Cystic fibrosis is a life-threatening genetic disease characterized by the altered secretion of electrolytes by the secretory epithelial cells of the body. It is caused by mutations in the CF gene, which encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a cell surface localized integral membrane protein that functions as chloride ion channel. The CF disease allele found in nearly 90% of cystic fibrosis patients in North America contains a trinucleotide deletion that removes the phenylalanine at position 508 of the CFTR peptide sequence. This protein, CFTR-deltaF508, is a conformational mutant that is recognized by the cellular protein quality control system and is eliminated via the ubiquitin-proteasome system in a process known as endoplasmic reticulum-associated degradation. However, the ability of CFTR-deltaF508 to function as a chloride channel under conditions that permit CFTR-deltaF508 to properly fold and traffic to the plasma membrane, such as at reduced incubation temperatures (<30?C), indicate that the rescue of CFTRdeltaF508 folding may serve as a feasible target for the treatment of cystic fibrosis. The identification of small molecules that bind to and stabilize the native fold of CFTR-deltaF508 (pharmacological chaperones) or of proteins involved in CFTR-deltaF508 processing by gene-knockdown studies have been hindered by the lack of technology to directly measure CFTR-delta508 present on the cell surface that is suited to high throughput screening (HTS) platforms. To overcome these limitations we have developed a rapid and robust cell-based system to directly detect the rescue of misfolded CFTR-deltaF508 to the cell surface that combines high affinity ligand binding, enzymatic complementation/signal amplification and a sensitive FRET based fluorescence detection system. The capability of this system is exemplified in a preliminary HTS study, in which we identified a novel small molecule derived from plant extracts that rescues CFTRdeltaF508 misfolding. Therefore, we propose to use this tool to 1) assess the ability of previously identified modulators of CFTR channel activity to rescue CFTR-deltaF508 misfolding , 2) perform screen of a set of natural products extracts for potential compounds that correct the folding of CFTR-deltaF508 and 3) set up a system in human cell line amenable to genetic studies to identify suppressors of CFTR-deltaF508 ER retention and degradation. The short-term goals of this proposal are to provide a greater understanding of the CFTR-deltaF508 biology, to identify CFTR-deltaF508 pharmacological chaperones and to discover proteins that could serve as drug targets for the rescue of CFTR-deltaF508 trafficking. The broad objective of our studies is to uncover leads that could translate into new treatments for cystic fibrosis.
