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ORIGINAL ARTICLE

Phenylglycine and Sulfonamide Correctors of Defective F508 and G551D Cystic Fibrosis Transmembrane Conductance Regulator Chloride-Channel Gating

Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California (N.P., N.D.S., J.H., A.S.V.); Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, Genova, Italy (N.P., A.T., O.Z.-M., L.J.V.G.); and Department of Chemistry, University of California, Davis, Davis, California (Y.F.S., L.I.R., C.W.D., D.W., M.H.N., M.J.K.)

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The F508 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 F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after F508-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 F508-CFTR gating ("potentiators") with nanomolar potency that were active in human F508 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 F508-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 F508-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 F508 subjects with significant F508-CFTR plasma-membrane expression.

Address correspondence to: Dr. Alan S. Verkman, 1246 Health Sciences East Tower, University of California, San Francisco, San Francisco, CA 94143-0521. E-mail: verkman{at}itsa.ucsf.edu

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