Cystic fibrosis (CF) is the most common genetic disease among Caucasians, and is caused by mutations of the chloride channel CFTR. 90% of CF patients carry at least one copy of the mutation encoding AF508-CFTR, which exhibits three major defects: ER retention, impaired channel function, and accelerated degradation. We hypothesize that selective inhibition of the CFTR-binding protein CAL will increase and stabilize cell-surface expression of AF508-CFTR. This proposal is based on the observations that (1) RNAi knock-down of endogenous CAL increases cell-surface expression of AF508-CFTR and transmembrane chloride currents in a polarized CF-patient airway epithelial cell line;(2) Localized, structurally conservative mutagenesis of the CAL binding pocket blocks CAL-mediated degradation of CFTR;and (3) Degradation can also be blocked by overexpression of another CFTR-binding protein, NHERF1. We have assembled a highly collaborative team with unique and complementary skills. Our goal is the targeted pharmacological disruption of a key CFTR trafficking interaction and its functional characterization.
The specific aims are: (1) To identify inhibitors of the CAL binding site with improved affinity, bioavailability, and selectivity vs. NHERF1. A first selective inhibitor has already been found. Peptide-based inhibitors will be detected using peptide-array and phage-display technologies. Biocompatible small-molecule inhibitors will be screened using a fluorescence-polarization binding assay. All """"""""hits"""""""" will be verified by secondary biochemical screens. (2) To characterize the effects of CAL inhibitors on cell-surface expression and chloride-channel activity of AF508-CFTR in polarized airway epithelial cells. For peptide inhibitors, delivery reagents, cell-penetrating peptide sequences, or side-chain cyclization will be used to facilitate delivery. Our small-molecule screens will focus on compounds with inherent permeability. Following delivery, we will use surface biotinylation and electrophysiological measurements to characterize AF508-CFTR rescue and co-immunoprecipitation experiments to quantify disruption of CFTR:PDZ interactions. The available inhibitor will allow us to begin functional studies and to develop a cell-based assay for tertiary compound screening of novel inhibitors. (3) To modify inhibitor lead compounds to enhance affinity and selectivity, and for peptide compounds, to optimize permeability and proteolytic stability. NMR will be used to determine binding stereochemistry of lead compounds, as a guide to chemical modification approaches. In addition to directed synthesis, combinatorial synthetic approaches will be used. Optimized compounds will be implemented in functional assays and will provide the basis for potential pharmaceutical development. Lay summary: In CF, genetic mutation prevents the CFTR protein from functioning correctly, leading to chronic lung infection and death. We seek chemicals that can correct this functional defect.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BST-Q (52))
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Mckeon, Catherine T
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Dartmouth College
Schools of Medicine
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Amacher, Jeanine F; Cushing, Patrick R; Brooks 3rd, Lionel et al. (2014) Stereochemical preferences modulate affinity and selectivity among five PDZ domains that bind CFTR: comparative structural and sequence analyses. Structure 22:82-93
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Kundu, Rituparna; Cushing, Patrick R; Popp, Brian V et al. (2012) Hybrid organic-inorganic inhibitors of a PDZ interaction that regulates the endocytic fate of CFTR. Angew Chem Int Ed Engl 51:7217-20
Bahl, Christopher D; Madden, Dean R (2012) Pseudomonas aeruginosa Cif defines a distinct class of ?/? epoxide hydrolases utilizing a His/Tyr ring-opening pair. Protein Pept Lett 19:186-93
Madden, Dean R; Swiatecka-Urban, Agnieszka (2012) Tissue-specific control of CFTR endocytosis by Dab2: Cargo recruitment as a therapeutic target. Commun Integr Biol 5:473-6
Amacher, Jeanine F; Cushing, Patrick R; Weiner, Joshua A et al. (2011) Crystallization and preliminary diffraction analysis of the CAL PDZ domain in complex with a selective peptide inhibitor. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:600-3

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