Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Research Articles

A bibliometric analysis of cystic fibrosis transmembrane conductance regulators

Cystic fibrosis (CF), a multisystem disease primarily affecting the lungs, arises due to pathogenic mutations in the CF transmembrane conductance regulator (CFTR) gene. This study embarked on a bibliometric analysis to survey the use of CFTR modulators in CF treatment. Utilizing the Scopus database, a comprehensive search was executed, incorporating terms related to CF and CFTR modulators. Various document types up to July 19, 2023, were included, with citation counts forming the basis of our analyses. Trends, contributor countries, leading institutions, top authors, journals, keywords, and annual citation trends were evaluated. Our search retrieved 2317 records, predominantly articles. The United States dominated in both publications and citations, followed by the United Kingdom. The University of Alabama, Birmingham, and Vertex Pharmaceuticals, Boston, were among the top institutions. Rowe S.M. was identified as a top-cited author. The Journal of Cystic Fibrosis emerged as the leading journal in terms of publication volume, while the New England Journal of Medicine had the highest citation count. The most-cited article addressed a CFTR potentiator's efficacy in patients with the G551D mutation. The keyword "Cystic fibrosis" appeared most frequently. This bibliometric analysis underscores the significant research focus on CF, especially concerning CFTR modulators. The results highlight the pivotal role of certain countries, institutions, authors, and journals in the progression of CF research, offering insights into current trends and future research directions.

Drug metabolism of ciprofloxacin, ivacaftor, and raloxifene by Pseudomonas aeruginosa cytochrome P450 CYP107S1

Drug metabolism is one of the main processes governing the pharmacokinetics and toxicity of drugs via their chemical biotransformation and elimination. In humans, the liver, enriched with cytochrome P450 (CYP) enzymes, plays a major metabolic and detoxification role. The gut microbiome and its complex community of microorganisms can also contribute to some extent to drug metabolism. However, during an infection when pathogenic microorganisms invade the host, our knowledge of the impact on drug metabolism by this pathobiome remains limited. The intrinsic resistance mechanisms and rapid metabolic adaptation to new environments often allow the human bacterial pathogens to persist, despite the many antibiotic therapies available. Here, we demonstrate that a bacterial CYP enzyme, CYP107S1, from Pseudomonas aeruginosa, a predominant bacterial pathogen in cystic fibrosis (CF) patients, can metabolize multiple drugs from different classes. CYP107S1 demonstrated high substrate promiscuity and allosteric properties much like human hepatic CYP3A4. Our findings demonstrated binding and metabolism by the recombinant CYP107S1 of fluoroquinolone antibiotics (ciprofloxacin and fleroxacin), a CF transmembrane conductance regulator potentiator (ivacaftor), and a SERM antimicrobial adjuvant (raloxifene). Our in vitro metabolism data were further corroborated by molecular docking of each drug to the heme active site using a CYP107S1 homology model. Our findings raise the potential for microbial pathogens modulating drug concentrations locally at the site of infection, if not systemically, via CYP-mediated biotransformation reactions. To our knowledge, this is the first report of a CYP enzyme from a known bacterial pathogen that is capable of metabolizing clinically utilized drugs.

Structure-based discovery of CFTR potentiators and inhibitors

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Recommended Tool Compounds for Modifying the Cystic Fibrosis Transmembrane Conductance Regulator Channel Variants.

Cystic fibrosis (CF) is a genetic disorder arising from variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to multiple organ system defects. CFTR tool compounds are molecules that can modify the activity of the CFTR channel. Especially, patients that are currently not able to benefit from approved CFTR modulators, such as patients with rare CFTR variants, benefit from further research in discovering novel tools to modulate CFTR. This Review explores the development and classification of CFTR tool compounds, including CFTR blockers (CFTRinh-172, GlyH-101), potentiators (VRT-532, Genistein), correctors (VRT-325, Corr-4a), and other approved and unapproved modulators, with detailed descriptions and discussions for each compound. The challenges and future directions in targeting rare variants and optimizing drug delivery, and the potential synergistic effects in combination therapies are outlined. CFTR modulation holds promise not only for CF treatment but also for generating CF models that contribute to CF research and potentially treating other diseases such as secretory diarrhea. Therefore, continued research on CFTR tool compounds is critical.

