Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists

Drug discovery by high-throughput screening is a promising approach to deve lop new therapies for the most common lethal genetic disease, cystic fibros is. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl- permeability, the goal of screening is to identify compounds that restore cell Cl- transport. We have developed a rapid, quantitative sc reening procedure for analysis of CFTR-mediated halide transport in cells w ith the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together w ith a yellow fluorescent protein (YFP)-based halide sensor. CFTR function w as assayed from the time course of cell fluorescence in response to extrace llular addition of 100 mM I- followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I- entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize a ssay sensitivity. In cells cultured on 96-well plastic dishes, the assay ga ve reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low c oncentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclop entyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl- channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the char acterization of alternative Cl- channels and transporters.