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Targeting CFTR endocytic interactions as a therapeutic strategy in cystic fibrosis / by Lemira Sahar Al Ayyoubi.

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Author	Ayyoubi, Lemira Sahar Al.
Title	Targeting CFTR endocytic interactions as a therapeutic strategy in cystic fibrosis / by Lemira Sahar Al Ayyoubi.
Date	2014
Abstract	Despite the recent FDA approval of the potentiator drug Kalydeco for treatment of cystic fibrosis (CF), only a small (3-5%) portion of patients benefit from this drug. Other functional classes of drugs including correctors are under clinical investigation for monotherapy and dual regimen therapies with potentiators. We are investigating a functional class of therapeutics referred to as "stabilizers". Stabilizers target the stability defect of CFTR at the apical membrane of epithelial cells. Mutant CFTR exhibits enhanced internalization and reduced recycling efficiency, resulting in CFTR instability at the apical membrane. An endocytic adaptor protein, Disabled-2 (Dab2), has been reported to promote the internalization of CFTR. The observation that RNAi- mediated knockdown of Dab2 in CF-derived bronchial epithelial cells increased apical CFTR, prompted investigating Dab2 as a therapeutic target. Dab2-internalization of CFTR requires a ?200 amino acid Dab-homology domain (DH) at the N-terminus of Dab2. A peptide targeting the DH domain inhibited CFTR internalization and resulted in increased apical CFTR. Using peptide arrays, we reengineered the low affinity peptide (K[subscript d]=20 [mu]M) into a high affinity binder (K[subscript d] = 150 nM). Using fluorescence anisotropy (FP) based competition assays and the newly developed high-affinity peptide as a reporter; we optimized and performed a high throughput screen of a 1600-compound library for DH domain binding small molecules. Our efforts resulted in five positive hits that are being currently biochemically and functionally investigated. To stabilize CFTR at the apical membrane, multiple approaches can be adopted. In addition to blocking the initial uptake, post-endocytic CFTR trafficking steps can be manipulated to promote recycling or inhibit targeting for lysosomal degradation. CFTR-associated ligand (CAL) is a protein that targets CFTR for lysosomal degradation. It was previously shown that peptide- and small-molecule based targeting of the CAL : CFTR interaction increased the stability of apical CFTR. Optimization of CAL targeting for CFTR stabilization necessitates understanding the CAL : CFTR interaction. We used immunostaining and subcellular fractionation techniques to determine the intracellular localization of CAL in CF-derived bronchial epithelial cells. We employed immuno-isolation techniques to investigate the role of CAL in CFTR trafficking through the Rab-GTPase regulated endosomal compartments.
Description	viii, 100 leaves : ill. (some col.)
Format	application/pdf
College	Dartmouth Medical School. Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.1255

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