Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds

The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been show n to activate the cystic fibrosis transmembrane conductance regulator (CFTR ), the protein that is defective in cystic fibrosis, Lead-based combinatori al and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to a ctivate CFTR at 50 muM concentration by measurement of the kinetics of iodi de influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR to gether with the green fluorescent protein-based halide indicator YFP-H148Q, Duplicate screenings revealed that 204 compounds did not significantly aff ect CFTR function. Compounds of the 7,8-benzoflavone class, which are struc turally intermediate between flavones and benzo[c]quinoliziniums, were effe ctive CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H -chromen-4-one bisulfate (UCCF-029). Compounds of the novel structural clas s of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl) -5-methoxy-l -phenylpyrazole-4-carbaldehyde (UCCF-180). A CFTR inhibitor was also identified. The active compounds did not induce iodide influx in null cells deficient in CFTR, Short-circuit cu rrent measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on p orous supports. Compared with genistein, the most active compounds had up t o 10 times greater potency in activating wildtype and/or G551D-CFTR. The ac tivators had low cellular toxicity and did not elevate cellular cAMP concen tration or inhibit phosphatase activity, suggesting that CFTR activation ma y involve a direct interaction. These results establish an efficient screen ing procedure to identify CFTR activators and inhibitors and have identifie d 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activ ators.