacología , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Interneuronas/patología , Proteína 2 de Unión a Metil-CpG/fisiología , Síndrome de Rett/patología , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos , Triazoles/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Factores de Transcripción/genéticaRESUMEN
Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.
Asunto(s)
Proteínas de Ciclo Celular , Oxindoles , Proteolisis , Ubiquitina-Proteína Ligasas , Humanos , Animales , Proteolisis/efectos de los fármacos , Ratones , Ubiquitina-Proteína Ligasas/metabolismo , Oxindoles/farmacología , Oxindoles/metabolismo , Oxindoles/química , Proteínas de Ciclo Celular/metabolismo , Factores de Transcripción/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Desnudos , Células HEK293 , Relación Estructura-Actividad , Complejo de la Endopetidasa Proteasomal/metabolismo , Azepinas/farmacología , Azepinas/química , Azepinas/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Femenino , Proteínas que Contienen Bromodominio , Receptores de Interleucina-17RESUMEN
A pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.
Asunto(s)
Azepinas/farmacología , Anticonceptivos Masculinos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Triazoles/farmacología , Animales , Azepinas/química , Barrera Hematotesticular , Anticonceptivos Masculinos/química , Femenino , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Modelos Moleculares , Proteínas Nucleares/química , Estructura Terciaria de Proteína , Recuento de Espermatozoides , Motilidad Espermática/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Triazoles/químicaRESUMEN
The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.
Asunto(s)
Azepinas/farmacología , Descubrimiento de Drogas , Leucemia Megacarioblástica Aguda/tratamiento farmacológico , Megacariocitos/metabolismo , Poliploidía , Pirimidinas/farmacología , Bibliotecas de Moléculas Pequeñas , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Aurora Quinasa A , Aurora Quinasas , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Megacarioblástica Aguda/genética , Megacariocitos/citología , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
Extrachromosomal DNA (ecDNA) is prevalent in human cancers and mediates high expression of oncogenes through gene amplification and altered gene regulation1. Gene induction typically involves cis-regulatory elements that contact and activate genes on the same chromosome2,3. Here we show that ecDNA hubs-clusters of around 10-100 ecDNAs within the nucleus-enable intermolecular enhancer-gene interactions to promote oncogene overexpression. ecDNAs that encode multiple distinct oncogenes form hubs in diverse cancer cell types and primary tumours. Each ecDNA is more likely to transcribe the oncogene when spatially clustered with additional ecDNAs. ecDNA hubs are tethered by the bromodomain and extraterminal domain (BET) protein BRD4 in a MYC-amplified colorectal cancer cell line. The BET inhibitor JQ1 disperses ecDNA hubs and preferentially inhibits ecDNA-derived-oncogene transcription. The BRD4-bound PVT1 promoter is ectopically fused to MYC and duplicated in ecDNA, receiving promiscuous enhancer input to drive potent expression of MYC. Furthermore, the PVT1 promoter on an exogenous episome suffices to mediate gene activation in trans by ecDNA hubs in a JQ1-sensitive manner. Systematic silencing of ecDNA enhancers by CRISPR interference reveals intermolecular enhancer-gene activation among multiple oncogene loci that are amplified on distinct ecDNAs. Thus, protein-tethered ecDNA hubs enable intermolecular transcriptional regulation and may serve as units of oncogene function and cooperative evolution and as potential targets for cancer therapy.
Asunto(s)
Neoplasias , Proteínas Nucleares , Azepinas/farmacología , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Proteínas Nucleares/genética , Oncogenes/genética , Factores de Transcripción/genéticaRESUMEN
Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.
