Dab2 Inhibitors as Therapeutic Stabilizers of F508-CFTR
Project Leader: Dean R. Madden, Ph.D.
Professor of Biochemistry
Geisel School of Medicine at Dartmouth
Our research focuses on the structure and function of proteins that regulate intracellular trafficking of ion channels. This pilot application tests a new therapeutic strategy targeting the underlying molecular defect of cystic fibrosis (CF). ~90% of patients carry a CFTR mutation known as ΔF508, which causes three defects: inefficient biosynthesis, low channel activity, and rapid post-endocytic degradation. Current therapeutic strategies focus on filling the 'pool' of functional CFTR, but not on blocking the 'drain' that depletes it. Thus, our overarching hypothesis is that blocking post-endocytic CFTR degradation will significantly increase the amount of apical ΔF508-CFTR in combination with correctors of the folding defect, compared with correctors alone. We recently showed that even modest Dab2 knock-down reduces CFTR endocytic uptake. Peptide inhibitors of Dab2 mimic this effect, but exhibit low affinity (10 μM) and work only at high (>100 μM) concentration. Using peptide arrays, we have engineered a 2nd generation 50 nM inhibitor. We have also established a collaboration with Dr. Nico Derichs at the Charité University Medical Center (Berlin) to test inhibitor peptides in CF patient biopsies. We propose to build on this system to test our hypothesis. In aim 1, we will engineer 3rd generation Dab2 inhibitors (Ki<10nM) and test CFTR rescue by 2nd and 3rd generation cell-permeable iDab2 peptides using primary airway cells and CF patient rectal biopsy samples. Surface-labeling and Ussing chamber experiments alone and in combination with VX-809 and/or iCAL will establish the magnitude and complementarity of CFTR rescue. In aim 2, we will develop and pilot a high-throughput screen for small-molecule Dab2 inhibitors. Coupled to fluorescein, our enhanced-affinity iDab2 peptide provides a robust fluorescence polarization (FP) displacement signal. Working at the Broad Institute, we will develop an FP assay to permit high-throughput screening for drug-like small molecules that inhibit Dab2:peptide binding. Our studies will validate Dab2 as a new therapeutic target for CFTR stabilization, demonstrate the potential benefits of stand-alone and combination therapy, and initiate drug screening efforts.
Dr. Madden collaborates actively with other Lung Biology Center researchers (Hogan, O'Toole, Stanton) and with an international team dedicated to the development of translational peptide reagents including laboratories in Berlin, Germany (Derichs, Volkmer) and Montpellier, France (Boisguérin).