RESUMEN
Mitochondrial diseases comprise a heterogeneous group of genetically inherited disorders that cause failures in energetic and metabolic function. Boosting residual oxidative phosphorylation (OXPHOS) activity can partially correct these failures. Herein, using a high-throughput chemical screen, we identified the bromodomain inhibitor I-BET 525762A as one of the top hits that increases COX5a protein levels in complex I (CI) mutant cybrid cells. In parallel, bromodomain-containing protein 4 (BRD4), a target of I-BET 525762A, was identified using a genome-wide CRISPR screen to search for genes whose loss of function rescues death of CI-impaired cybrids grown under conditions requiring OXPHOS activity for survival. We show that I-BET525762A or loss of BRD4 remodeled the mitochondrial proteome to increase the levels and activity of OXPHOS protein complexes, leading to rescue of the bioenergetic defects and cell death caused by mutations or chemical inhibition of CI. These studies show that BRD4 inhibition may have therapeutic implications for the treatment of mitochondrial diseases.
Asunto(s)
Benzodiazepinas/farmacología , Grupo Citocromo c/genética , Complejo I de Transporte de Electrón/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Proteínas de Ciclo Celular , Fusión Celular , Línea Celular , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Grupo Citocromo c/metabolismo , Complejo I de Transporte de Electrón/deficiencia , Complejo IV de Transporte de Electrones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Metaboloma , Metabolómica , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismoRESUMEN
High-throughput assays generate immense quantities of data that require sophisticated data analysis tools. We have created a freely available software tool, SLIMS (Small Laboratory Information Management System), for chemical genetics which facilitates the collection and analysis of large-scale chemical screening data. Compound structures, physical locations, and raw data can be loaded into SLIMS. Raw data from high-throughput assays are normalized using flexible analysis protocols, and systematic spatial errors are automatically identified and corrected. Various computational analyses are performed on tested compounds, and dilution-series data are processed using standard or user-defined algorithms. Finally, published literature associated with active compounds is automatically retrieved from Medline and processed to yield potential mechanisms of actions. SLIMS provides a framework for analyzing high-throughput assay data both as a laboratory information management system and as a platform for experimental analysis.
Asunto(s)
Evaluación Preclínica de Medicamentos , Programas Informáticos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Bases de Datos Genéticas , Flavonas , Flavonoides/farmacología , Genes Reporteros , Humanos , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Unión al ARN , Proteínas del Complejo SMNRESUMEN
Most patients with the pediatric neurodegenerative disease spinal muscular atrophy have a homozygous deletion of the survival motor neuron 1 (SMN1) gene, but retain one or more copies of the closely related SMN2 gene. The SMN2 gene encodes the same protein (SMN) but produces it at a low efficiency compared with the SMN1 gene. We performed a high-throughput screen of approximately 47,000 compounds to identify those that increase production of an SMN2-luciferase reporter protein, but not an SMN1-luciferase reporter protein. Indoprofen, a nonsteroidal anti-inflammatory drug (NSAID) and cyclooxygenase (COX) inhibitor, selectively increased SMN2-luciferase reporter protein and endogenous SMN protein and caused a 5-fold increase in the number of nuclear gems in fibroblasts from SMA patients. No other NSAIDs or COX inhibitors tested exhibited this activity.