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 IC50 values of the best analogs down to ∼1 μM. Small-molecule CaCC inhibitors may be useful in pharmacological dissection of CaCC functions and in reducing intestinal fluid losses in CaCC-mediated secretory diarrheas.
Footnotes
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This work was supported by grants DK72517, HL73854, EB00415, EY13574, DK35124, and DK43840 from the National Institutes of Health, and Drug Discovery and Research Development Program grants from the Cystic Fibrosis Foundation.
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W.N. and A.M. contributed equally to this work.
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ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; CaCC, calcium-activated Cl- channel; YFP, yellow fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; NMDG, N-methyl-d-glucamine chloride; CaMKII, calcium-calmodulin protein kinase 2.
- The American Society for Pharmacology and Experimental Therapeutics
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