Discovery Of G551D-CFTR Mutant Chloride Channel Activators And GlpF E.coli Glycerol Channel Inhibitors By High-throughput Screening

The purpose of this thesis is to develop cell-based assay models of mutant CFTR chloride channels and glycerol-selective transporters. Cystic fibrosis (CF) is one of the most common lethal autosomal recessive genetic diseases caused by over 1,300 mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-dependent chloride channel expressed in various fluid-transporting epithelia. The glycine to aspartic acid missence mutation at codon 551 of CFTR (G551D) is one of the five most frequent CF mutations with a frequency of 6% and is associated with severe CF phenotype. The G551D-CFTR belongs to class III CF mutations in which the processing, maturation, and assigned apical location of G551D-CFTR are not affected. However, chloride transport in cells expressing the G551D mutant CFTR cannot be activated by cAMP-elevating agents, resulting in deficiency of CFTR function.Although numerous activators of wild type CFTR have been identified, few of them can efficiently activate G551D-CFTR. Compounds such as isoflavone Genistein and benzo[c]quinolizinium compound MPB-91 can activate G551D-CFTR, but they act at high concentrations, nonspecific and sometimes have dual effects.One purpose of this study is to develop a cell-based fluorescent assay of G551D-CFTR function using halide-sensitive yellow fluorescent protein mutant (EYFP-H148Q-I152L) as intracellular indicator and to discover by high throughput screening high-affinity, specific and nontoxic small molecule G551D-CFTR activators as candidate drugs to ameliorate the chloride transport defect associated with class III CF. A stably transfected CHO cell line coexpressing G551D-CFTR and an iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established as assay model for G551D-CFTR chloride channel function. Isoflavone compound Genistein, a known low affinity G551D-CFTR activator, stimulatedG551D-CFTR-mediated chloride transport in the established CHO/G551D-CFTR/EYFP-H148Q-I152L assay system. A high-affinity (Kd~5 uM) bicyclooctane compound (G551D-CFTRBco) was identified to activate G551D-CFTR chloride channel by screening of 100,000 diverse small molecule compounds. The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. G551D-CFTReco will be useful in studies of structure and function of G551D-CFTR and of therapeutic correction of CF phenotypes caused by G551D-CFTR mutation.The second purpose of this study is to explore the feasibility of functional expression of E. coli glycerol transporter GlpF in mammalian cells and of high throughput screening using mammalian cell-based radioactive assay to identify small molecule inhibitors of glycerol transporters. A stably transfected CHO cell line with high expression of GlpF mRNA and protein was obtained and functionally characterized. Although GlpF protein was strongly expressed in cell membrane, it was poorly functional in transporting 14C-labled glycerol. Further work is needed to optimize the expression and assay procedure to develop a mammalian cell-based assay suitable for high throughput screening of specific glycerol transport inhibitors.