Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs
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Research Article Cell biology Pulmonology

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination

Abstract

Based on its clinical benefits, Trikafta — the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) — was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.

Authors

Guido Veit, Ariel Roldan, Mark A. Hancock, Dillon F. Da Fonte, Haijin Xu, Maytham Hussein, Saul Frenkiel, Elias Matouk, Tony Velkov, Gergely L. Lukacs

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Figure 2

VX-445 binds to and changes the unfolding trajectory of CFTR-NBD1.

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VX-445 binds to and changes the unfolding trajectory of CFTR-NBD1. (A) R...
(A) Representative surface plasmon resonance (SPR) sensorgram for the binding of VX-445 (0–200 μM) to immobilized F508del–NBD1-1S. (B) Binding isotherms for VX-445 binding to immobilized F508del–NBD1-1S or WT–NBD1-1S as determined by SPR (n = 3). Curve fitting was performed as described in Methods. (C) Aggregation rates observed in NBD1 aggregation assays between 30 and 50 minutes for the different compounds were normalized by the rate observed for F508del–NBD1-1S in 1% DMSO (n = 4). Compound/chaperone concentrations were 50 or 100 μM VX-445, 10% glycerol, 100 μM VX-661, 100 μM 3151, 10 μM DnaK, or DnaK-DnaJ-GrpE at 10, 2, and 10 μM, respectively. (D) Protein secondary structure stability was studied by far-UV CD spectra of F508del–NBD1-1S. CD scans between 250 and 195 nm were taken every minute at 32°C in the presence of vehicle control (1% 1,4-Dioxane), 100 μM VX-661, or 100 μM VX-445. CD scans obtained at different time intervals of 1 representative experiment were overlaid. (E) Quantification of the ellipticity values (in mDeg) observed at 207 nm. Values were plotted as a function of time in the presence of vehicle control (1% 1,4-Dioxane); 50, 100, or 200 μM VX-445; or 100 μM VX-661 (n = 2–3). Continuous lines were derived by 4-point smooth iteration. Data in B, C, and E are means ± SEM of the indicated number of independent experiments. *P < 0.05, **P < 0.01 by 1-way ANOVA followed by Turkey’s post hoc test.