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This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.
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Inhibidores Enzimáticos/farmacología , Proteína 2 de Unión a Retinoblastoma/antagonistas & inhibidores , Animales , Sitios de Unión , Western Blotting , Línea Celular , Descubrimiento de Drogas , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Humanos , Concentración 50 Inhibidora , Ratones , Microsomas Hepáticos/enzimología , Modelos Moleculares , RatasRESUMEN
In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.
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Azepinas/farmacología , Diseño de Fármacos , Proteínas Nucleares/antagonistas & inhibidores , Oxazoles/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Azepinas/síntesis química , Azepinas/química , Proteínas de Ciclo Celular , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Oxazoles/síntesis química , Oxazoles/química , Relación Estructura-ActividadRESUMEN
The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G(1) arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.
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Apoptosis/fisiología , Linfoma de Burkitt/tratamiento farmacológico , Ciclo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción/metabolismo , Animales , Apoptosis/genética , Azepinas/farmacología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
The mammalian DAF-16-like transcription factors, FKHR, FKHRL1, and AFX, function as key regulators of insulin signaling, cell cycle progression, and apoptosis downstream of phosphoinositide 3-kinase. Gene activation through binding to insulin response sequences (IRS) has been thought to be essential for mediating these functions. However, using transcriptional profiling, chromatin immunoprecipitation, and functional experiments, we demonstrate that rather than activation of IRS regulated genes (Class I transcripts), transcriptional repression of D-type cyclins (in Class III) is required for FKHR mediated inhibition of cell cycle progression and transformation. These data suggest that a novel mechanism of FKHR-mediated gene regulation is linked to its activity as a suppressor of tumor growth.
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Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Proteínas Serina-Treonina Quinasas , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Adenoviridae/genética , Carcinoma/patología , Ciclo Celular/fisiología , Línea Celular , Ciclinas/genética , Ciclinas/fisiología , Regulación hacia Abajo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead , Perfilación de la Expresión Génica , Glioblastoma/patología , Humanos , Neoplasias Renales/patología , Cinética , Fosfohidrolasa PTEN , Monoéster Fosfórico Hidrolasas/deficiencia , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-akt , Activación Transcripcional , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.
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Proteínas Cullin , Simulación de Dinámica Molecular , Proteínas Cullin/metabolismo , Deuterio , Lisina/metabolismo , Espectrometría de Masas , Proteolisis , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
OBJECTIVE: To develop recommendations for the assessment of people with systemic lupus erythematosus (SLE) in Canada. METHODS: Recommendations were developed using the GRADE (Grading of Recommendations Assessment, Development, and Evaluation) approach. The Canadian SLE Working Group (panel of Canadian rheumatologists and a patient representative from Canadian Arthritis Patient Alliance) was created. Questions for recommendation development were identified based on the results of a previous survey of SLE practice patterns of members of the Canadian Rheumatology Association. Systematic literature reviews of randomized trials and observational studies were conducted. Evidence to Decision tables were prepared and presented to the panel at 2 face-to-face meetings and online. RESULTS: There are 15 recommendations for assessing and monitoring SLE, with varying applicability to adult and pediatric patients. Three recommendations focus on diagnosis, disease activity, and damage assessment, suggesting the use of a validated disease activity score per visit and annual damage score. Strong recommendations were made for cardiovascular risk assessment and measuring anti-Ro and anti-La antibodies in the peripartum period and conditional recommendations for osteoporosis and osteonecrosis. Two conditional recommendations were made for peripartum assessments, 1 for cervical cancer screening and 2 for hepatitis B and C screening. A strong recommendation was made for annual influenza vaccination. CONCLUSION: These are considered the first guidelines using the GRADE method for the monitoring of SLE. Existing evidence is largely of low to moderate quality, resulting in more conditional than strong recommendations. Additional rigorous studies and special attention to pediatric SLE populations and patient preferences are needed.
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Directrices para la Planificación en Salud , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/terapia , Tamizaje Masivo , Adulto , Canadá , Enfermedades Cardiovasculares/diagnóstico , Enfermedades Cardiovasculares/etiología , Niño , Femenino , Personal de Salud , Hepatitis C/diagnóstico , Hepatitis C/etiología , Humanos , Infecciones/diagnóstico , Infecciones/etiología , Lupus Eritematoso Sistémico/complicaciones , Masculino , Osteonecrosis/diagnóstico , Osteonecrosis/etiología , Osteoporosis/diagnóstico , Osteoporosis/etiología , Periodo Periparto/sangre , Embarazo , Reumatólogos , Medición de Riesgo , Índice de Severidad de la Enfermedad , Revisiones Sistemáticas como Asunto , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/etiología , VacunaciónRESUMEN
In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).
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Antineoplásicos/química , Antineoplásicos/uso terapéutico , Azepinas/química , Azepinas/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Azepinas/farmacocinética , Azepinas/farmacología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Perros , Genes myc/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ratas , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The identification and development of a novel series of small molecule Enhancer of Zeste Homologue 2 (EZH2) inhibitors is described. A concise and modular synthesis enabled the rapid development of structure-activity relationships, which led to the identification of 44 as a potent, SAM-competitive inhibitor of EZH2 that dose-dependently decreased global H3K27me3 in KARPAS-422 lymphoma cells.