Cystic Fibrosis Modulator Therapies: Bridging Insights from CF to other Membrane Protein Misfolding Diseases

AbstractCystic Fibrosis (CF) is a genetic disorder resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to a faulty CFTR protein. Dysfunctional CFTR causes chloride ion imbalance, resulting in dense mucus accumulation in various organs, particularly the lungs. CF treatments focus on symptom management and addressing CFTR′s functional defects. Notably, development of CFTR modulator therapies has significantly advanced CF treatment. These drugs target CFTR protein structural defects induced by mutations, restoring its function and improving CF symptoms. VX‐770, a CFTR potentiator, and CFTR correctors like VX‐809, VX‐661, and VX‐445, have gained FDA approval and widespread clinical use, greatly enhancing the health and survival of many CF patients. However, some CFTR mutations lack effective targeted therapies, leaving approximately 6 % of CF patients without suitable options. CFTR modulator therapies have proven essential for combating the underlying causes of protein misfolding diseases, serving as a blueprint for similar treatments in other membrane protein misfolding diseases. This review explores current and future CFTR modulator therapies, and applications of established paradigms to membrane protein misfolding diseases. Ongoing research and innovation hold the potential for further improvements in CF management and the treatment of protein misfolding diseases.

Neutrophil bactericidal activity and host defenses in cystic fibrosis: a narrative review.

Cystic fibrosis (CF) is a disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR). Without properly functioning CFTR channels, chloride does not exit respiratory epithelial cells, and consequently the mucus lining the surface of the cells becomes thick. This viscous mucus accumulates and causes abnormal function of the mucociliary apparatus, which can lead to bacterial colonization, infections with Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and eventually lung damage. Recent studies have shown that the increased susceptibility to respiratory infections in CF patients may also be due to defects in neutrophil function, but the exact mechanism is uncertain. The PubMed database was searched on February 10, 2023 and again on July 23, 2023 to compile a comprehensive list of clinical and experimental studies to evaluate neutrophil function in CF. The first search included a combination of MeSH terms: "cystic fibrosis" and "neutrophils/physiology". A separate second search included a combination of the MeSH terms: "neutrophils" and "cystic fibrosis transmembrane conductance regulator". Neutrophils from patients with CF have decreased transfer of chloride into phagolysosomes after bacterial ingestion and have dysregulated degranulation. This reduces the production of toxic oxidative radicals, especially hypochlorous acid (HOCl), and reduces bactericidal activity. CFTR potentiators correct the dysregulated degranulation in patients with CF and increased neutrophil killing activity. A reduced concentration of chloride in in vitro assays also reduces neutrophil killing activity; these observations are relevant to the reduced chloride concentrations in respiratory secretions in patients with CF. This literature review summarizes studies that demonstrate that an important defect in CF neutrophils lies in the oxygen-dependent pathway in phagolysosomes and studies with ivacaftor demonstrate that this drug corrects CF neutrophil function. These studies demonstrate the potential utility of using easily available neutrophils to study drug effects in CF patients.

A Novel 7H-[1,2,4]Triazolo[3,4-b]thiadiazine-based Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Directed toward Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal genetic disorder caused by disrupted anion transport in epithelial cells lining tissues in the human airways and digestive system. While cystic fibrosis transmembrane conductance regulator (CFTR) modulator compounds have provided transformative improvement in CF respiratory function, certain patients exhibit marginal clinical benefit or detrimental effects or have a form of the disease not approved or unlikely to respond using CFTR modulation. We tested hit compounds from a 300,000-drug screen for their ability to augment CFTR transepithelial transport alone or in combination with the FDA-approved CFTR potentiator ivacaftor (VX-770). A subsequent SAR campaign led us to a class of 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines that in combination with VX-770 rescued function of G551D mutant CFTR channels to approximately 400% above the activity of VX-770 alone and to nearly wild-type CFTR levels in the same Fischer rat thyroid model system.

