title: Synergy-based small-molecule screen using a human lung epithelial cell line yields ΔF508-CFTR correctors that augment VX-809 maximal efficacy.
abstract: The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Puay-Wah
authors.initials: PW
authors.lastname: Phuan
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Guido
authors.initials: G
authors.lastname: Veit
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Tan
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Ariel
authors.initials: A
authors.lastname: Roldan
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Walter E
authors.initials: WE
authors.lastname: Finkbeiner
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
authors.forename: Gergely L
authors.initials: GL
authors.lastname: Lukacs
authors.affiliation: Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada Alan.Verkman@ucsf.edu.
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000631:Aminopyridines / Q000494:pharmacology*; D000818:Animals; D052117:Benzodioxoles / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000188:drug therapy / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D010539:Permeability / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D000631:Aminopyridines; D052117:Benzodioxoles; D002712:Chlorides; D054852:Small Molecule Libraries; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006735:Horseradish Peroxidase; C569105:lumacaftor
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