Substituted 4-methylcoumarin inhibitors of SLC26A3 (DRA) for treatment of constipation and hyperoxaluria.

SLC26A3, also known as downregulated in adenoma (DRA), is an anion (Cl-, HCO3- and oxalate) exchanger in the luminal membrane of intestinal epithelial cells. Loss of DRA function in mice and humans causes congenital chloride-losing diarrhea and reduces urinary excretion of oxalate, a major constituent of kidney stones. Thus, inhibition of DRA is a potential treatment approach for constipation and calcium oxalate kidney stones. High-throughput screening previously identified 4,8-dimethylcoumarins (4a-4c) as DRA inhibitors, with lead candidate 4b having an IC50 of 40-50 nM for DRA inhibition. Here, we explored the effects of varying substituents at the 8-position, and replacing 8-methyl by 5-methyl (4e-4h). A focused library of 17 substituted compounds (4d-4t) was synthesized with good yield and purity. Compounds were tested for DRA inhibition potency using Fischer rat thyroid cells stably expressing DRA and a halide-sensitive YFP. Structure-activity analysis revealed that 8-bromo- (4m-4p) and 8-fluoro-coumarins (4q-4t) were slightly less potent than the corresponding 8-chloro analogs, demonstrating that the size of methyl or chloro substituents at the coumarin 8 position affects the potency. An analog containing 8-chlorocoumarin (4k) had ∼2-fold improved potency (IC50 25 nM) compared with the original lead candidate 4b. 5,8-Dimethylcoumarins were active against DRA, but with much lower potency than 4,8-disubstituted coumarins. In mice, orally administered 4k at 10 mg kg-1 reduced constipation and normalized stool water content in a loperamide-induced constipation model with comparable efficacy to 4b. Pharmacokinetic analysis of orally administered 4k at 10 mg kg-1 in mice indicated serum levels of >10 µM for at least six hours after single dose. This study expands SAR knowledge of 4,8-disubstituted coumarin inhibitors of DRA as novel drug candidates for constipation and kidney stones.

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function.

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.