Phenylglycine and Sulfonamide Correctors of Defective ΔF508 and G551D Cystic Fibrosis Transmembrane Conductance Regulator Chloride-Channel Gating

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The ΔF508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 μM (with forskolin, 20 μM) by an iodide uptake assay in epithelial cells coexpressing ΔF508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after ΔF508-CFTR rescue by 24-h culture at 27°C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective ΔF508-CFTR gating (“potentiators”) with nanomolar potency that were active in human ΔF508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2–1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylacetamide, reversibly activated ΔF508-CFTR in the presence of forskolin with K_a ∼ 70 nM and also activated the CFTR gating mutants G551D and G1349D with K_a values of ∼1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K_a ∼ 20 nM for activation of ΔF508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by gating mutants and possibly for a subset of ΔF508 subjects with significant ΔF508-CFTR plasma-membrane expression.