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. 2016 May 23;11(5):e0155882.
doi: 10.1371/journal.pone.0155882. eCollection 2016.

Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis

Vedrana Bali  1 , Ahmed Lazrak  2   3 , Purushotham Guroji  1   4 , Sadis Matalon  2   4   3 , Zsuzsanna Bebok  1   4   3
Affiliations

Mechanistic Approaches to Improve Correction of the Most Common Disease-Causing Mutation in Cystic Fibrosis

Vedrana Bali et al. PLoS One. .

Abstract

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to deletion of the phenylalanine at position 508 (ΔF508) in the CFTR protein and causes multiple folding and functional defects. Contrary to large-scale efforts by industry and academia, no significant therapeutic benefit has been achieved with a single "corrector". Therefore, investigations concentrate on drug combinations. Orkambi (Vertex Pharmaceuticals), the first FDA-approved drug for treatment of cystic fibrosis (CF) caused by this mutation, is a combination of a corrector (VX-809) that facilitates ΔF508 CFTR biogenesis and a potentiator (VX-770), which improves its function. Yet, clinical trials utilizing this combination showed only modest therapeutic benefit. The low efficacy Orkambi has been attributed to VX-770-mediated destabilization of VX-809-rescued ΔF508 CFTR. Here we report that the negative effects of VX-770 can be reversed by increasing the half-life of the endoplasmic reticulum (ER) form (band B) of ΔF508 CFTR with another corrector (Corr-4a.) Although Corr-4a alone has only minimal effects on ΔF508 CFTR rescue, it increases the half-life of ΔF508 CFTR band B when it is present during half-life measurements. Our data shows that stabilization of band B ΔF508 CFTR with Corr-4a and simultaneous rescue with VX-809, leads to a >2-fold increase in cAMP-activated, CFTRinh-172-inhibited currents compared to VX-809 alone, or VX-809+VX-770. The negative effects of VX-770 and the Corr-4a protection are specific to the native I507-ATT ΔF508 CFTR without affecting the inherently more stable, synonymous variant I507-ATC ΔF508 CFTR. Our studies emphasize that stabilization of ΔF508 CFTR band B in the ER might improve its functional rescue by Orkambi.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Corr-4a diminishes negative effects of VX-770 on VX-809-rescued I507-ATT ΔF508 CFTR.
A. Mechanism of action of CFTR modulators used in our studies. B. Western blot analysis of I507-ATT and I507-ATC ΔF508 CFTR expressing HEK-293 cell lysates following vehicle control (0.15% DMSO, 16 h, 37°C) or corrector treatment (5μM VX-809, 5μM VX-770 and 5μM VX-809+5μM VX-770, 16 h, 37°C). Loading control: β-actin. C. Densitometry of band B and band C I507-ATT and I507-ATC ΔF508 CFTR levels. CFTR band B was plotted relative to vehicle control (DMSO). Band C was plotted relative to VX-809 (37°C). DMSO control, corrector VX-809 (VX-809), potentiator VX-770 (VX-770), combination (VX-809+VX-770), n = 9,9,6,3. D. Western blot analysis of I507-ATT and I507-ATC ΔF508 CFTR expressing HEK-293 cell lysates following vehicle control (0.2% DMSO, 24 h, 37°C), Corr-4a (10μM, 24 h, 37°C) or drug combination treatments (5μM VX-809+10 μM Corr-4a and 5μM VX-809+10 μM Corr-4a+5μM VX-770, 16 h, 37°C). Loading control: β-actin. E. Quantification of I507-ATT and I507-ATC ΔF508 CFTR levels. CFTR band B levels were plotted relative to vehicle control (DMSO). Band C levels were plotted relative to VX-809 (37°C). DMSO control (Ctr), corrector combination (VX-809+Corr-4a), combination of correctors and potentiator VX-770 (VX-809+Corr-4a+VX-770), n = 4. All values are means ± 1 SD. *p<0.05; **: p<0.02, significantly different from VX-809-treated sample by ANOVA, Turkey-Kramer procedure.
Fig 2
Fig 2. Corr-4a reverses the negative effects of VX-770 on VX-809-corrected ΔF508 CFTR half-life.
A. I507-ATT ΔF508 CFTR turnover in VX-809 (5μM), VX-809+VX-770 (5μM+5μM) and VX-809+VX-770+Corr-4a (5μM +5μM+10μM) treated cells. B. I507-ATC ΔF508 CFTR turnover in VX-809 (5μM), VX-809+VX-770 (5μM+5μM) and VX-809+VX-770+Corr-4a (5μM +5μM+10μM) treated cells. Cycloheximide (CHX) (200μg/ml, 37°C) was used to inhibit protein synthesis. Cells were lysed at 0, 1 and 3 hours post CHX treatment. Representative gels are shown on top. Loading control: β-actin. CFTR band B (left) and band C (right) half-lives are plotted in the lower panels. Please see text for half-life calculation explanation. All values are means ± 1 SD. *p<0.05; **: p<0.02, significantly different by ANOVA, n = 3.
Fig 3
Fig 3. Corr-4a increases cAMP-activated, CFTRinh-172-inhibited whole-cell currents in VX-809+VX-770 treated I507-ATT ΔF508 CFTR expressing cells.
Whole-cell patch clamp studies were performed following treatment with CFTR modulators as specified (5μM VX-809 + 10μM Corr-4a+ 5μM VX-770, 16h, 37°C). Corr-4a and VX-809 were present during recordings, but VX-770 was washed out prior to experiments. A: Results are plotted as maximum forskolin+IBMX-induced and CFTRinh-172-inhibited maximum currents (ΔpA/pF), n = 21 (Control, VX-809 and VX-809+Corr-4a), n = 13 (VX-770, VX-809+VX-770 and VX-809+VX-770+Corr-4a), *: p<0.01; **: p<0.001 by ANOVA; error bars: means± SE B: Representative I/V relationships obtained from cells treated with VX-809+Corr-4a and VX-809+Corr-4a+VX-770, n = 8, error bars: means± SE.
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