Elexacaftor Mediates the Rescue of F508del CFTR Functional Expression Interacting with MSD2

Abstract

Cystic fibrosis (CF) is one of the most frequent lethal autosomal recessive diseases affecting the Caucasian population. It is caused by loss of function variants of the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein located on the apical side of epithelial cells. The most prevalent CF-causing mutation, the deletion of phenylalanine at position 508 (F508del), is characterized by folding and trafficking defects, resulting in the decreased functional expression of the protein on the plasma membrane. Two classes of small-molecule modulators, termed potentiators and correctors, respectively, have been developed to rescue either the gating or the cellular processing of defective F508del CFTR. Kaftrio, a next-generation triple-combination drug, consisting of the potentiator ivacaftor (VX770) and the two correctors tezacaftor (VX661) and elexacaftor (VX445), has been demonstrated to be a life-changing therapeutic modality for the majority of people with CF worldwide. While the mechanism of action of VX770 and VX661 is almost known, the precise mechanism of action and binding site of VX445 have not been conclusively determined. We investigated the activity of VX445 on mutant F508del to identify the protein domains whose expression is mostly affected by this corrector and to disclose its mechanisms of action. Our biochemical analyses revealed that VX445 specifically improves the expression and the maturation of MSD2, heterologously expressed in HEK 293 cells, and confirmed that its effect on the functional expression of defective F508del CFTR is additive either with type I or type II CFTR correctors. We are confident that our study will help to make a step forward in the comprehension of the etiopathology of the CF disease, as well as to give new information for the development and testing of combinations of even more effective correctors able to target mutation-specific defects of the CFTR protein.

In Silico and In Vitro Evaluation of the Mechanism of Action of Three VX809-Based Hybrid Derivatives as Correctors of the F508del CFTR Protein

“…MSD2 and NBD2 were detected as bands of ~47 and ~30 kDa, respectively. Controls in untransfected HEK-t cells showed that the primary As expected [38,[48][49][50][51], treatment with VX661 increased the expression of MSD1. Indeed, in HEK-t cells treated with VX661, the expression of this domain was 2.19 times higher than in control DMSO-treated cells.…”

“…Treatment with the corrector, VX661, or with 2a, 7m, and 7a did not change the expression of MSD2. On the contrary, as observed elsewhere [40,51], VX445 increased the expression of this domain to 2.51 times that observed in untreated control HEK-t cells (Figure 8E). Treatment with VX661 or VX445, or with each of the three hybrids, did not modify the expression level of NBD2 (Figure 8F).…”

“…Then, we analysed the effect of 2a, 7m, and 7a on the expression of full-length WT and F508del CFTR, and on different domains of the CFTR protein, transfected individually in HEK-t cells, a heterologous expression system widely used because of its higher transfectability. Since the CFTR domains, whose expression is most affected by VX445 and VX661 are known [38,40,[48][49][50][51], we used these correctors as reference compounds to compare results. Our analysis showed that in full-length WT CFTR lysates, neither the total (B + C bands) nor the mature (C/(C + B) band ratio) expression level of WT CFTR protein was increased after treatment with the correctors VX661 and VX445, or each of the three hybrids under study (Figure 9A).…”

Organic Synthesis and Current Understanding of the Mechanisms of CFTR Modulator Drugs Ivacaftor, Tezacaftor, and Elexacaftor

“…These findings suggested that elexacaftor most likely requires interaction with more than a single domain to exert its rescue effects, in an MoA probably affecting domain interfaces such as TMD1 and TMD2 assembly and their interface with NBD1 [ 125 ]. Bongiorno and co-authors [ 126 ] further explored the MoA of elexacaftor by assessing its effects on p.Phe508del-CFTR to identify the protein domains affected by this modulator. Using different biochemical approaches and plasmid constructs expressing the isolated domains, the authors suggested that different correctors can act on the expression and stability of distinct p.Phe508del-CFTR regions, identifying TMD2 as the primarily CFTR domain involved in p.Phe508del rescue by elexacaftor [ 126 ].…”

“…Bongiorno and co-authors [ 126 ] further explored the MoA of elexacaftor by assessing its effects on p.Phe508del-CFTR to identify the protein domains affected by this modulator. Using different biochemical approaches and plasmid constructs expressing the isolated domains, the authors suggested that different correctors can act on the expression and stability of distinct p.Phe508del-CFTR regions, identifying TMD2 as the primarily CFTR domain involved in p.Phe508del rescue by elexacaftor [ 126 ].…”