Principal Investigator/Program Director (Last, First, Middle): MADDEN, Dean R. Cystic fibrosis (CF) is the most common fatal genetic disorder among Caucasians and is caused by mutations in the gene encoding the chloride channel CFTR. Ninety percent of CF patients carry at least one copy of the mutation encoding F508-CFTR, which exhibits three major defects: inefficient biosynthesis, impaired channel function, and accelerated degradation. While the first two have been the focus of extensive pharmacological investigation, the third defect is poorly characterized. We have now shown that the CFTR-Associated Ligand CAL is implicated in regulating the post-maturational stability of F508-CFTR. CAL contains a PDZ (PSD-95/DLG/ZO-1) protein-protein interaction domain that binds to the CFTR C-terminus and is required for CAL-mediated degradation of CFTR. Correspondingly, CAL inhibition increases the apical membrane half-life and net functional expression level of F508-CFTR in polarized human airway epithelial cells. Given a lack of small-molecule probes for the CAL PDZ domain, we engineered peptides that selectively inhibit CAL binding for these proof-of-principle studies. However, a mechanistic understanding of CAL's effect will require dissection of the competing roles of CAL and other PDZ proteins in the endocytic pathways that modulate F508-CFTR stability. Inhibitors that would allow us to address this question should avoid the long incubation times required for CAL- specific RNAi, and also avoid the in vivo efficacy issues associated with peptide delivery and stability. To identify drug-like small-molecule inhibitors, we have now established orthogonal peptide-based FRET and AlphaScreen reporter assays. Here, we propose to combine these assays to develop a platform for identifying selective CAL inhibitors in high-throughput screening mode and to validate the platform using a sample array of 5600 bioactive compounds. Compounds identified by these techniques in single-point assays will be subjected to dose-response analysis. The validity and site(s) of interaction of dose-responding hits will be determined using an established NMR assay, both as a tertiary screen and to facilitate combinatorial chemistry approaches if needed. Confirmed hits will be counterscreened for lack of inhibition of the NHERF1 and NHERF2 PDZ domains, whose interaction with CFTR stabilizes cell-surface expression of functional chloride channels, and tested for biocompatibility. Our goal is to configure a robust protocol to screen large compound libraries for CAL inhibitors for downstream testing in our polarized human airway epithelial-cell system.
In CF, genetic mutation prevents the CFTR protein from functioning correctly, leading to chronic lung infection and death. We seek chemical compounds that can correct this functional defect.
| Cushing, Patrick R; Vouilleme, Lars; Pellegrini, Maria et al. (2010) A stabilizing influence: CAL PDZ inhibition extends the half-life of ýýF508-CFTR. Angew Chem Int Ed Engl 49:9907-11 |
