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SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation
Peter M. Haggie, … , Puay-Wah Phuan, Alan S. Verkman
Peter M. Haggie, … , Puay-Wah Phuan, Alan S. Verkman
Published July 26, 2018
Citation Information: JCI Insight. 2018;3(14):e121370. https://doi.org/10.1172/jci.insight.121370.
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Research Article Gastroenterology

SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation

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Abstract

SLC26A3 (downregulated in adenoma; DRA) is a Cl–/anion exchanger expressed in the luminal membrane of intestinal epithelial cells, where it facilitates electroneutral NaCl absorption. SLC26A3 loss of function in humans or mice causes chloride-losing diarrhea. Here, we identified slc26a3 inhibitors in a screen of 50,000 synthetic small molecules done in Fischer rat thyroid (FRT) cells coexpressing slc26a3 and a genetically encoded halide sensor. Structure-activity relationship studies were done on the most potent inhibitor classes identified in the screen: 4,8-dimethylcoumarins and acetamide-thioimidazoles. The dimethylcoumarin DRAinh-A250 fully and reversibly inhibited slc26a3-mediated Cl– exchange with HCO3–, I–, and thiocyanate (SCN–), with an IC50 of ~0.2 μM. DRAinh-A250 did not inhibit the homologous anion exchangers slc26a4 (pendrin) or slc26a6 (PAT-1), nor did it alter activity of other related proteins or intestinal ion channels. In mice, intraluminal DRAinh-A250 blocked fluid absorption in closed colonic loops but not in jejunal loops, while the NHE3 (SLC9A3) inhibitor tenapanor blocked absorption only in the jejunum. Oral DRAinh-A250 and tenapanor comparably reduced signs of constipation in loperamide-treated mice, with additive effects found on coadministration. DRAinh-A250 was also effective in loperamide-treated cystic fibrosis mice. These studies support a major role of slc26a3 in colonic fluid absorption and suggest the therapeutic utility of SLC26A3 inhibition in constipation.

Authors

Peter M. Haggie, Onur Cil, Sujin Lee, Joseph-Anthony Tan, Amber A. Rivera, Puay-Wah Phuan, Alan S. Verkman

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Figure 1

Assay for high-throughput identification of SLC26A3 inhibitors.

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Assay for high-throughput identification of SLC26A3 inhibitors.
(A) Assa...
(A) Assay schematic. Extracellular addition of an I–-containing solution drives slc26a3-mediated Cl–/I– exchange, resulting in YFP fluorescence quenching. (B) Representative fluorescence time course data for nontransfected cells, and slc26a3-transfected cells for vehicle control and inactive and active compounds. (C) Absence of slc26a3 regulation by common second messengers, and membrane depolarization. Fluorescence time course data (top) and summary (bottom) for slc26a-mediated Cl–/I– exchange under control conditions and after cell treatments to activate cAMP, cGMP, Ca2+, or phorbol ester signaling, or depolarization (mean ± SEM, n = 5, differences not significant by one-way ANOVA).

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