Abstract
The flavonoid genistein and the benzo[c]quinolizinium MPB-07 have been shown to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the protein that is defective in cystic fibrosis. Lead-based combinatorial and parallel synthesis yielded 223 flavonoid, quinolizinium, and related heterocyclic compounds. The compounds were screened for their ability to activate CFTR at 50 microm concentration by measurement of the kinetics of iodide influx in Fisher rat thyroid cells expressing wild-type or G551D CFTR together with the green fluorescent protein-based halide indicator YFP-H148Q. Duplicate screenings revealed that 204 compounds did not significantly affect CFTR function. Compounds of the 7,8-benzoflavone class, which are structurally intermediate between flavones and benzo[c]quinoliziniums, were effective CFTR activators with the most potent being 2-(4-pyridinium)benzo[h]4H-chromen-4-one bisulfate (UCcf-029). Compounds of the novel structural class of fused pyrazolo heterocycles were also strong CFTR activators with the most potent being 3-(3-butynyl)-5-methoxy-1-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 current measurements showed that the CFTR activators identified by screening induced strong anion currents in the transfected cell monolayers grown on porous supports. Compared with genistein, the most active compounds had up to 10 times greater potency in activating wild-type and/or G551D-CFTR. The activators had low cellular toxicity and did not elevate cellular cAMP concentration or inhibit phosphatase activity, suggesting that CFTR activation may involve a direct interaction. These results establish an efficient screening procedure to identify CFTR activators and inhibitors and have identified 7,8-benzoflavones and pyrazolo derivatives as novel classes of CFTR activators.
Highlights
The most common lethal genetic disease, cystic fibrosis (CF),1 is caused by mutations in the cystic fibrosis transmembrane conductance regulator protein CFTR [1]
The compounds are referred to as UCCF-01 (University of California-cystic fibrosis) through UCCF-223 and have been grouped in the following structural classes: flavonoids, quinoliziniums, pyridiniums, azacyanines, isoxazoles, and fused pyrazole heterocycles
FRT cells coexpressing human wild-type CFTR or G551D CFTR and YFP-H148Q were cultured on 96-well plates for the monitoring of YFP fluorescence in a plate reader
Summary
The most common lethal genetic disease, cystic fibrosis (CF), is caused by mutations in the cystic fibrosis transmembrane conductance regulator protein CFTR [1]. Activators of CFTR chloride permeability can function by a number of direct and indirect mechanisms including increased cAMP production, inhibition of phosphodiesterase or phosphatase activities, or direct interactions with CFTR. Preliminary combinatorial libraries were synthesized based on the flavone and benzo[c]quinolizinium structures We chose these lead compounds because they are the most likely of the known activators to interact directly with CFTR, and they share a common structural motif amenable to the design of hybrid structures. Identification of Novel CFTR Activators compounds for CFTR-activating potency by a cell-based halide transport assay that utilized fluorescent epithelial cells stably expressing wild-type or G551D CFTR together with the green fluorescent protein halide indicator YFP-H148Q. The screen revealed new classes of CFTR activators and a CFTR inhibitor, which were characterized further in terms of potency, CFTR specificity, activation mechanism, and the ability to induce transepithelial chloride currents in polarized epithelial cells
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