Partial Rescue of F508del-CFTR Stability and Trafficking Defects by Double Corrector Treatment

Abstract

Deletion of phenylalanine at position 508 (F508del) in the CFTR chloride channel is the most frequent mutation in cystic fibrosis (CF) patients. F508del impairs the stability and folding of the CFTR protein, thus resulting in mistrafficking and premature degradation. F508del-CFTR defects can be overcome with small molecules termed correctors. We investigated the efficacy and properties of VX-445, a newly developed corrector, which is one of the three active principles present in a drug (Trikafta^®/Kaftrio^®) recently approved for the treatment of CF patients with F508del mutation. We found that VX-445, particularly in combination with type I (VX-809, VX-661) and type II (corr-4a) correctors, elicits a large rescue of F508del-CFTR function. In particular, in primary bronchial epithelial cells of CF patients, the maximal rescue obtained with corrector combinations including VX-445 was close to 60-70% of CFTR function in non-CF cells. Despite this high efficacy, analysis of ubiquitylation, resistance to thermoaggregation, protein half-life, and subcellular localization revealed that corrector combinations did not fully normalize F508del-CFTR behavior. Our study indicates that it is still possible to further improve mutant CFTR rescue with the development of corrector combinations having maximal effects on mutant CFTR structural and functional properties.

Keywords: VX-445; allosteric folding correction; chloride secretion; conformational stability; elexacaftor; primary bronchial cells.

Figure 1

Functional evaluation of VX-445 corrector activity in heterologous expression systems. Compounds were tested on CFBE41o- and FRT cells expressing F508del-CFTR and the halide-sensitive yellow fluorescent protein (HS-YFP). (A) Dose-responses relationships for VX-445 and its (R)-enantiomer (i.e., distomer), as single agents or in the presence of VX-809 (1 µM) on CFBE41o- cells. (B) Evaluation of corrector combination. The graphs report F508del-CFTR activity in CFBE41o- (left) and FRT (right) cells treated with vehicle alone (DMSO) or VX-809 (3 µM), VX-661 (10 µM), corr-4a (5 µM), 3151 (10 µM), 4172 (10 µM), racVX-445 (3 µM), or their combinations. Symbols indicate statistical significance versus treatment with racVX-445 alone: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 2

Functional evaluation of VX-445 corrector activity in human bronchial epithelial cells. (A) Effect of 24 h cell treatment with vehicle alone (DMSO), VX-445 (5 µM), VX-809 (1 µM), or both correctors together. Experiments were done on F508del/F508del bronchial epithelial cells derived from 5 different subjects (BE86, BE91, BE93, BE111, and BE115) with the short-circuit current technique. Representative recordings are from BE115. As parameters of CFTR function, we measured the total current sensitive to inh-172 (I_TOT) and the current elicited by the cAMP analog alone (I_cAMP). (B) Representative trace obtained from short-circuit current recordings on non-CF bronchial epithelial cells (BE99). (C) Summary of I_TOT (mean ± SD, n = 26–31) measured in F508del/F508del epithelia, derived from 5 different subjects (BE86, BE91, BE93, BE111, and BE115), exposed to indicated treatments. The dashed line reports the value of I_TOT in non-CF bronchial cells. *, p < 0.05; ***, p < 0.001 between indicated groups of data. (D) Ratio between the current elicited by cAMP stimulation (I_cAMP) and total CFTR current (I_TOT) in F508del/F508del epithelia treated with the indicated compounds (data obtained from experiments shown in A and C). *, p < 0.05; ***, p < 0.001 between indicated groups of data. (E) Evaluation of VX-445 as potentiator on F508del-CFTR CFBE41o- cells rescued at 32 °C for 24 h (mean ± SD, n = 5). ***, p < 0.001 vs. control.

