Date of Award

9-16-2011

Embargo Period

10-2-2012

Degree Type

Dissertation/Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

Advisor(s)

Jonathan W. Jarvik

Abstract

Cystic fibrosis is caused by mutations in the membrane chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, disrupts protein folding resulting in premature degradation which precludes expression at the cell surface. Therapeutic strategies have been developed to rescue ΔF508 by using small molecules, called correctors, which promote folding and trafficking to the surface. Currently, the discovery and evaluation of these correctors requires indirect functional measurements or time intensive biochemical methods. In order to facilitate faster analysis of corrector compounds and provide a screening assay that directly monitors rescue of ΔF508 trafficking, we developed a rapid fluorescence detection platform using fluorogen activating proteins (FAPs) capable of labeling of CFTR at the cell surface. We created two chimeric reporter constructs by fusing the FAP to the N-terminus, or by insertion of the FAP into the fourth extracellular loop. We expressed these constructs in HEK293 cells and verified that the ion transport function, biochemical properties and cellular localization reproduced the native behavior of CFTR. Under normal conditions, CFTR ΔF508 is absent from the surface, however incubation in the presence of correctors restored trafficking to the plasma membrane that was robustly detected by FAP fluorescence. Using this approach we have characterized the efficacy of two new corrector compounds C548, C951 and the well-studied reference corrector, C4. The most potent corrector identified was C951 which performed 2 fold better than the previously described, C4 corrector. Other studies have shown that combinations of correctors exhibit an additive or synergistic effect, therefore, we tested combinations of correctors using FAP-CFTR constructs and found they had a synergistic effect on the rescue of ΔF508, improving the density of protein at the cell surface 4 fold greater than C4 alone. These results correlated closely with functional data obtained from polarized human bronchial epithelia that endogenously express ΔF508, suggesting that the FAP tagged CFTR reporters represent a physiologically faithful model of corrector rescue.

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