Selective isoxazolopyrimidine PAT1 (SLC26A6) inhibitors for therapy of intestinal disorders

Abstract

A loss of prosecretory Cl⁻ channel CFTR activity in the intestine is considered as the key cause of gastrointestinal problems in cystic fibrosis (CF): meconium ileus, distal intestinal obstruction syndrome (DIOS) and constipation. Since CFTR modulators have minimal effects on gastrointestinal symptoms, there is an unmet need for novel treatments for CF-associated gastrointestinal disorders. Meconium ileus and DIOS mainly affect the ileum (distal small intestine). SLC26A6 (putative anion transporter 1, PAT1) is a Cl⁻/HCO₃⁻ exchanger at the luminal membrane of small intestinal epithelial cells which facilitates Cl⁻ and fluid absorption. We recently identified first-in-class PAT1 inhibitors by high-throughput screening. Isoxazolopyrimidine PAT1_inh-A01 was a hit compound, which had low potency (IC₅₀ 5.2 μM) for SLC26A6 inhibition precluding further preclinical development. Here we performed structure–activity relationship studies to optimize isoxazolopyrimidine SLC26A6 inhibitors and tested a potent inhibitor in mouse models of intestinal fluid absorption. Structure–activity studies of 377 isoxazolopyrimidine analogs identified PAT1_inh-A0030 (ethyl 4-(benzyl(methyl)amino)-3-methylisoxazolo[5,4-d]pyrimidine-6-carboxylate) as the most potent SLC26A6 inhibitor with a 1.0 μM IC₅₀. Selectivity studies showed that PAT1_inh-A030 has no activity on relevant ion transporters/channels (SLC26A3, SLC26A4, SLC26A9, CFTR, TMEM16A). In a closed-loop model of intestinal fluid absorption, intraluminal PAT1_inh-A0030 treatment inhibited fluid absorption in the ileum of wild-type and CF mice (Cftr^{delF508/delF508}) with >90% prevention of a decrease in loop fluid volume and loop weight/length ratio at 30 minutes. These results suggest that SLC26A6 is the key transporter mediating Cl⁻ and fluid absorption in the ileum and SLC26A6 inhibitors are novel drug candidates for treatment of CF-associated small intestinal disorders.