F508del Cystic Fibrosis Transmembrane Conductance Regulator Research Articles

Structural determinants of protein kinase A essential for CFTR channel activation

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

Next generation triplex-forming PNAs for site-specific genome editing of the F508del CFTR mutation

BackgroundCystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. MethodsHere, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). ResultsTreatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. ConclusionsThese findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.

Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.

Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE-engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal 'dead' single-guide RNAs-we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Impact of CFTR Correctors on the Channel Activity of Rescued F508del CFTR in Airway Epithelial Cells

Cystic fibrosis (CF) is caused by the functional deficiency of a cAMP-activated anion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). Over 90% of CF patients carry at least one copy of the F508del CFTR gene. The F508del mutation causes a global conformational change that prevents the nascent protein from exiting the endoplasmic reticulum, leading to loss of function at the cell surface. CFTR correctors have been developed to bind to the nascent F508del CFTR, change its conformation, and mobilize it to the plasma membrane. Rescued F508del CFTR (rF508del) does not fully regain channel activity. Lumacaftor, the first CFTR corrector approved by the FDA, acutely inhibits rF508del channel opening (Ambrosetti et al. Pediatric Pulmonology 54(S2): 190-191). Our objective is to evaluate the impact of the other two FDA-approved CFTR correctors, tezacaftor and elexacaftor, on rF508del channel activity. We hypothesize that different CFTR correctors impact rF508del channel activity differently. We utilized an electrode-based, real-time iodide efflux assay to evaluate the acute impact of CFTR correctors on the cAMP-dependent anion channel activity of rF508del in a CF bronchial epithelial cell line stably expressing F508del CFTR (CFBE-DF) at 37°C. CFBE-DF cells were incubated with a corrector for over 24 hours before iodide efflux assay was performed in the acute presence or absence of the same corrector. Genistein was used as a potentiator. F508del processing was assessed by quantitative immunoblotting. The lack of acute exposure to 5μM lumacaftor results in a 2% increase in the mean F508del processing (n=3, p=0.94) and a 48% increase in channel activity (n=3, p=0.04). F508del processing peaks at 1μM lumacaftor but the channel activity peaks at a much lower 0.05μM lumacaftor. In contrast, the lack of acute exposure to 5μM tezacaftor results in an 8% reduction in the mean channel activity (n>3, p=0.79) and a 17% reduction in the mean F508del processing (n>3, p=0.43). Both F508del processing and channel activity peak at 5μM tezacaftor. The lack of acute exposure to 2μM elexacaftor results in an 11% decrease in the mean F508del channel activity (n=4. p=0.54). F508del channel activity peaks at 2μM elexacaftor. We conclude that, unlike lumacaftor, tezacaftor and elexacaftor do not inhibit the channel activity of rescued F508del CFTR in CFBE-DF cells. A better understanding of the impact of CFTR correctors on the channel activity of rescued CFTR processing mutants will inform the development of highly effcacious CFTR modulator combinations for the treatment of CF patients. This work is supported by NIH (5P30DK072482), Samford University, and Cystic Fibrosis Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Discovery of GLPG2737, a Potent Type 2 Corrector of CFTR for the Treatment of Cystic Fibrosis in Combination with a Potentiator and a Type 1 Co-corrector.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. This epithelial anion channel regulates the active transport of chloride and bicarbonate ions across membranes. Mutations result in reduced surface expression of CFTR channels with impaired functionality. Correctors are small molecules that support the trafficking of CFTR to increase its membrane expression. Such correctors can have different mechanisms of action. Combinations may result in a further improved therapeutic benefit. We describe the identification and optimization of a new pyrazolol3,4-bl pyridine-6-carboxylic acid series with high potency and efficacy in rescuing CFTR from the cell surface. Investigations showed that carboxylic acid group replacement with acylsulfonamides and acylsulfonylureas improved ADMET and PK properties, leading to the discovery of the structurally novel co-corrector GLPG2737. The addition of GLPG2737 to the combination of the potentiator GLPG1837 and C1 corrector 4 led to an 8-fold increase in the F508del CFTR activity.

A uniquely efficacious type of CFTR corrector with complementary mode of action.

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.

SUMOylation Inhibition Enhances Protein Transcription under CMV Promoter: A Lesson from a Study with the F508del-CFTR Mutant.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

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.

The Anion Channel TMEM16a/Ano1 Modulates CFTR Activity, but Does Not Function as an Apical Anion Channel in Colonic Epithelium from Cystic Fibrosis Patients and Healthy Individuals.

