Elexacaftor/VX-445-mediated CFTR interactome remodeling reveals differential correction driven by mutation-specific translational dynamics.

Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as lumacaftor (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report that variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry multiplexed with isobaric tandem mass tags was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize nonresponsive CFTR variants to known and available correctors.

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

The monogenic rare disease Cystic Fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance (CFTR) protein, an anion channel expressed at the apical plasma membrane of epithelial cells. The discovery and subsequent development of CFTR modulators-small molecules acting on the basic molecular defect in CF-have revolutionized the standard of care for people with CF (PwCF), thus drastically improving their clinical features, prognosis, and quality of life. Currently, four of these drugs are approved for clinical use: potentiator ivacaftor (VX-770) alone or in combination with correctors lumacaftor, (VX-809), tezacaftor (VX-661), and elexacaftor (VX-445). Noteworthily, the triple combinatorial therapy composed of ivacaftor, tezacaftor, and elexacaftor constitutes the most effective modulator therapy nowadays for the majority of PwCF. In this review, we exploit the organic synthesis of ivacaftor, tezacaftor, and elexacaftor by providing a retrosynthetic drug analysis for these CFTR modulators. Furthermore, we describe the current understanding of the mechanisms of action (MoA's) of these compounds by discussing several studies that report the key findings on the molecular mechanisms underlying their action on the CFTR protein.

KCa3.1 potentiation stimulates Cl- secretion in F508del and G551D CFTR-corrected primary human bronchial epithelial cells.

We previously identified potentiators of KCa3.1 (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one; DCEBIO) that stimulate Cl- secretion across human bronchial epithelial cells (HBEs) expressing wild-type (WT) cystic fibrosis transmembrane conductance regulator (CFTR). However, these compounds failed to stimulate Cl- secretion in F508del CFTR HBEs. Drug discovery efforts identified CFTR potentiators (VX-770) and correctors (VX-445, VX-661) for cystic fibrosis (CF) disease-causing mutations, including F508del and G551D. Herein, we evaluated the effect of KCa3.1 potentiation on Cl- equivalent current (ICl) across primary HBEs expressing WT, F508del, and G551D CFTR. Transepithelial impedance analysis was used to obtain estimates of apical (Ra) and basolateral membrane (BLM; Rb) resistances. In WT CFTR HBEs, DCEBIO stimulated ICl, which was increased by forskolin. Similarly, forskolin stimulated ICl, and this was increased by DCEBIO. The KCa3.1 blocker, TRAM-34 inhibited ICl. DCEBIO decreased Rb, whereas TRAM-34 increased Rb, consistent with BLM localization of KCa3.1. Following correction of F508del CFTR with VX-445 + VX-661, DCEBIO failed to stimulate ICl, although the subsequent addition of forskolin + VX-770 increased ICl. Importantly, following stimulation of ICl with forskolin + VX-770, DCEBIO induced a further significant increase in ICl. As above, DCEBIO reduced Rb, whereas TRAM-34 increased Rb, consistent with BLM localized KCa3.1. Finally, we assessed KCa3.1 potentiation on ICl in G551D/F508del CFTR HBEs in the absence or presence of VX-445 + VX-661. In both cases, DCEBIO failed to stimulate ICl. However, following stimulation with forskolin + VX-770, DCEBIO nearly doubled ICl. Our results demonstrate that following correction/potentiation of F508del and G551D CFTR, potentiation of KCa3.1 increases the Cl- secretory response, suggesting this class of compounds may represent a novel means of further increasing Cl- secretion across CF airway.

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

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.

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

Cystic fibrosis (CF), the most common autosomal recessive fatal genetic disease in the Caucasian population, is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel that regulates salt and water transport across a variety of secretory epithelia. Deletion of phenylalanine at position 508, F508del, the most common CF-causing mutation, destabilises the CFTR protein, causing folding and trafficking defects that lead to a dramatic reduction in its functional expression. Small molecules called correctors have been developed to rescue processing-defective F508del CFTR. We have combined in silico and in vitro approaches to investigate the mechanism of action and potential as CFTR correctors of three hybrid derivatives (2a, 7a, and 7m) obtained by merging the amino-arylthiazole core with the benzodioxole carboxamide moiety characterising the corrector lumacaftor. Molecular modelling analyses suggested that the three hybrids interact with a putative region located at the MSD1/NBD1 interface. Biochemical analyses confirmed these results, showing that the three molecules affect the expression and stability of the F508del NBD1. Finally, the YFP assay was used to evaluate the influence of the three hybrid derivatives on F508del CFTR function, assessing that their effect is additive to that of the correctors VX661 and VX445. Our study shows that the development and testing of optimised compounds targeting different structural and functional defects of mutant CFTR is the best strategy to provide more effective correctors that could be used alone or in combination as a valuable therapeutic option to treat an even larger cohort of people affected by CF.