Drug Development and Conservation of Biodiversity in West and Central Africa: Performance of Neurochemical and Radio Receptor Assays of Plant Extracts Drug Discovery for the Central Nervous System PRINCIPAL INVESTIGATOR: Simon Efange, Ph.D

Background: Cystic fibrosis (CF) results from mutationsin the cystic fibrosis transmembrane conductance regula-tor (CFTR) gene, which encodes a chloride channel local-ized at the plasma membrane of diverse epithelia. Themost common mutation leading to CF, F508, occurs inthe first nucleotide-binding domain (NBD1) of CFTR. TheF508 mutation disrupts protein processing, leading to adecreased level of mutant channels at the plasma mem-brane and reduced transepithelial chloride permeability.Partial correction of the F508 molecular defect in vitro isachieved by incubation of cells with several classes ofchemical chaperones, indicating that further investigationof novel small molecules is warranted as a means for pro-ducing new therapies for CF.Materials and Methods: The yeast two-hybrid assay wasused to study the effect of CF-causing mutations on theability of NBD1 to self-associate and form dimers. A yeaststrain demonstrating defective growth as a result of im-paired NBD1 dimerization due to F508 was used as adrug discovery bioassay for the identification of plantnatural product compounds restoring mutant NBD1 in-teraction. Active compounds were purified and thechemical structures determined. The purified compoundswere tested in epithelial cells expressing CFTR F508 Address correspondence and reprint requests to: John Teem,Department of Biological Science, Biounit-238, Florida StateUniversity, Tallahassee, Florida 32306, USA. Phone: (850) 644-5121;fax: (850) 644-0481; e-mail: [email protected] the resulting effect on transepithelial chloride perme-ability was assessed using short-circuit chloride currentmeasurements.Results: Wild-type NBD1 of CFTR forms homodimers ina yeast two-hybrid assay. CF-causing mutations withinNBD1 that result in defective processing of CFTR ( F508,I507, and S549R) disrupted NBD1 interaction in yeast.In contrast, a CF-causing mutation that does not impairCFTR processing (G551D) had no effect on NBD1 dimer-ization. Using the yeast-based assay, we identified a novellimonoid compound (TS3) that corrected the F508 NBD1dimerization defect in yeast and also increased the chlo-ride permeability of Fisher Rat Thyroid (FRT) cells stablyexpressing CFTR F508.Conclusion: The establishment of a phenotype for theF508 mutation in the yeast two-hybrid system yielded asimple assay for the identification of small molecules thatinteract with the mutant NBD1 and restore dimerization.The natural product compound identified using the sys-tem (TS3) was found to increase chloride conductance inepithelial cells to an extent comparable to genistein, aknown CFTR activator. The yeast system will thus be use-ful for further identification of compounds with potentialfor CF drug therapy.