RESUMEN
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
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
The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.
Asunto(s)
Antivirales , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/farmacología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/farmacología , Anticuerpos Antivirales/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/genética , COVID-19/inmunología , COVID-19/virología , Cricetinae , Citidina/análogos & derivados , Combinación de Medicamentos , Hidroxilaminas , Indazoles , Lactamas , Leucina , Ratones , Nitrilos , Prolina , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genética , Triazinas , TriazolesRESUMEN
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
The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Inmunoterapia/métodos , Linfocitos/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiocinas/inmunología , Quimiocinas/metabolismo , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Femenino , Citometría de Flujo , Linfocitos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/genética , Pirazinas/farmacología , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Fosfato de Sitagliptina , Triazoles/farmacologíaRESUMEN
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
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
CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokine interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.
Asunto(s)
Quimiocina CCL5/química , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , VIH-1/fisiología , Modelos Moleculares , Imitación Molecular , Receptores CCR5/química , Animales , Antagonistas de los Receptores CCR5/química , Antagonistas de los Receptores CCR5/farmacología , Quimiocina CCL5/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Ciclohexanos/química , Ciclohexanos/farmacología , Proteína gp120 de Envoltorio del VIH/metabolismo , Inhibidores de Fusión de VIH/química , Infecciones por VIH/tratamiento farmacológico , Humanos , Maraviroc , Unión Proteica , Conformación Proteica , Receptores CCR5/metabolismo , Células Sf9 , Spodoptera , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacología , Internalización del Virus/efectos de los fármacosRESUMEN
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
Automated synthesis platforms accelerate and simplify the preparation of molecules by removing the physical barriers to organic synthesis. This provides unrestricted access to biopolymers and small molecules via reproducible and directly comparable chemical processes. Current automated multistep syntheses rely on either iterative1-4 or linear processes5-9, and require compromises in terms of versatility and the use of equipment. Here we report an approach towards the automated synthesis of small molecules, based on a series of continuous flow modules that are radially arranged around a central switching station. Using this approach, concise volumes can be exposed to any reaction conditions required for a desired transformation. Sequential, non-simultaneous reactions can be combined to perform multistep processes, enabling the use of variable flow rates, reuse of reactors under different conditions, and the storage of intermediates. This fully automated instrument is capable of both linear and convergent syntheses and does not require manual reconfiguration between different processes. The capabilities of this approach are demonstrated by performing optimizations and multistep syntheses of targets, varying concentrations via inline dilutions, exploring several strategies for the multistep synthesis of the anticonvulsant drug rufinamide10, synthesizing eighteen compounds of two derivative libraries that are prepared using different reaction pathways and chemistries, and using the same reagents to perform metallaphotoredox carbon-nitrogen cross-couplings11 in a photochemical module-all without instrument reconfiguration.
Asunto(s)
Técnicas de Química Sintética/instrumentación , Técnicas de Química Sintética/métodos , Triazoles/síntesis química , Anticonvulsivantes/síntesis química , Anticonvulsivantes/química , Automatización/instrumentación , Automatización/métodos , Carbono/química , Indicadores y Reactivos/química , Nitrógeno/química , Oxidación-Reducción , Procesos Fotoquímicos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Programas Informáticos , Soluciones/química , Triazoles/químicaRESUMEN
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
The recently developed double-click reaction sequence [G. Meng et al., Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón , Péptidos , Regulación Alostérica , Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Péptidos/química , Triazoles/químicaRESUMEN
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.
Asunto(s)
Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Animales , Antineoplásicos/farmacología , Azepinas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Mutación , Nitrilos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas , Interferencia de ARN , Receptores de Citocinas/genética , Transcriptoma , Triazoles/farmacologíaRESUMEN
We previously showed that orexin neurons are activated by hypoxia and facilitate the peripheral chemoreflex (PCR)-mediated hypoxic ventilatory response (HVR), mostly by promoting the respiratory frequency response. Orexin neurons project to the nucleus of the solitary tract (nTS) and the paraventricular nucleus of the hypothalamus (PVN). The PVN contributes significantly to the PCR and contains nTS-projecting corticotropin-releasing hormone (CRH) neurons. We hypothesized that in male rats, orexin neurons contribute to the PCR by activating nTS-projecting CRH neurons. We used neuronal tract tracing and immunohistochemistry (IHC) to quantify the degree that hypoxia activates PVN-projecting orexin neurons. We coupled this with orexin receptor (OxR) blockade with suvorexant (Suvo, 20â mg/kg, i.p.) to assess the degree that orexin facilitates the hypoxia-induced activation of CRH neurons in the PVN, including those projecting to the nTS. In separate groups of rats, we measured the PCR following systemic orexin 1 receptor (Ox1R) blockade (SB-334867; 1â mg/kg) and specific Ox1R knockdown in PVN. OxR blockade with Suvo reduced the number of nTS and PVN neurons activated by hypoxia, including those CRH neurons projecting to nTS. Hypoxia increased the number of activated PVN-projecting orexin neurons but had no effect on the number of activated nTS-projecting orexin neurons. Global Ox1R blockade and partial Ox1R knockdown in the PVN significantly reduced the PCR. Ox1R knockdown also reduced the number of activated PVN neurons and the number of activated tyrosine hydroxylase neurons in the nTS. Our findings suggest orexin facilitates the PCR via nTS-projecting CRH neurons expressing Ox1R.