Increasing the Endoplasmic Reticulum Pool of the F508del Allele of the Cystic Fibrosis Transmembrane Conductance Regulator Leads to Greater Folding Correction by Small Molecule Therapeutics

• Authors: Wook Chung, Emory University; Jennifer L. Goeckeler-Fried, University of Pittsburgh; Viktoria Havasi, University of Alabama at Birmingham; Annette Chiang, University of Pittsburgh; Steven M. Rowe, University of Alabama at Birmingham; Zackery E. Plyler, University of Alabama at Birmingham; Jeong Hong, Emory University; Marina Mazur, University of Alabama at Birmingham; Gary A Piazza, University of South Alabama; Adam B. Keeton, University of South Alabama; E. Lucile White, Southern Research Institute; Lynn Rasmussen, Southern Research Institute; Allan M. Weissman, National Institutes of Health; R. Aldrin Denny, Pfizer; Jeffrey L. Brodsky, University of Pittsburgh; Eric Sorscher, Emory University
• Journal Title: PLoS ONE
• Volume: Volume 11, Number 10
• Publisher: Public Library of Science | 2016-10-12, Pages e0163615-e0163615
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1371/journal.pone.0163615
• Subjects: Biology, Bioinformatics; Biology, Genetics; Health Sciences, General
• Keywords: Science & Technology; Multidisciplinary Sciences; DELTA-F508 CFTR; IN-VITRO; MUTANT PROTEIN; PHE508DEL CFTR; MESSENGER-RNA; DEGRADATION; MUTATION; PROTEASOME; IVACAFTOR; RESCUE; Library screening; Small molecules; Epithelial cells; Cystic fibrosis; Cell membranes; Messenger RNA; HEK 293 cells; Cell cultures

Abstract

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ∼2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ∼10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41 - a compound that inhibits the E1 ubiquitin activating enzyme - was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.