Studies in human colonic cell lines and murine intestine suggest the presence of a Ca2+-activated anion channel, presumably TMEM16a. Is there a potential for fluid secretion in patients with severe cystic fibrosis transmembrane conductance regulator (CFTR) mutations by activating this alternative pathway? Two-dimensional nondifferentiated colonoid-myofibroblast cocultures resembling transit amplifying/progenitor (TA/PE) cells, as well as differentiated monolayer (DM) cultures resembling near-surface cells, were established from both healthy controls (HLs) and patients with severe functional defects in the CFTR gene (PwCF). F508del mutant and CFTR knockout (null) mice ileal and colonic mucosa was also studied. HL TA/PE monolayers displayed a robust short-circuit current response (ΔIeq) to UTP (100 µM), forskolin (Fsk, 10 µM) and carbachol (CCH, 100 µM), while ΔIeq was much smaller in differentiated monolayers. The selective TMEM16a inhibitor Ani9 (up to 30 µM) did not alter the response to luminal UTP, significantly decreased Fsk-induced ΔIeq, and significantly increased CCH-induced ΔIeq in HL TA/PE colonoid monolayers. The PwCF TA/PE and the PwCF differentiated monolayers displayed negligible agonist-induced ΔIeq, without a significant effect of Ani9. When TMEM16a was localized in intracellular structures, a staining in the apical membrane was not detected. TMEM16a is highly expressed in human colonoid monolayers resembling transit amplifying cells of the colonic cryptal neck zone, from both HL and PwCF. While it may play a role in modulating agonist-induced CFTR-mediated anion currents, it is not localized in the apical membrane, and it has no function as an apical anion channel in cystic fibrosis (CF) and healthy human colonic epithelium.

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

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.

WS10.01 The French Compassionate Programme of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis with advanced lung disease and no F508del CFTR variant

Objective: The European Medicines Agency has approved the cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination elexacaftor-tezacaftor-ivacaftor (ETI) for people with cystic fibrosis (pwCF) carrying at least one F508del variant. The United States Food and Drug Administration (FDA) also approved ETI for pwCF carrying one of 177 rare variants. Our objective was to evaluate ETI in pwCF with advance lung disease and no F508del variant.

Gaps in Cystic Fibrosis Care Are Associated with Reduced Lung Function in the U.S. Cystic Fibrosis Foundation Patient Registry.

Rationale: Cystic fibrosis (CF) is a genetic disease leading to progressive lung function loss and early mortality. Many clinical and demographic variables are associated with lung function decline, but little is known about the effects of prolonged periods of missed care. Objectives: To determine if missed care in the Cystic Fibrosis Foundation Patient Registry (CFFPR) is associated with decreased lung function at follow-up visits. Methods: Deidentified CFFPR data for 2004-2016 were analyzed, with the exposure of interest being ⩾12-month gap in CFFPR data. We modeled percentage predicted forced expiratory volume in 1 second using longitudinal semiparametric modeling with natural cubic splines for age (knots at quantiles) and with subject-specific random effects, adjusted for sex and CFTR (cysticfibrosis transmembrane conductance regulator) genotype, race, and ethnicity and included time-varying covariates for gaps in care, insurance type, underweight body mass index, CF-related diabetes status, and chronic infections. Results: A total of 24,328 individuals with 1,082,899 encounters in the CFFPR met inclusion criteria. In the cohort, 8,413 (35%) individuals had at least a single ⩾12-month episode of discontinuity, whereas 15,915 (65%) had continuous care. Of the encounters preceded by a 12-month gap, 75.8% occurred in patients 18 years and older. Compared with those with continuous care, those with a discontinuous care episode had a lower follow-up percentage predicted forced expiratory volume in 1 second at the index visit (-0.81%; 95% confidence interval, -1.00, -0.61) after adjustment for other variables. The magnitude of this difference was much greater (-2.1%; 95% confidence interval, -1.5, -2.7) in young adult F508del homozygotes. Conclusions: There was a high rate of ⩾12-month gap in care, especially in adults, documented in the CFFPR. Discontinuous care identified in the CFFPR was strongly associated with decreased lung function, especially in adolescents and young adults homozygous for the F508del CFTR mutation. This may have implications for identifying and treating people with lengthy gaps in care and may have implications for CFF care recommendations.

The French Compassionate Program of elexacaftor-tezacaftor-ivacaftor in people with cystic fibrosis with advanced lung disease and no F508del CFTR variant.

