Cystic fibrosis (CF) is the most common lethal pediatric genetic disease in the Caucasian population. CF is caused by mutations in the CF gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel that belongs to the ATP-binding cassette family. A large number of mutations, including the most common deletion of phenylalanine 508 (deltaF508), cause folding and processing defect of CFTR. Using a functional high-throughput screening (HTS) assay, we have identified four classes of small molecules ('correctors') that partially revert the processing defect in transfected non-polarized and polarized cells, as well as in primary cultures of human airway epithelia. Our long-term goal is to identify clinically useful small molecules for CF therapy. Toward this goal we have three specific aims. 1) A comprehensive cell biological and biochemical analysis will establish the molecular mechanisms of deltaF508-CFTR corrector action. Potential sites of action to be analyzed include: translation, posttranslational folding, chaperone interactions, ER stability, cell surface stability, internalization and recycling efficiency and ubiquitination at the ER and post-Golgi compartments. Understanding the corrector mechanism is important to prioritize small molecule classes for further clinical development and in selecting synergistic combinations. 2) Additional HTS assays will be performed using a collection of >100,000 drug-like small molecules with maximum chemical diversity to identify highly efficient correctors. Hits will be optimized by medicinal chemistry and validated by biochemical and electrophysiological assays. A series of secondary and tertiary screens will be subsequently carried out to prioritize potential lead compounds for further development: efficacy studies on primary and immortalized cultures of human airway epithelia, pharmacological and efficacy analysis in rodents. 3) To identify novel targets for small molecule discovery, functional genetics for correction of the deltaF508-CFTR processing defect will be perfromed. Genome-wide loss-of-function (LOF) screens by siRNA knock-down and gain-of function (GOF) screens by cDNA transduction will be carried out using the biochemical/functional HTS assays. The proposed studies will provide an understanding of the small molecule correctors mechanism, as well as provide new classes of lead compounds for development and novel targets for small-molecule discovery in CF.
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