RESUMEN
Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.
Asunto(s)
Codón sin Sentido/antagonistas & inhibidores , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/efectos de los fármacos , Degradación de ARNm Mediada por Codón sin Sentido , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Factores de Terminación de Péptidos/metabolismo , Aminoglicósidos/metabolismo , Codón sin Sentido/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Genes Reporteros , Gentamicinas/farmacología , Células HEK293 , Humanos , Microsomas Hepáticos/efectos de los fármacos , Factores de Terminación de Péptidos/genética , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , Ribosomas/metabolismo , Relación Estructura-ActividadRESUMEN
Cystic fibrosis (CF) is a hereditary disease caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). A large number of nearly 2000 reported mutations, including the premature termination codon (PTC) mutations, urgently require new and personalized medicines. We have developed cell-based assays for readthrough modulators of CFTR PTC mutations (or nonsense mutation suppressors), based on the trafficking and surface expression of CFTR. Approximately 85,000 compounds have been screened for two PTC mutations (Y122X and W1282X). The hit rates at the threshold of 50% greater than vehicle response are 2% and 1.4% for CFTR Y122X and CFTR W1282X, respectively. The overlap of the two hit sets at this stringent hit threshold is relatively small. Only ~28% of the hits from the W1282X screen were also hits in the Y122X screen. The overlap increases to ~50% if compounds are included that in the second screen achieve only a less stringent hit criterion, that is, horseradish peroxidase (HRP) activity greater than three standard deviations above the mean of the vehicle. Our data suggest that personalization may not need to address individual genotypes, but that patients with different CFTR PTC mutations could benefit from the same medicines.