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.
Highlights
The most common cause of cystic fibrosis (CF) is the out-of-frame deletion of three nucleotides (CTT) in the CFTR gene, resulting in loss of phenylalanine at position 508 (ΔF508) of the CFTR protein and a synonymous mutation (ATC/ATT) at codon encoding isoleucine 507 [1,2,3]
When cells were treated with VX-770 and VX-809 combination (5μM each for 16h) we observed a considerable reduction (40%) in both band B and C levels of native, I507-ATT ΔF508 CFTR (Fig 1B and 1C, left)
Corr-4a co-treatment significantly enhances the functionality of the rescued ΔF508 CFTR when it is co-administered with VX-809 and VX-770
Summary
The most common cause of cystic fibrosis (CF) is the out-of-frame deletion of three nucleotides (CTT) in the CFTR gene, resulting in loss of phenylalanine at position 508 (ΔF508) of the CFTR protein and a synonymous mutation (ATC/ATT) at codon encoding isoleucine 507 [1,2,3]. When rescued from ERAD, ΔF508 CFTR demonstrates reduced plasma membrane stability and functional abnormalities [5]. Efforts to treat CF caused by the ΔF508 mutation focus on finding small molecular correctors that enhance ΔF508 CFTR folding coand/or post-translationally and potentiators to improve its function (Fig 1A) [5, 6]. Orkambi (Vertex Pharmaceuticals), the first FDA approved combinational treatment for CF contains the cyclopropane carboxamide derivative corrector (VX-809, Lumacaftor) and the N-(2,4-Ditert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide potentiator (VX770, Ivacaftor). It is estimated that 50% of CF patients will benefit from combination therapy [7]
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