The European Medicines Agency has approved the cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination elexacaftor-tezacaftor-ivacaftor (ETI) for people with cystic fibrosis (pwCF) carrying at least one F508del variant. The United States Food and Drug Administration (FDA) also approved ETI for pwCF carrying one of 177 rare variants. An observational study was conducted to evaluate the effectiveness of ETI in pwCF with advanced lung disease that were not eligible to ETI in Europe. All patients with no F508del variant and advanced lung disease (defined as having a percent predicted forced expiratory volume (ppFEV1)<40 and/or being under evaluation for lung transplantation) and enrolled in the French Compassionate Program initiated ETI at recommended doses. Effectiveness was evaluated by a centralized adjudication committee at 4-6 weeks in terms of clinical manifestations, sweat chloride concentration and ppFEV1. Among the first 84 pwCF included in the program, ETI was effective in 45 (54%) and 39 (46%) were considered to be non-responders. Among the responders 22/45 (49%) carried a CFTR variant that is not currently approved by FDA for ETI eligibility. Important clinical benefits, including suspending the indication for lung transplantation, a significant decrease in sweat chloride concentration by a median [IQR] -30 [-14;-43]mmol·l-1 (n=42; p<0.0001) and an improvement in ppFEV1 by+10.0 [6.0; 20.5] (n=44, p<0.0001), were observed in those for whom treatment was effective. Clinical benefits were observed in a large subset of pwCF with advanced lung disease and CFTR variants not currently approved for ETI.

Phenotypic Alteration of an Established Human Airway Cell Line by Media Selection.

Cystic Fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), a chloride/bicarbonate channel. Many studies utilize human airway cell models (cell lines and primary cells) to study different aspects of CFTR biology. Media selection can alter the growth and differentiation of primary cells, yet the impact on stable airway cell lines is unclear. To determine the impact of media and growth conditions on CFBE41o- cells stably transduced with wild-type or F508del CFTR, we examined four commonly used growth media, measuring epithelial and mesenchymal markers, as well as CFTR expression, maturation, and function. The selection of growth media altered the expression of epithelial and mesenchymal markers in the cell lines, and significantly impacted CFTR expression and subsequent function. These results highlight the importance of media selection to CFTR and cell line behavior and should be considered in both studies of primary human airway cells and stable cell lines.

High-throughput functional assay in cystic fibrosis patient-derived organoids allows drug repurposing.

Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Here, we miniaturised the previously described forskolin-induced swelling (FIS) assay in intestinal organoids from a 96-well to a 384-well plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound Food and Drug Administration (FDA)-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. The 384-well FIS assay demonstrated uniformity and robustness based on coefficient of variation and Z'-factor calculations. In the primary screen, CFTR induction was limited overall, yet interestingly, the top five compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins (mevastatin, lovastatin, simvastatin and fluvastatin) increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was concentration-dependent and W1282X-specific. Future studies should focus on elucidating genotype specificity and mode-of-action of statins in more detail. This study exemplifies proof of principle of large-scale compound screening in a functional assay using patient-derived organoids.

656 Genome-wide functional genomics analyses of F508del–cystic fibrosis transmembrane conductance regulator degradation

656 Genome-wide functional genomics analyses of F508del–cystic fibrosis transmembrane conductance regulator degradation

The CFTR Amplifier Nesolicaftor Rescues TGF-β1 Inhibition of Modulator-Corrected F508del CFTR Function.

Highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have led to dramatic improvements in lung function in many people with cystic fibrosis (PwCF). However, the efficacy of CFTR modulators may be hindered by persistent airway inflammation. The cytokine transforming growth factor-beta1 (TGF-β1) is associated with worse pulmonary disease in PwCF and can diminish modulator efficacy. Thus, strategies to augment the CFTR response to modulators in an inflammatory environment are needed. Here, we tested whether the CFTR amplifier nesolicaftor (or PTI-428) could rescue the effects of TGF-β1 on CFTR function and ciliary beating in primary human CF bronchial epithelial (CFBE) cells. CFBE cells homozygous for F508del were treated with the combination of elexacaftor/tezacaftor/ivacaftor (ETI) and TGF-β1 in the presence and absence of nesolicaftor. Nesolicaftor augmented the F508del CFTR response to ETI and reversed TGF-β1-induced reductions in CFTR conductance by increasing the expression of CFTR mRNA. Nesolicaftor further rescued the reduced ciliary beating and increased expression of the cytokines IL-6 and IL-8 caused by TGF-β1. Finally, nesolicaftor augmented the F508del CFTR response to ETI in CFBE cells overexpressing miR-145, a negative regulator of CFTR expression. Thus, CFTR amplifiers, but only when used with highly effective modulators, may provide benefit in an inflamed environment.

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.

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation

Cirrhosis and Portal Hypertension in Compound Heterozygous People With Cystic Fibrosis Harboring One F508del CFTR Gene Mutation