title: Identification of new small molecule inhibitors of cystic fibrosis transmembrane conductance regulator protein: in vitro and in vivo studies.
abstract: 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) amino]-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 Kis of 15 and 20 microM, 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 microM, respectively. Calcium-activated chloride secretion in the T84 cells was markedly inhibited by 100 microM of INH 1, but was unaffected by 100 microM 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.
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Chatchai
authors.initials: C
authors.lastname: Muanprasat
authors.forename: Santi
authors.initials: S
authors.lastname: Kaewmokul
authors.forename: Varanuj
authors.initials: V
authors.lastname: Chatsudthipong
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D047310:Apigenin / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D045744:Cell Line, Tumor; D018118:Chloride Channels / Q000502:physiology; D002772:Cholera Toxin / Q000633:toxicity; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects / Q000473:pathology; D007583:Jejunum / Q000187:drug effects / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D015394:Molecular Structure; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D013449:Sulfonamides / Q000737:chemistry / Q000494:pharmacology*; D013764:Tetrahydronaphthalenes / Q000737:chemistry / Q000494:pharmacology; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C522103:1-acetyl-5-bromo-2,3-dihydro-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1H-Indole-7-sulfonamide; C522102:2-(N-(3-hydroxy-4-carboxyphenyl)amino)-4-(4-methylphenyl)thiazole; D018118:Chloride Channels; D008164:Luminescent Proteins; D010726:Phosphodiesterase Inhibitors; D013449:Sulfonamides; D013764:Tetrahydronaphthalenes; D013844:Thiazoles; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D003994:Bucladesine; D000584:Amiloride; D047310:Apigenin; D002217:Carbachol; D002772:Cholera Toxin; D015056:1-Methyl-3-isobutylxanthine