Inhibitors of pendrin anion exchange identified in a small molecule screen increase airway surface liquid volume in cystic fibrosis

Abstract

Pendrin (SLC26A4) is a Cl 2 /anion exchanger expressed in the epithelium of inflamed airways where it is thought to facilitate Cl 2 absorption and HCO 3 2 secretion. Studies using pendrin knockout mice and airway epithelial cells from hearing-impaired subjects with pendrin loss of function suggest involvement of pendrin in inflammatory lung diseases, including cystic fibrosis (CF), perhaps by regulation of airway surface liquid (ASL) volume. Here we identified small-molecule pendrin inhibitors and demonstrated their efficacy in increasing ASL volume. A cell-based, functional high-throughput screen of ∼36,000 synthetic small molecules produced 3 chemical classes of inhibitors of human pendrin. After structure-activity studies, tetrahydropyrazolopyridine and pyrazolothiophenesulfonamide compounds reversibly inhibited pendrin-facilitated Cl 2 exchange with SCN 2 , I 2 , NO 3 2 , and HCO 3 2 with drug concentration causing 50% inhibition down to ∼2.5 mM. In well-differentiated primary cultures of human airway epithelial cells from non-CF and CF subjects, treatment with IL-13, which causes inflammation with strong pendrin up-regulation, strongly increased Cl 2 /HCO 3 2 exchange and the increase was blocked by pendrin inhibition. Pendrin inhibition significantly increased ASL depth (by ∼8 mm) in IL-13-treated non-CF and CF cells but not in untreated cells. These studies implicate the involvement of pendrin-facilitated Cl 2 / HCO 3 2 in the regulation of ASL volume and suggest the utility of pendrin inhibitors in inflammatory lung diseases, including CF

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

“…FRT epithelial cells (UCSF Cell Culture Facility), HEK cells (UCSF Cell Culture Facility), and HBE cell cultures (UCSF Cystic Fibrosis Research Development Program) were cultured using standard methods (19). For slc26a3 inhibitor screening, an FRT cell line virally transduced to express YFP (46) was transfected with pIRESpuro3-slc26a3, selected using 0.15 μg/ml puromycin, and a clonal cell line (termed FRT-YFP-slc26a3) was isolated.…”

“…The closest SLC26A3 homolog, SLC26A4 (pendrin; 45% amino acid identity), is an anion exchanger in the inner ear, thyroid, inflamed airways, and kidney (6,9,18). We previously identified small-molecule pendrin inhibitors that increased airway surface liquid depth in airway epithelial cell cultures and produced a diuretic response in mice, suggesting potential therapeutic utility in inflammatory lung disorders, such as CF and asthma, and in volume-overload edema (19,20). The second most closely related SLC26A3 homolog, SLC26A6 (putative anion transporter-1 [PAT-1]; 34% amino acid identity), is also an anion exchanger expressed at the apical membrane in the small intestine that, together with SLC26A3, facilitates electroneutral NaCl absorption (21,22).…”

Small-Molecule Inhibitors of Pendrin Potentiate the Diuretic Action of Furosemide

“…Amiloride-sensitive ENaC activity was measured by short-circuit current analysis of human bronchial epithelial cell cultures. 13 Specificity assays were done with 25 mM PDS inh -C01. …”

“…The inhibitors were identified in a high-throughput screen against human pendrin, which was recently reported. 13 Here, compounds were characterized and optimized for inhibition of murine pendrin and were tested in mice alone and in combination with short-or long-term diuretic therapy. Although no effect of pendrin inhibition alone was seen, as predicted from data in pendrin knockout mice and humans with Pendred syndrome, we found significant potentiation of the diuretic response to furosemide.…”

Pendrin—A New Target for Diuretic Therapy?

“…Verkman and colleagues 14 identified novel compounds selectively inhibiting pendrin with high affinity using a functional chemical library screen. As reported here in this issue of the Journal of the American Society of Nephrology, these compounds given to mice had, per se, no effect on diuresis or acid-base parameters in urine and blood.…”