title: Searching for combinations of small-molecule correctors to restore f508del-cystic fibrosis transmembrane conductance regulator function and processing.
abstract: The mutated protein F508del-cystic fibrosis transmembrane conductance regulator (CFTR) failed to traffic properly as a result of its retention in the endoplasmic reticulum and functions as a chloride (Cl(-)) channel with abnormal gating and endocytosis. Small chemicals (called correctors) individually restore F508del-CFTR trafficking and Cl(-) transport function, but recent findings indicate that synergistic pharmacology should be considered to address CFTR defects more clearly. We studied the function and maturation of F508del-CFTR expressed in HeLa cells using a combination of five correctors [miglustat, IsoLAB (1,4-dideoxy-2-hydroxymethyl-1,4-imino-l-threitol), Corr4a (N-[2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']bithiazolyl-2'-yl]-benzamide), VX-809 [3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid], and suberoylamilide hydroxamic acid (SAHA)]. Using the whole-cell patch-clamp technique, the current density recorded in response to CFTR activators (forskolin + genistein) was significantly increased in the presence of the following combinations: VX-809 + IsoLAB; VX-809 + miglustat + SAHA; VX-809 + miglustat + IsoLAB; VX-809 + IsoLAB + SAHA; VX-809 + miglustat + IsoLAB + SAHA. These combinations restored the activity of F508del-CFTR but with a differential effect on the appearance of mature c-band of F508del-CFTR proteins. Focusing on the VX-809 + IsoLAB cocktail, we recorded a level of correction higher at 37°C versus room temperature, but without amelioration of the thermal instability of CFTR. The level of functional rescue with VX-809 + IsoLAB after 4 hours of incubation was maximal and similar to that obtained in optimal conditions of use for each compound (i.e., 24 hours for VX-809 + 4 hours for IsoLAB). Finally, we compared the stimulation of F508del-CFTR by forskolin or forskolin + VX-770 [N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide] with cells corrected by VX-809 + IsoLAB. Our results open new perspectives for the development of a synergistic polypharmacology to rescue F508del-CFTR and show the importance of temperature on the effect of correctors and on the level of correction, suggesting that optimized combination of correctors could lead to a better rescue of F508del-CFTR function.
authors: Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France.
authors: Clément
authors: C
authors: Boinot
authors: Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France.
authors: Mathilde
authors: M
authors: Jollivet Souchet
authors: Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France.
authors: Romain
authors: R
authors: Ferru-Clément
authors: Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France frederic.becq@univ-poitiers.fr.
authors: Frédéric
authors: F
authors: Becq
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000631:Aminopyridines / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D052117:Benzodioxoles / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D013844:Thiazoles / Q000494:pharmacology
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D052117:Benzodioxoles; C505032:CFTR protein, human; C561871:N-(2-5-chloro-2-methoxyphenylamino)4'-methyl(4,5''bithiazolyl-2'-yl)benzamide; D013844:Thiazoles; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017485:1-Deoxynojirimycin; D005576:Colforsin; C059896:miglustat; C569105:lumacaftor