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An improved understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein structure and the consequences of gene mutations have allowed the development of novel therapies targeting specific defects underlying CF. Some strategies are mutation specific and have already reached clinical development; some strategies include a read-through of the specific premature termination codons (read-through therapies, nonsense mediated decay pathway inhibitors for Class I mutations); correction of CFTR folding and trafficking to the apical plasma membrane (correctors for Class II mutations); and an increase in the function of CFTR channel (potentiators therapy for Class III mutations and any mutant with a residual function located at the membrane). Other therapies that are in preclinical development are not mutation specific and include gene therapy to edit the genome and stem cell therapy to repair the airway tissue. These strategies that are directed at the basic CF defects are now revolutionizing the treatment for patients and should positively impact their survival rates.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50–100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application. Targeting different aspects of mutant CFTR structural defects with combination therapy leads to more potent rescue of function than that following single therapy.

Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein–lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules.

Clinical studies with modulators of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein have demonstrated that functional restoration of the mutated CFTR can lead to substantial clinical benefit. However, studies have shown highly variable patient responses. The objective of this study was to determine a biomarker predictive of the clinical response. CFTR function was assessed in vivo via nasal potential difference (NPD) and in human nasal epithelial (HNE) cultures by the response to Forskolin/IBMX and the CFTR potentiator VX-770 in short-circuit-current (∆I scF/I+V ) experiments. CFTR expression was evaluated by apical membrane fluorescence semi-quantification. I sc measurements discriminated CFTR function between controls, healthy heterozygotes, patients homozygous for the severe F508del mutation and patients with genotypes leading to absent or residual function. ∆I scF/I+V correlated with CFTR cellular apical expression and NPD measurements. The CFTR correctors lumacaftor and tezacaftor significantly increased the ∆I scF/I+V response to about 25% (SEM = 4.4) of the WT-CFTR level and the CFTR apical expression to about 22% (SEM = 4.6) of the WT-CFTR level in F508del/F508del HNE cells. The level of CFTR correction in HNE cultures significantly correlated with the FEV 1 change at 6 months in 8 patients treated with CFTR modulators. We provide the first evidence that correction of CFTR function in HNE cell cultures can predict respiratory improvement by CFTR modulators.

Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. Morbidity is mainly due to early airway infection. We hypothesized that S. aureus clearance during the first hours of infection was impaired in CF human Airway Surface Liquid (ASL) because of a lowered pH. The ASL pH of human bronchial epithelial cell lines and primary respiratory cells from healthy controls (WT) and patients with CF was measured with a pH microelectrode. The antimicrobial capacity of airway cells was studied after S. aureus apical infection by counting surviving bacteria. ASL was significantly more acidic in CF than in WT respiratory cells. This was consistent with a defect in bicarbonate secretion involving CFTR and SLC26A4 (pendrin) and a persistent proton secretion by ATP12A. ASL demonstrated a defect in S. aureus clearance which was improved by pH normalization. Pendrin inhibition in WT airways recapitulated the CF airway defect and increased S. aureus proliferation. ATP12A inhibition by ouabain decreased bacterial proliferation. Antimicrobial peptides LL-37 and hBD1 demonstrated a pH-dependent activity. Normalizing ASL pH might improve innate airway defense in newborns with CF during onset of S. aureus infection. Pendrin activation and ATP12A inhibition could represent novel therapeutic strategies to normalize pH in CF airways.

C407 is a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein carrying the p.Phe508del (F508del) mutation. We investigated the corrector effect of c407 and its derivatives on F508del-CFTR protein. Molecular docking and dynamics simulations combined with site-directed mutagenesis suggested that c407 stabilizes the F508del-Nucleotide Binding Domain 1 (NBD1) during the co-translational folding process by occupying the position of the p.Phe1068 side chain located at the fourth intracellular loop (ICL4). After CFTR domains assembly, c407 occupies the position of the missing p.Phe508 side chain. C407 alone or in combination with the F508del-CFTR corrector VX-809, increased CFTR activity in cell lines but not in primary respiratory cells carrying the F508del mutation. A structure-based approach resulted in the synthesis of an extended c407 analog G1, designed to improve the interaction with ICL4. G1 significantly increased CFTR activity and response to VX-809 in primary nasal cells of F508del homozygous patients. Our data demonstrate that in-silico optimized c407 derivative G1 acts by a mechanism different from the reference VX-809 corrector and provide insights into its possible molecular mode of action. These results pave the way for novel strategies aiming to optimize the flawed ICL4–NBD1 interface.

Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene ( CFTR ) are responsible for Cystic Fibrosis (CF). The most common CF-causing mutation is the deletion of the 508th amino-acid of CFTR (F508del), leading to dysregulation of the epithelial fluid transport in the airway’s epithelium and the production of a thickened mucus favoring chronic bacterial colonization, sustained inflammation and ultimately respiratory failure. c407 is a bis-phosphinic acid derivative which corrects CFTR dysfunction in epithelial cells carrying the F508del mutation. This study aimed to investigate c407 in vivo activity in the F508del Cftr tm1Eur murine model of CF. Using nasal potential difference measurement, we showed that in vivo administration of c407 by topical, short-term intraperitoneal and long-term subcutaneous route significantly increased the CFTR dependent chloride (Cl − ) conductance in F508del Cftr tm1Eur mice. This functional improvement was correlated with a relocalization of F508del-cftr to the apical membrane in nasal epithelial cells. Importantly, c407 long-term administration was well tolerated and in vitro ADME toxicologic studies did not evidence any obvious issue. Our data provide the first in vivo preclinical evidence of c407 efficacy and absence of toxicity after systemic administration for the treatment of Cystic Fibrosis.

The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal) enzyme and the anti-cytokeratin8 (K8) antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium)-tren-cholesterol (BGTC) (bis (guanidinium)-tren-cholesterol) combined to the colipid dioleoyl phosphatidylethanolamine (DOPE) (dioleoyl phosphatidylethanolamine) was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP) associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP)-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

Introduction: Cystic fibrosis (CF) is caused by defective Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) proteins. CFTR controls chloride (Cl − ) and bicarbonate (HCO 3 − ) transport into the Airway Surface Liquid (ASL). We investigated the impact of F508del-CFTR correction on HCO 3 − secretion by studying transepithelial HCO 3 − fluxes. Methods: HCO 3 − secretion was measured by pH-stat technique in primary human respiratory epithelial cells from healthy subjects (WT) and people with CF (pwCF) carrying at least one F508del variant. Its changes after CFTR modulation by the triple combination VX445/661/770 and in the context of TNF-α+IL-17 induced inflammation were correlated to ASL pH and transcriptional levels of CFTR and other HCO 3 − transporters of airway epithelia such as SLC26A4 (Pendrin), SLC26A9 and NBCe1. Results: CFTR-mediated HCO 3 − secretion was not detected in F508del primary human respiratory epithelial cells. It was rescued up to ∼ 80% of the WT level by VX-445/661/770. In contrast, TNF-α+IL-17 normalized transepithelial HCO 3 − transport and increased ASL pH. This was related to an increase in SLC26A4 and CFTR transcript levels. VX-445/661/770 induced an increase in pH only in the context of inflammation. Effects on HCO 3 − transport were not different between F508del homozygous and F508del compound heterozygous CF airway epithelia. Conclusion: Our studies show that correction of F508del-CFTR HCO 3 − is not sufficient to buffer acidic ASL and inflammation is a key regulator of HCO 3 − secretion in CF airways. Prediction of the response to CFTR modulators by theratyping should take into account airway inflammation.