Broad Institute Small-molecule targeting of CFTR: PDZ Trafficking Interactions Inhibitor Probe Project
The primary target of our project is to identify cellular probes that will (1) enable us to identify and characterize the intracellular Cystic fibrosis transmembrane conductance regulator (CFTR) trafficking pathways regulated by CFTR-Associated Ligand (CAL) and CAL PDZ inhibitors; and ..more
Dean Madden, Dartmouth University, firstname.lastname@example.org, 603-650-1164
Patrick Cushing, Dartmouth University, email@example.com, 603-748-9843
The primary target of our project is to identify cellular probes that will (1) enable us to identify and characterize the intracellular Cystic fibrosis transmembrane conductance regulator (CFTR) trafficking pathways regulated by CFTR-Associated Ligand (CAL) and CAL PDZ inhibitors; and
(2) serve as a scaffold for the development of potential therapeutics aimed at restoration of mucociliary clearance in Cystic Fibrosis (CF) patients.
We will execute a high-throughput screening effort for inhibitors and molecular probes of the PDZ domain of the CFTR-Associated Ligand (CAL). A major goal of our research is to understand the stereochemistry of PDZ:peptide interactions and to use that information to identify probes that can restore mucociliary clearance in the airways of patients with cystic fibrosis (CF).
CFTR, CAL, PDZ, Cystic Fibrosis
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that maintains the hydration of airway surface liquid in the lung. One mutation, the deletion of Phe508 (DF508), is present in ~90% of patients. The resulting protein is inefficiently folded, shows limited (but non-zero) channel activity, and is rapidly degraded. While compounds are being developed to address the first two defects, little has been done to address post-maturational stability. Previously, it has been shown that the PDZ (PSD-95, Discs large, ZO-1) protein CFTR-Associated Ligand (CAL) is critical for the degradation of CFTR. Knockdown of CAL prolongs the half-life of DF508-CFTR and increases its cell-surface abundance. The rescued DF508- CFTR protein is functional, as evidenced by increased chloride efflux in polarized airway epithelial cells. CAL contains a single PDZ domain that binds the CFTR C-terminus, an interaction that is required for CAL-mediated degradation of CFTR. CFTR also interacts with the PDZ proteins NHERF1 and NHERF2, which mediate favorable effects on CFTR activity. Our experiments have been designed to evaluate the importance of inhibitor selectivity on CFTR activity. Peptide inhibitor studies suggest that a non-selective inhibitor that binds the PDZ domains of CAL and the two NHERF proteins still provides significant rescue of CFTR-mediated chloride efflux. However, a CAL-selective inhibitor provides enhanced rescue and acts additively with a known corrector of the DF508-CFTR folding defect. Unfortunately, these peptide inhibitors act only for a short period (<3.5 hours) before losing efficacy. As a result, we have decided to investigate the CFTR stability effects of targeted inhibition of this domain by small molecules.