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Received for publication January 4, 2005.
Revised February 18, 2005.
Accepted for publication February 18, 2005.
F508- AND G551D-CFTR CHLORIDE CHANNEL GATING
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 co-expressing F508-CFTR and a fluorescent halide indicator (YFP-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 Ka ~70 nM, and also activated the CFTR gating mutants G551D and G1349D with Ka ~1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had Ka ~20 nM for activation of F508-CFTR. In cell-attached patch-clamp experiments, PG-01 and SF-01 increased channel open probability >5-fold by 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 mono-therapy of cystic fibrosis caused by gating mutants and possibly for a subset of F508 subjects with significant F508-CFTR plasma membrane expression.
Key words:
Ion transporters (SERCA, Na/K ATPase, CFTR), Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants, Fluorescence techniques, Single channel kinetics
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