CFTR potentiator ivacaftor protects against noise-induced hair cell loss by increasing Nrf2 and reducing oxidative stress

Over-production of reactive oxygen species (ROS) in the inner ear can be triggered by a variety of pathological events identified in animal models after traumatic noise exposure. Our previous research found that inhibition of the AMP-activated protein kinase alpha subunit (AMPKα) protects against noise-induced cochlear hair cell loss and hearing loss by reducing ROS accumulation. However, the molecular pathway through which AMPKα exerts its antioxidative effect is still unclear. In this study, we have investigated a potential target of AMPKα and ROS, cystic fibrosis transmembrane conductance regulator (CFTR), and the protective effect against noise-induced hair cell loss of an FDA-approved CFTR potentiator, ivacaftor, in FVB/NJ mice, mouse explant cultures, and HEI-OC1 cells. We found that noise exposure increases phosphorylation of CFTR at serine 737 (p-CFTR, S737), which reduces wildtype CFTR function, resulting in oxidative stress in cochlear sensory hair cells. Pretreatment with a single dose of ivacaftor maintains CFTR function by preventing noise-increased p-CFTR (S737). Furthermore, ivacaftor treatment increases nuclear factor E2-related factor 2 (Nrf2) expression, diminishes ROS formation, and attenuates noise-induced hair cell loss and hearing loss. Additionally, inhibition of noise-induced AMPKα activation by compound C also diminishes p-CFTR (S737) expression. In line with these in-vivo results, administration of hydrogen peroxide to cochlear explants or HEI-OC1 cells increases p-CFTR (S737) expression and induces sensory hair cell or HEI-OC1 cell damage, while application of ivacaftor halts these effects. Although ivacaftor increases Nrf2 expression and reduces ROS accumulation, cotreatment with ML385, an Nrf2 inhibitor, abolishes the protective effects of ivacaftor against hydrogen-peroxide-induced HEI-OC1 cell death. Our results indicate that noise-induced sensory hair cell damage is associated with p-CFTR. Ivacaftor has potential for treatment of noise-induced hearing loss by maintaining CFTR function and increasing Nrf2 expression for support of redox homeostasis in sensory hair cells.

Cystic fibrosis transmembrane conductance regulator potentiators attenuate platelet activation and aggregation in blood of healthy donors and COVID-19 patients

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl - channel and ABC transporter; its mutations cause the clinical picture of cystic fibrosis (CF). Of late, CFTR has emerged as an important regulator of platelet function, as CFTR dysfunction causes agonist-induced platelet hyperactivation. These findings are reminiscent of platelets from SARS-CoV-2 infected patients since thromboembolic complications represent hallmarks of severe COVID-19 that may critically contribute to morbidity and mortality. CFTR modulators have recently been introduced as a treatment for patients with various CFTR mutations, but have also been reported by us and others to enhance channel function of wild type CFTR. We therefore postulated that CFTR modulators may exert anti-coagulant effects on platelets of healthy donors (HD) and COVID-19 patients.We recruited 36 COVID-19 patients with moderate, and 34 COVID-19 patients with severe disease course (all w/o anti-platelet drugs), and 38 HDs. In line with our hypothesis, we observed significant reductions in platelet agonists adenosine diphosphate (ADP)- or thrombin receptor activating protein-6 (TRAP6)-induced CD62p/CD63 expression, Ca 2+ -mobilization, aggregation, and adhesion of platelets from HDs by pre-treatment with ivacaftor. In blood from COVID-19 patients, platelet activation correlates with disease severity, as demonstrated by a 5-fold and 8-fold increase in the proportion of CD62p + platelets from patients with moderate and severe disease, respectively, relative to HDs. Similarly, the proportion of CD63 + platelets in patients with severe COVID-19 was 2-fold higher than in HDs. Retrospective analysis of clinical data from a total of 4,050 CF patients with COVID-19 receiving single or combination therapy of ivacaftor, lumacaftor, tezacaftor, or elexacaftor in comparison to an untreated cohort revealed that CF therapy reduced the relative risk to suffer thromboembolism-associated cardiovascular events such as heart attack or deep vein thrombosis by 50.0% or 61.1%, respectively, suggesting an anti-thrombotic effect of CFTR modulators in CF COVID-19 patients. In line with this observation, ex vivo pre-treatment of platelets from acute COVID-19 patients with ivacaftor reduced Ca 2+ mobilization, adhesion, and aggregation of platelets .Our results demonstrate an anticoagulant effect of CFTR potentiators on platelets from HDs and severe COVID-19 patients and thus, suggest CFTR potentiators as a promising strategy to reduce the risk of thrombotic events in the clinical management of COVID-19 and similar pro-thrombotic disease states. F. Behrens received funding from the Berlin Institute of Health (BIH). L. Michalick reports grants from the BIH and the German Centre for Cardiovascular Research (DZHK). A. Haghikia is participant in the BIH-Charité Advanced Clinician Scientist Pilotprogram funded by the Charité - Universitätsmedizin Berlin and the BIH and reports a research grant within the BIH & MDC Focus Area Translational Vascular Biomedicine. R. Preissner reports partial funding of this work by the German Research Foundation (KFO339, TRR295). M. Witzenrath reports grants from the German Research Foundation (SFB-TR84 C06 and C09, SFB 1449 B02), and from the German Ministry of Education and Research (BMBF) in the framework of CAPSyS (01ZX1604B, 01ZX1304B), SYMPATH (01ZX1906A), PROVID (01KI20160A), Phage4Cure (16GW0141), MAPVAP (16GW0247) and NUM-NAPKON. W. M. Kuebler reports grants from the German Research Foundation (SFB-TR84 A2 and C9, SFB 1449 B1, SFB 1470 A4, KU1218/9-1, KU1218/11-1, and KU1218/12-1), the BMBF in the framework of SYMPATH (01ZX1906A) and PROVID (01KI20160A), and the DZHK. S. Simmons reports grants from the DZHK and the German Foundation for Heart Research (F-09-19). This is the full abstract presented at the American Physiology Summit 2023 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.

