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Mechanism-based corrector combination restores ΔF508-CFTR folding and function

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Abstract

The most common cystic fibrosis mutation, ΔF508 in nucleotide binding domain 1 (NBD1), impairs cystic fibrosis transmembrane conductance regulator (CFTR)-coupled domain folding, plasma membrane expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and an incompletely understood mechanism, hampering drug development. Given the effect of second-site suppressor mutations, robust ΔF508-CFTR correction most likely requires stabilization of NBD1 energetics and the interface between membrane-spanning domains (MSDs) and NBD1, which are both established primary conformational defects. Here we elucidate the molecular targets of available correctors: class I stabilizes the NBD1-MSD1 and NBD1-MSD2 interfaces, and class II targets NBD2. Only chemical chaperones, surrogates of class III correctors, stabilize human ΔF508-NBD1. Although VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional plasma membrane expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in cystic fibrosis bronchial epithelial cells and intestinal organoids. Thus, the combination of structure-guided correctors represents an effective approach for cystic fibrosis therapy.

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Figure 1: Combination of corrector and suppressor mutations restores ΔF508-CFTR folding, plasma membrane expression and function.
Figure 2: VX-809 functions as a pharmacological chaperone of CFTR.
Figure 3: Evaluating corrector mechanism by using CFTR variants.
Figure 4: Effect of correctors on the isolated NBD1 stability in vitro and in vivo.
Figure 5: Combination of correctors targeting distinct structural defects completely restores ΔF508-CFTR folding, plasma membrane expression and stability in BHK cells.
Figure 6: Corrector combination restores ΔF508-CFTR functional expression in polarized epithelial cell lines and rectal organoids from ΔF508 cystic fibrosis patients.

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Acknowledgements

We thank L. Konerman, T. Wales, J. Engen, M.J. Chalmers and P.R. Griffin for valuable advice on setting up the HDX-MS technique; D. Gruenert (University of California–San Francisco) for the parental CFBE41o- cell line; R.J. Bridges (Rosalind Franklin University of Medicine and Science) and Cystic Fibrosis Foundation Therapeutics (CFFT) Inc. for CFTR modulator panels; D. Thomas (McGill University, Canada) for kindly providing RDR compounds; and W.R. Skach for advice. We are grateful for the financial support of the Dutch Cystic Fibrosis foundation and the Wilhelmina Children's Hospital Research fund to J.M.B., the Hungarian National Science Foundation (MB08C-80039) and the European Union (FP7-IRG 239270) to T.H., the European Research Area (ERA)-Chemistry Hungarian Scientific Research Fund (OTKA) (102166) to A.S., the Tara K. Telford Fund for Cystic Fibrosis Research at the University of California–Davis and the US National Institutes of Health (NIH; grants DK072517 and GM089153) to M.K., NIH–National Institute of Diabetes and Digestive and Kidney Diseases (R01DK75302), CFFT Inc., Cystic Fibrosis Canada, Canadian Institutes of Health Research and Canada Foundation for Innovation to G.L.L. T.H. is a Bolyai Fellow of the Hungarian Academy of Sciences. G.V. was partly supported by a European Molecular Biology Association and Fonds de Recherche Santé Québec Fellowship. G.L.L. is a Canada Research Chair.

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T.O. designed and carried out the biochemical studies, including the cell-surface ELISA, pulse-chase and immunoblotting assays, and analyzed the data. G.V. generated the inducible epithelial cell lines and carried out the ICl(apical) measurements. J.F.D. performed the intestinal organoid transport assay under the direction of J.M.B. M.B. carried out the bilayer measurements. N.S. measured the HDX of NBDs. H.X. engineered the CFTR mutants and generated the BHK cell lines. A.R. purified the NBDs and characterized their thermal stability. A.S.V. and M.K. provided reagents. A.S. and T.H. performed the in silico docking. G.L.L. conceived and directed the study and wrote the manuscript with T.O.

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Correspondence to Gergely L Lukacs.

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Okiyoneda, T., Veit, G., Dekkers, J. et al. Mechanism-based corrector combination restores ΔF508-CFTR folding and function. Nat Chem Biol 9, 444–454 (2013). https://doi.org/10.1038/nchembio.1253

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