Figure 3

Functional evaluation of VX-445-based combinations on F508del/F508del human bronchial epithelial cells. (A) Representative traces of the effect of vehicle alone (DMSO) or VX-445 (3 µM), VX-809 (3 µM), or VX-661 (10 µM) as single agents or combinations in F508del/F508del bronchial epithelial cells (BE91 and BE86) with the short-circuit current technique. (B) Summary of results obtained from short-circuit current recordings on F508del/F508del bronchial epithelial cells derived from four different CF patients. Data reported are the amplitude of the current blocked by 10 µM inh-172 (I_TOT; mean ± SD, n = 5). Asterisks indicate the statistical significance of single correctors vs. control (DMSO-treated): *, p < 0.05; ***, p < 0.001. Other symbols indicate the statistical significance of combinations of correctors vs. VX-445 alone: §, p < 0.05; ns, not significant.

Figure 4

Effect of VX-445-based treatments on mutant CFTR ubiquitylation. (A) Biochemical analysis of CFTR ubiquitylation and expression pattern in CFTR immunoprecipitates from wild-type or F508del-CFTR expressing CFBE41o- cells after 24 h treatment with vehicle alone (DMSO), or (for mutant CFTR only) VX-445 (3 µM), VX-809 (3 µM), or VX-661 (10 µM) as single agents or combinations in the absence or in the presence of the proteasome inhibitor MG-132 (10 µM; last 4 hr) to block proteasomal degradation. For comparison, whole lysates derived from CFBE41o- cells not expressing CFTR (null cells) are also shown as controls for antibody specificity. (B–D) Representative density profiles (left graphs) and corresponding quantification (right graphs) of CFTR and ubiquitin in the absence (B) or in the presence of MG-132 (C,D) Quantification of the density profiles was performed by integrating the profile curves in the selected intervals of molecular weight (see left graphs). *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs. DMSO; §§, p < 0.01; §§§, p < 0.001 vs. VX-445 alone.

Figure 5

Effect of VX-445-based treatments on mutant CFTR half-life. (A) Immunoblot detection of CFTR in whole lysates derived from wild-type or F508del-CFTR expressing CFBE41o- cells treated with vehicle alone (DMSO), or (for mutant CFTR only) with VX-445 (3 µM), VX-809 (3 µM), or VX-661 (10 µM) as single agents or combinations, at different time points following CHX-induced block of protein synthesis. For comparison, whole lysates derived from CFBE41o- cells not expressing CFTR (null cells) are also shown as controls for antibody specificity. (B) Quantification of wild-type or mutant CFTR (band B and band C) half-life in experiments detailed in (A), normalized by the value at time = 0. Data are means ± SD (n = 3). Dashed lines indicate 50% of the protein remaining (y-axis) and the corresponding intercepts on the x-axis, indicating the estimated half-life.

Figure 6

Effect of VX-445-based treatments on the conformational stability of mutant CFTR. (A) Immunoblot detection of CFTR in whole lysates derived from wild-type or F508del-CFTR expressing CFBE41o- cells treated with vehicle alone (DMSO), or (for mutant CFTR only) with VX-445 (3 µM), VX-809 (3 µM), or VX-661 (10 µM) as single agents or combinations, following heat-denaturation at 28–70 °C. For comparison, whole lysates derived from parental CFBE41o- cells (null cells) are also shown as controls for antibody specificity. (B) Quantification of aggregation-resistant (soluble) CFTR band C by densitometry, normalized by HSP90AB1 expression (means ± SD; n = 3).

Figure 7

Analysis of CFTR subcellular localization. (A,B) Representative images showing the detection of F508del-CFTR or wild type CFTR protein in CFBE41o- cells by immunofluorescence. Cells were incubated with vehicle (DMSO) or with the corrector combination (5 µM VX-445 plus 1 µM VX-809) for 24 h. Cells were immediately fixed or treated for the indicated time (1–6 h) with CHX and then fixed (scale bar: 15 µm). (C) Analysis of CFTR protein expression in the plasma membrane at different times following CHX addition. **, p < 0.01; ***, p < 0.001.