RESUMEN
Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.
Asunto(s)
Neoplasias , Reparación del ADN por Recombinación , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , ADN , Daño del ADN , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Serina-Treonina QuinasasRESUMEN
Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.
Asunto(s)
MicroARNs/genética , Proteínas de Unión al ARN/metabolismo , Línea Celular , Línea Celular Tumoral , Haploinsuficiencia/genética , Humanos , MicroARNs/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Esquizofrenia/genéticaRESUMEN
Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl-l-lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/Cmax) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/Cmax>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Ácidos y Sales Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Canalículos Biliares/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Ciclosporina/farmacología , Expresión Génica , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Ratones , Ratones TransgénicosRESUMEN
Recent developments of novel targeted therapies are contributing to the increased long-term survival of cancer patients; however, drug-induced cardiotoxicity induced by cancer drugs remains a serious problem in clinical settings. Nevertheless, there are few in vitro cell-based assays available to predict this toxicity, especially from the aspect of morphology. Here, we developed a simple two-dimensional (2D) morphological assessment system, 2DMA, to predict drug-induced cardiotoxicity in cancer patients using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with image-based high-content analysis in a high-throughput manner. To assess the effects of drugs on cardiomyocytes, we treated iPSC-CMs with 28 marketed pharmaceuticals and measured two key parameters: number of cell nuclei and sarcomere morphology. Drugs that significantly perturbed these two parameters at concentrations ≤30 times the human Cmax value were regarded as positive in the test. Based on these criteria, the sensitivity and specificity of the 2DMA system were 81% and 100%, respectively. Moreover, the translational predictability of 2DMA was comparable with that of a three-dimensional cardiotoxicity assay. RNA sequencing further revealed that the expression levels of several genes related to sarcomere components decreased following treatment with sunitinib, suggesting that inhibition of the synthesis of proteins that comprise the sarcomere contributes to drug-induced sarcomere disruption. Based on these features, the 2DMA system provides mechanistic insight with high predictability of cancer drug-induced cardiotoxicity in humans, and could thus contribute to the reduction of drug attrition rates at early stages of drug development.
Asunto(s)
Antineoplásicos/toxicidad , Cardiotoxinas/toxicidad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Microscopía Electrónica/métodos , Miocitos Cardíacos/efectos de los fármacos , Cardiotoxicidad/patología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Colorantes Fluorescentes/análisis , Predicción , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/patologíaRESUMEN
Dysferlinopathies, which are muscular diseases caused by mutations in the dysferlin gene, remain serious medical problems due to the lack of therapeutic agents. Herein, we report the design, synthesis, and structure-activity relationships of a 2,6-disubstituted 3H-imidazo[4,5-b]pyridine series, which was identified from the phenotypic screening of chemicals that increase the level of dysferlin in myocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs). Optimization studies with cell-based phenotypic assay led to the identification of a highly potent compound, 19, with dysferlin elevation effects at double-digit nanomolar concentrations. In addition, the molecular target of our chemical series was identified as tubulin, through a tubulin polymerization assay and a competitive binding assay using a photoaffinity labeling probe.
Asunto(s)
Imidazoles/química , Distrofia Muscular de Cinturas/tratamiento farmacológico , Piridinas/química , Moduladores de Tubulina/uso terapéutico , Sitios de Unión , Diferenciación Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Disferlina/metabolismo , Células Hep G2 , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Simulación del Acoplamiento Molecular , Distrofia Muscular de Cinturas/patología , Proteína MioD/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Estructura Terciaria de Proteína , Piridinas/farmacología , Piridinas/uso terapéutico , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin ß, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cardiotónicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Miocitos Cardíacos/citología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Células Cultivadas , Complejo IV de Transporte de Electrones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ivermectina/farmacología , Ratones , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , beta Carioferinas/metabolismoRESUMEN
Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.