Trans-NIH Research Support

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DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. When CFTR is absent, airway surface liquid is depleted, the clearance of mucus and bacteria from the lungs is impaired, and inflammation and bronchiectasis lead to respiratory failure. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, eliminating its trafficking to the epithelial cell apical membrane. Although High Throughput Screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, primary CF drug discovery assays have employed measurements of CFTR's anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Until now, there has been no simple method to directly determine correction of the primary defect in CF: trafficking of the mutant protein to the cell surface. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules and have extended this assay to the 96-well format. Using this approach, we will: 1) Perform HTS in collaboration with the MLSCN to identify new correctors of F508del CFTR trafficking, 2) Employ secondary screens to verify hits from HTS and evaluate their selectivity, and 3) Determine hit efficacy using human airway cell primary cultures from F508del patients, the most relevant assay available currently for advancing CF drugs to the clinic. This new and simple assay of F508del CFTR at the cell surface should permit the discovery more efficacious drugs and thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform will make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins.