title: 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.
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.
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.|Department of Neurobiology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: W Joon
authors.initials: WJ
authors.lastname: Chung
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Goeckeler-Fried
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Viktoria
authors.initials: V
authors.lastname: Havasi
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Annette
authors.initials: A
authors.lastname: Chiang
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.|Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.|Department of Cellular, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Rowe
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Zackery E
authors.initials: ZE
authors.lastname: Plyler
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.|Department of Cellular, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Jeong S
authors.initials: JS
authors.lastname: Hong
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.
authors.forename: Marina
authors.initials: M
authors.lastname: Mazur
authors.affiliation: Oncologic Sciences, USA Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, United States of America.
authors.forename: Gary A
authors.initials: GA
authors.lastname: Piazza
authors.affiliation: Oncologic Sciences, USA Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama, United States of America.
authors.forename: Adam B
authors.initials: AB
authors.lastname: Keeton
authors.affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.
authors.forename: E Lucile
authors.initials: EL
authors.lastname: White
authors.affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.
authors.forename: Lynn
authors.initials: L
authors.lastname: Rasmussen
authors.affiliation: Center for Cancer Research, National Institutes of Health, Frederick, Maryland, United States of America.
authors.forename: Allan M
authors.initials: AM
authors.lastname: Weissman
authors.affiliation: Worldwide Medicinal Chemistry, Pfizer, Cambridge, Massachusetts, United States of America.
authors.forename: R Aldrin
authors.initials: RA
authors.lastname: Denny
authors.affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
authors.affiliation: Department of Pediatrics, Emory University, Atlanta, Georgia, United States of America.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
mesh_terms: D000483:Alleles; D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005663:Furans / Q000737:chemistry / Q000494:pharmacology; D017353:Gene Deletion; D057809:HEK293 Cells; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects; D000077337:Vorinostat
chemical_list: C523934:4(4-(5-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl)-benzoic acid ethyl ester; D001565:Benzoates; D005663:Furans; D006877:Hydroxamic Acids; D011720:Pyrazoles; D012333:RNA, Messenger; D054852:Small Molecule Libraries; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000077337:Vorinostat