Identification of new small molecule inhibitors of cystic fibrosis transmembrane conductance regulator protein: In vitro and in vivo studies

Cystic fibrosis transmembrane conductance regulator (CFTR) protein is a cAMP-regulated chloride channel that has been proposed as a pharmacological target to reduce intestinal fluid loss in cholera. The aim of this study was to identify new CFTR inhibitors by high-throughput screening. Screening of 50,000 drug-like small molecules was performed using a cell-based assay of iodide influx in Fisher rat thyroid (FRT) cells co-expressing human CFTR and halide-sensitive yellow fluorescent protein (YFP-H148Q). Two new CFTR inhibitors, 2-[N-(3-hydroxy-4-carboxyphenyl) aminol-4-(4-methylphenyl)-thiazole (INH 1) and 1-acetyl-5-bromo-2,3-dihydro-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1H-Indole-7-sulfonamide (INH 2), were identified. They were then determined for potency, reversibility and specificity by electrophysiological methods, and for in vivo efficacy in mouse model of cholera toxin-induced intestinal fluid secretion. INH 1 and INH 2 reversibly inhibited cAMP-activated apical chloride current in FRT cells with K(i)s of 15 and 20 mu m, respectively. Similarly, in short-circuit current analysis in human colonic epithelial cell lines (T84 cells), cAMP-activated chloride secretion was inhibited by INH 1 and INH 2 with Kis of 24.5 and 25.3 mu m, respectively. Calcium-activated chloride secretion in the T84 cells was markedly inhibited by 100 mu m of INH 1, but was unaffected by 100 mu m of INH 2. In vivo studies in mice showed that a single intraperitoneal injection of INH 1 (3 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 40%, whereas INH 2 produced no effect. Our results indicate that INH 1 could be a new class candidate for a blocker of cholera toxin-induced intestinal fluid secretion as well as a CFTR inhibitor.