Asunto(s)
Antineoplásicos/farmacología , Azepinas/farmacología , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacología , Antineoplásicos/química , Azepinas/química , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Células K562 , Modelos Moleculares , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/químicaRESUMEN
Brain rhythms provide the timing for recruitment of brain activity required for linking together neuronal ensembles engaged in specific tasks. The γ-oscillations (30 to 120 Hz) orchestrate neuronal circuits underlying cognitive processes and working memory. These oscillations are reduced in numerous neurological and psychiatric disorders, including early cognitive decline in Alzheimer's disease (AD). Here, we report on a potent brain-permeable small molecule, DDL-920 that increases γ-oscillations and improves cognition/memory in a mouse model of AD, thus showing promise as a class of therapeutics for AD. We employed anatomical, in vitro and in vivo electrophysiological, and behavioral methods to examine the effects of our lead therapeutic candidate small molecule. As a novel in central nervous system pharmacotherapy, our lead molecule acts as a potent, efficacious, and selective negative allosteric modulator of the γ-aminobutyric acid type A receptors most likely assembled from α1ß2δ subunits. These receptors, identified through anatomical and pharmacological means, underlie the tonic inhibition of parvalbumin (PV) expressing interneurons (PV+INs) critically involved in the generation of γ-oscillations. When orally administered twice daily for 2 wk, DDL-920 restored the cognitive/memory impairments of 3- to 4-mo-old AD model mice as measured by their performance in the Barnes maze. Our approach is unique as it is meant to enhance cognitive performance and working memory in a state-dependent manner by engaging and amplifying the brain's endogenous γ-oscillations through enhancing the function of PV+INs.
Asunto(s)
Enfermedad de Alzheimer , Cognición , Modelos Animales de Enfermedad , Ritmo Gamma , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Ratones , Cognición/efectos de los fármacos , Ritmo Gamma/efectos de los fármacos , Memoria/efectos de los fármacos , Receptores de GABA-A/metabolismo , Ratones Transgénicos , Humanos , Masculino , Memoria a Corto Plazo/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Alanina/análogos & derivados , AzepinasRESUMEN
MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Mieloma Múltiple/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos/química , Azepinas/química , Azepinas/farmacología , Benzodiazepinas/química , Benzodiazepinas/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Activación Transcripcional/efectos de los fármacos , Triazoles/química , Triazoles/farmacologíaRESUMEN
The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds to BRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 in vitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitor in a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.
Asunto(s)
Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Endopeptidasas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Factores de Transcripción/genética , Animales , Antineoplásicos/farmacología , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Endopeptidasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear/deficiencia , Co-Represor 2 de Receptor Nuclear/genética , Piperazinas/farmacología , Próstata/efectos de los fármacos , Próstata/enzimología , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Neoplasias de la Próstata Resistentes a la Castración/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis , Piridinas/farmacología , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Transcripción Genética , Triazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.
Asunto(s)
Azepinas , Receptor X de Pregnano , Triazoles , Azepinas/química , Azepinas/farmacología , Línea Celular Tumoral , Proliferación Celular , Citocromo P-450 CYP3A/genética , Proteínas Nucleares/metabolismo , Receptor X de Pregnano/química , Proteínas Proto-Oncogénicas c-myc/genética , Receptores Citoplasmáticos y Nucleares , Triazoles/química , Triazoles/farmacología , HumanosRESUMEN
Bromodomain and extraterminal (BET) domain inhibitors (BETis) show efficacy on NUT midline carcinoma (NMC). However, not all NMC patients respond, and responders eventually develop resistance and relapse. Using CRISPR and ORF expression screens, we systematically examined the ability of cancer drivers to mediate resistance of NMC to BETis and uncovered six general classes/pathways mediating resistance. Among these, we showed that RRAS2 attenuated the effect of JQ1 in part by sustaining ERK pathway function during BRD4 inhibition. Furthermore, overexpression of Kruppel-like factor 4 (KLF4), mediated BETi resistance in NMC cells through restoration of the E2F and MYC gene expression program. Finally, we found that expression of cyclin D1 or an oncogenic cyclin D3 mutant or RB1 loss protected NMC cells from BETi-induced cell cycle arrest. Consistent with these findings, cyclin-dependent kinase 4/6 (CDK4/6) inhibitors showed synergistic effects with BETis on NMC in vitro as well as in vivo, thereby establishing a potential two-drug therapy for NMC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azepinas/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Triazoles/uso terapéutico , Animales , Azepinas/farmacología , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Ciclinas/metabolismo , Resistencia a Antineoplásicos , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Proteínas de Unión al GTP Monoméricas/genética , Mutación , Proteínas de Neoplasias , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Oncogénicas/antagonistas & inhibidores , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Piridinas/uso terapéutico , Factores de Transcripción/antagonistas & inhibidores , Triazoles/farmacologíaRESUMEN
Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.