SLC26A6-selective inhibitor identified in a small-molecule screen blocks fluid absorption in small intestine.

SLC26A6 (also known as putative anion transporter 1 [PAT1]) is a Cl-/HCO3- exchanger expressed at the luminal membrane of enterocytes where it facilitates intestinal Cl- and fluid absorption. Here, high-throughput screening of 50,000 synthetic small molecules in cells expressing PAT1 and a halide-sensing fluorescent protein identified several classes of inhibitors. The most potent compound, the pyrazolo-pyrido-pyrimidinone PAT1inh-B01, fully inhibited PAT1-mediated anion exchange (IC50 ~350 nM), without inhibition of the related intestinal transporter SLC26A3 (also known as DRA). In closed midjejunal loops in mice, PAT1inh-B01 inhibited fluid absorption by 50%, which increased to >90% when coadministered with DRA inhibitor DRAinh-A270. In ileal loops, PAT1inh-B01 blocked fluid absorption by >80%, whereas DRAinh-A270 was without effect. In colonic loops, PAT1inh-B01 was without effect, whereas DRAinh-A270 completely blocked fluid absorption. In a loperamide constipation model, coadministration of PAT1inh-B01 with DRAinh-A270 increased stool output compared with DRAinh-A270 alone. These results provide functional evidence for complementary and region-specific roles of PAT1 and DRA in intestinal fluid absorption, with PAT1 as the predominant anion exchanger in mouse ileum. We believe that PAT1inh-B01 is a novel tool to study intestinal ion and fluid transport and perhaps a drug candidate for small intestinal hyposecretory disorders such as cystic fibrosis-related meconium ileus and distal intestinal obstruction syndrome.

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

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.

Hollow Micropillar Array Method for High-Capacity Drug Screening on Filter-Grown Epithelial Cells.

New high-throughput assay formats and innovative screening technologies are needed for miniaturized screens using small quantities of near-native, patient-derived cells. Here, we developed a hollow micropillar array method to screen compounds using epithelial cells cultured on a porous support, with the goal of screening thousands of compounds using a single 24 mm diameter transwell filter containing cultured cells. Test compounds (∼1 nL) in an alginate hydrogel were printed by microinjection in hollow cylindrical micropillars (height = 150 µm, inner diameter = 100 µm) spaced 300 µm apart in a square array configuration. Compounds were delivered by positioning the array near the surface of a cell layer, with 5-10 µm of distance between the micropillars and cell surface. Micropillar array geometry, and the viscosity of the hydrogel and overlying solutions, were optimized computationally and experimentally to produce sustained exposure of cells to test compounds with minimal cross-talk from compounds in neighboring micropillar wells. The method was implemented using a 10 × 10 micropillar array (size = 3 × 3 mm) on CFTR-expressing epithelial cells, in which CFTR chloride channel function was measured from fluorescence in response to iodide addition using a genetically encoded cytoplasmic yellow fluorescent protein halide indicator. The hollow micropillar array platform developed here should be generally applicable for high-capacity drug screening using small numbers of cells cultured on solid or porous supports.

Salt-sparing diuretic action of a water-soluble urea analog inhibitor of urea transporters UT-A and UT-B in rats.

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.

Antidiarrheal efficacy and cellular mechanisms of a Thai herbal remedy.

Screening of herbal remedies for Cl(-) channel inhibition identified Krisanaklan, a herbal extract used in Thailand for treatment of diarrhea, as an effective antidiarrheal in mouse models of secretory diarrheas with inhibition activity against three Cl(-) channel targets. Krisanaklan fully inhibited cholera toxin-induced intestinal fluid secretion in a closed-loop mouse model with ∼50% inhibition at a 1 ∶ 50 dilution of the extract. Orally administered Krisanaklan (5 µL/g) prevented rotavirus-induced diarrhea in neonatal mice. Short-circuit current measurements showed full inhibition of cAMP and Ca(2+) agonist-induced Cl(-) conductance in human colonic epithelial T84 cells, with ∼ 50% inhibition at a 1 ∶ 5,000 dilution of the extract. Krisanaklan also strongly inhibited intestinal smooth muscle contraction in an ex vivo preparation. Together with measurements using specific inhibitors, we conclude that the antidiarrheal actions of Krisanaklan include inhibition of luminal CFTR and Ca(2+)-activated Cl(-) channels in enterocytes. HPLC fractionation indicated that the three Cl(-) inhibition actions of Krisanaklan are produced by different components in the herbal extract. Testing of individual herbs comprising Krisanaklan indicated that agarwood and clove extracts as primarily responsible for Cl(-) channel inhibition. The low cost, broad antidiarrheal efficacy, and defined cellular mechanisms of Krisanaklan suggests its potential application for antisecretory therapy of cholera and other enterotoxin-mediated secretory diarrheas in developing countries.