Abstract
Cystic fibrosis (CF) is a genetic disease associated with the defective function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that causes obstructive disease and chronic bacterial infections in airway epithelia. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), leads to CFTR misfolding, trafficking defects and premature degradation. A number of correctors that are able to partially rescue F508del-CFTR processing defects have been identified. Clinical trials have demonstrated that, unfortunately, mono-therapy with the best correctors identified to date does not ameliorate lung function or sweat chloride concentration in homozygous F508del patients. Understanding the mechanisms exerted by currently available correctors to increase mutant F508del-CFTR expression is essential for the development of new CF-therapeutics. We investigated the activity of correctors on the mutant F508del and wild type (WT) CFTR to identify the protein domains whose expression is mostly affected by the action of correctors, and we investigated their mechanisms of action. We found that the four correctors under study, lumacaftor (VX809), the quinazoline derivative VX325, the bithiazole compound corr4a, and the new molecule tezacaftor (VX661), do not influence either the total expression or the maturation of the WT-CFTR transiently expressed in human embryonic kidney 293 (HEK293) cells. Contrarily, they significantly enhance the expression and the maturation of the full length F508del molecule. Three out of four correctors, VX809, VX661 and VX325, seem to specifically improve the expression and the maturation of the mutant CFTR N-half (M1N1, residues 1–633). By contrast, the CFTR C-half (M2N2, residues 837–1480) appears to be the region mainly affected by corr4a. VX809 was shown to stabilize both the WT- and F508del-CFTR N-half isoforms, while VX661 and VX325 demonstrated the ability to enhance the stability only of the mutant F508del polypeptide.
Highlights
Cystic fibrosis (CF) is a fatal autosomal recessive inherited disease caused by loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride and bicarbonate channel expressed at the apical membrane of epithelial cells in the airways, pancreas, testis, and other tissues [1]
We found that all assayed correctors were able to increase the functional expression of the F508del-CFTR that was heterologously expressed in highly transfectable human embryonic kidney 293 (HEK-t) cells
The relative abundance of constructs codifying for the full length wild type (WT)- or F508del-CFTR, WTor F508del-M1N1, and M2N2, WT- or F508del-∆NBD2 was evaluated in HEK-t transfected cells by quantitative real-time polymerase chain reaction (qRT-PCR), using specific primer pair sets
Summary
Cystic fibrosis (CF) is a fatal autosomal recessive inherited disease caused by loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride and bicarbonate channel expressed at the apical membrane of epithelial cells in the airways, pancreas, testis, and other tissues [1]. F508del-CFTR consists of a thick tenacious mucus that obstructs distal airways and sub-mucosal glands in the lungs. As a consequence of the alterations in airway surface liquid regulation and mucus consistence, the lungs are colonized by opportunistic bacteria and suffer from a rapid functional decline of respiratory function, eventually culminating in lung failure [3]. The 1480-amino acid CFTR protein shares structural and folding features with the other members of the ATP-binding cassette (ABC) transporter family. It is comprised of two homologous repeats, each consisting of six transmembrane (TMD1 and TMD2) regions followed by a cytoplasmic nucleotide binding domain (NBD1 and NBD2). A little of the F508del CFTR that is successfully and properly delivered to its native destination retains some functional activity, albeit with altered gating with respect to wild type (WT) CFTR [11,12,13,14]
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