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BACKGROUND: To estimate rheumatoid arthritis (RA) prevalence in Quebec using administrative health data, comparing across regions. METHODS: Cases of RA were ascertained from physician billing and hospitalization data, 1992-2008. We used three case definitions: 1) ≥ 2 billing diagnoses, submitted by any physician, ≥ 2 months apart, but within 2 years; 2) ≥ 1 diagnosis, by a rheumatologist; 3) ≥1 hospitalization diagnosis (all based on ICD-9 code 714, and ICD-10 code M05). We combined data across these three case definitions, using Bayesian hierarchical latent class models to estimate RA prevalence, adjusting for the imperfect sensitivity and specificity of the data. We compared urban versus rural regions. RESULTS: Using our case definitions and no adjustment for error, we defined 75,760 cases for an over-all RA prevalence of 9.9 per thousand residents. After adjusting for the imperfect sensitivity and specificity of our case definition algorithms, we estimated Quebec RA prevalence at 5.6 per 1000 females and 4.1 per 1000 males. The adjusted RA prevalence estimates for older females were the highest for any demographic group (9.9 cases per 1,000), and were similar in rural and urban regions. In younger males and females, and in older males, RA prevalence estimates were lower in rural versus urban areas. CONCLUSIONS: Without adjustment for error inherent in administrative databases, RA prevalence in Quebec was approximately 1%, while adjusted estimates are approximately half that. The lower prevalence in rural areas, seen for most demographic groups, may suggest either true regional variations in RA risk, or under-ascertainment of cases in rural Quebec.
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Artritis Reumatoide/epidemiología , Encuestas Epidemiológicas/estadística & datos numéricos , Salud Rural/estadística & datos numéricos , Salud Urbana/estadística & datos numéricos , Adulto , Teorema de Bayes , Femenino , Encuestas Epidemiológicas/métodos , Hospitalización/estadística & datos numéricos , Humanos , Formulario de Reclamación de Seguro/estadística & datos numéricos , Clasificación Internacional de Enfermedades/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Visita a Consultorio Médico/estadística & datos numéricos , Alta del Paciente/estadística & datos numéricos , Prevalencia , Quebec/epidemiología , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To estimate systemic autoimmune rheumatic disease (SARD) prevalence across 7 Canadian provinces using population-based administrative data evaluating both regional variations and the effects of age and sex. METHODS: Using provincial physician billing and hospitalization data, cases of SARD (systemic lupus erythematosus, scleroderma, primary Sjögren syndrome, polymyositis/dermatomyositis) were ascertained. Three case definitions (rheumatology billing, 2-code physician billing, and hospital diagnosis) were combined to derive a SARD prevalence estimate for each province, categorized by age, sex, and rural/urban status. A hierarchical Bayesian latent class regression model was fit to account for the imperfect sensitivity and specificity of each case definition. The model also provided sensitivity estimates of different case definition approaches. RESULTS: Prevalence estimates for overall SARD ranged between 2 and 5 cases per 1000 residents across provinces. Similar demographic trends were evident across provinces, with greater prevalence in women and in persons over 45 years old. SARD prevalence in women over 45 was close to 1%. Overall sensitivity was poor, but estimates for each of the 3 case definitions improved within older populations and were slightly higher for men compared to women. CONCLUSION: Our results are consistent with previous estimates and other North American findings, and provide results from coast to coast, as well as useful information about the degree of regional and demographic variations that can be seen within a single country. Our work demonstrates the usefulness of using multiple data sources, adjusting for the error in each, and providing estimates of the sensitivity of different case definition approaches.
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Lupus Eritematoso Sistémico/epidemiología , Enfermedades Reumáticas/epidemiología , Esclerodermia Sistémica/epidemiología , Síndrome de Sjögren/epidemiología , Adulto , Distribución por Edad , Anciano , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/epidemiología , Teorema de Bayes , Canadá/epidemiología , Comorbilidad , Bases de Datos Factuales , Femenino , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Masculino , Persona de Mediana Edad , Prevalencia , Enfermedades Reumáticas/diagnóstico , Población Rural , Esclerodermia Sistémica/diagnóstico , Distribución por Sexo , Síndrome de Sjögren/diagnóstico , Población UrbanaRESUMEN
The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) is a candidate oncogene due to its prevalent overexpression in malignant diseases, including late stage prostate and breast cancers. The dependency of cancer cells on EZH2 activity is also predicated by recurrent missense mutations residing in the catalytic domain of EZH2 that have been identified in subtypes of diffuse large B cell lymphoma, follicular lymphoma and melanoma. Herein, we report the identification of a highly selective small molecule inhibitor series of EZH2 and EZH1. These compounds inhibit wild-type and mutant versions of EZH2 with nanomolar potency, suppress global histone H3-lysine 27 methylation, affect gene expression, and cause selective proliferation defects. These compounds represent a structurally distinct EZH2 inhibitor chemotype for the exploration of the role of Polycomb Repressive Complex 2-mediated H3K27 methylation in various biological contexts.
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Antineoplásicos/farmacología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/patología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Proteína Potenciadora del Homólogo Zeste 2 , Células HeLa , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Estructura Molecular , Complejo Represivo Polycomb 2/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.