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 F508, 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 F508-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 M) and work only at high (>100 M) 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).
Sahar Al-Ayyoubi, Graduate Student Sahar has developed peptide-arrays and is pioneering the FP screening assay.
Moises Chavez, Graduate Student Moises is developing a FRET-based assay for detection of Dab2 binding.
Jeanine Amacher, Graduate Student Jeanine has uncovered the basis for selective peptide inhibition of the CFTR-Associated Ligand (CAL), another target for the post-endocytic stabilization of CFTR.
Yu Zhao, Graduate Student Yu is developing and optimizing small-molecule and peptidomimetic inhibitors of CAL.
Kelli Hvorecny, Graduate Student Kelli is focused on understanding the mechanism of action of a Pseudomonas aeruginosa virulence factor Cif, which triggers degradation of CFTR and the transporter associated with antigen presentation (TAP1).
Chris Pennil, Research Associate Chris studies the role of a transcriptional regulator of Cif, known as CifR, which controls Cif expression and thus represents an additional therapeutic target.
Ruth Sinnamon, Graduate Student Ruth is investigating the binding specificity of the PDZ domains in the Multiple PDZ Protein MPDZ/MUPP1, a neuronal scaffolding protein implicated in addiction processes.