Related Experiment Video
Updated: Apr 27, 2026
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
Searching for combinations of small-molecule correctors to restore f508del-cystic fibrosis transmembrane conductance
Clément Boinot1, Mathilde Jollivet Souchet1, Romain Ferru-Clément1
1Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, Centre National de la Recherche Scientifique (CNRS), Poitiers, France.
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.
Insights
This study explored combinations of correctors to improve F508del-cystic fibrosis transmembrane conductance regulator (CFTR) protein function. Synergistic drug combinations, particularly VX-809 + IsoLAB, showed significant rescue of chloride channel activity, highlighting potential for improved CFTR therapies.
Area of Science:
- Molecular Biology
- Pharmacology
- Cell Physiology
Background:
- The F508del mutation in cystic fibrosis transmembrane conductance regulator (CFTR) causes protein misfolding, endoplasmic reticulum retention, and impaired chloride (Cl(-)) channel function.
- Individual CFTR correctors can partially restore F508del-CFTR trafficking and function, but synergistic approaches are needed for more effective rescue.
- Understanding the combined effects of correctors is crucial for developing improved therapeutic strategies for cystic fibrosis.
Purpose of the Study:
- To investigate the synergistic effects of multiple CFTR correctors on the function and maturation of F508del-CFTR.
- To evaluate the efficacy of specific corrector combinations, including VX-809 and IsoLAB, in rescuing F508del-CFTR chloride channel activity.
- To determine the influence of temperature on the effectiveness of CFTR corrector combinations.
Main Methods:
- Utilized HeLa cells expressing F508del-CFTR.
- Employed whole-cell patch-clamp technique to measure CFTR-mediated current density.
- Tested combinations of five correctors: miglustat, IsoLAB, Corr4a, VX-809, and suberoylamilide hydroxamic acid (SAHA).
Main Results:
- Several combinations, notably VX-809 + IsoLAB, significantly increased F508del-CFTR current density in response to activators.
- The VX-809 + IsoLAB combination demonstrated functional rescue of F508del-CFTR activity, with enhanced correction observed at 37°C compared to room temperature.
- Optimized incubation times for VX-809 + IsoLAB achieved maximal functional rescue comparable to individual optimal conditions.
Conclusions:
- Synergistic polypharmacology using combinations of correctors offers a promising strategy for rescuing F508del-CFTR function.
- Temperature plays a significant role in the efficacy of CFTR correctors and the level of functional rescue achieved.
- Optimized corrector combinations hold potential for developing more effective therapeutic interventions for cystic fibrosis.
More Related Videos
14:56Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae
Published on: March 10, 2012
07:04Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients
Published on: February 11, 2017
Related Concept Videos
01:24Cystic Fibrosis: Management
Sinus disease and chronic...
01:23Cystic Fibrosis: Pathogenesis
CF is primarily caused by a genetic mutation in a chromosome 7 gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common gene mutation leading to CF is the ΔF508 mutation,...
01:29Pharmacogenomics: Identification of New Drug Targets