title: Small-molecule screen identifies inhibitors of a human intestinal calcium-activated chloride channel.
abstract: Calcium-activated chloride channels (CaCCs) are widely expressed in mammalian tissues, including intestinal epithelia, where they facilitate fluid secretion. Potent, selective CaCC inhibitors have not been available. We established a high-throughput screen for identification of inhibitors of a human intestinal CaCC based on inhibition of ATP/carbachol-stimulated iodide influx in HT-29 cells after lentiviral infection with the yellow fluorescent halide-sensing protein YFP-H148Q/I152L. Screening of 50,000 diverse, drug-like compounds yielded six classes of putative CaCC inhibitors, two of which, 3-acyl-2-aminothiophenes and 5-aryl-2-aminothiazoles, inhibited by >95% iodide influx in HT-29 cells in response to multiple calcium-elevating agonists, including thapsigargin, without inhibition of calcium elevation, calcium-calmodulin kinase II activation, or cystic fibrosis transmembrane conductance regulator chloride channels. These compounds also inhibited calcium-dependent chloride secretion in T84 human intestinal epithelial cells. Patch-clamp analysis indicated inhibition of CaCC gating, which, together with the calcium-calmodulin data, suggests that the inhibitors target the CaCC directly. Structure-activity relationships were established from analysis of more than 1800 analogs, with IC(50) values of the best analogs down to approximately 1 muM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.
authors: 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco CA 94143-0521, USA.
authors: Ricardo
authors: R
authors: De La Fuente
authors: Wan
authors: W
authors: Namkung
authors: Aaron
authors: A
authors: Mills
authors: A S
authors: AS
authors: Verkman
mesh_terms: D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D002217:Carbachol / Q000037:antagonists & inhibitors / Q000494:pharmacology; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D002712:Chlorides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D005822:Genetic Vectors; D019073:HT29 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects / Q000473:pathology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics; D008954:Models, Biological; D015394:Molecular Structure; D018408:Patch-Clamp Techniques; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship
chemical_list: D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D002217:Carbachol; D002118:Calcium; D016257:Fura-2