Cystic fibrosis transmembrane conductance regulator in COPD: a role in respiratory epithelium and beyond.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel for transport of chloride and bicarbonate anions. Functional roles of CFTR have been identified in a broad range of cell types including epithelial, endothelial, immune and structural cells. While CFTR has been investigated largely in the context of inborn dysfunction in cystic fibrosis, recent evidence shows that CFTR is also affected by acquired dysfunction in COPD. In patients with COPD and smokers, CFTR impairment has been demonstrated in the upper and lower airways, sweat glands and intestines, suggesting both pulmonary and systemic defects. Cigarette smoke, a key factor in COPD development, is the major cause of acquired CFTR dysfunction. Inflammation, bacterial byproducts and reactive oxygen species can further impair CFTR expression and function. CFTR dysfunction could contribute directly to disease manifestation and progression of COPD including disturbed airway surface liquid homeostasis, airway mucus obstruction, pathogen colonisation and inflammation. Mucus plugging and neutrophilic inflammation contribute to tissue destruction, development of dysfunction at the level of the small airways and COPD progression. Acquired CFTR dysfunction in extrapulmonary organs could add to common comorbidities and the disease burden. This review explores how CFTR dysfunction may be acquired and its potential effects on patients with COPD, particularly those with chronic bronchitis. The development of CFTR potentiators and the probable benefits of CFTR potentiation to improve tissue homeostasis, reduce inflammation, improve host defence and potentially reduce remodelling in the lungs will be discussed.

Loss of endothelial CFTR drives barrier failure and edema formation in lung infection and can be targeted by CFTR potentiation

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl-]i and [Ca2+]i). Inhibition of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i, whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/-) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae. The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.

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.

Plasma and cellular ivacaftor concentrations in patients with cystic fibrosis.

Access to cystic fibrosis transmembrane conductance regulator(CFTR) modulators has been gradually increasing for people with cystic fibrosis, the first of which was ivacaftor, a CFTR potentiator that is part of all clinically available modulator treatments. In this study, we hypothesized that the steady-state concentrations in blood and tissue are highly variable in patients taking ivacaftor in a real-world context, which may have an impacton the treatment approach. We collected nasal epithelial cells to estimate target site concentrations and blood samples to estimate pharmacokinetic parameters at a steady state. We found that patients on ivacaftor monotherapy have variable concentrations well above the maximal effective concentration and may maintain concentrations necessary for the clinical benefit even if dosing is reduced. We also are the first to provide detailed target site concentration data over time, which shows that tissue concentrations do not fluctuate significantly and do not correlate with plasma concentrations. These findings show that some patients may have higher-than-expected concentrations and may benefit from tailored dosing to balance clinical response with side effects or adherence needs.

Loss of Endothelial CFTR Drives Barrier Failure and Edema Formation in Lung Infection and Can Be Targeted by CFTR Potentiation

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS), a potentially fatal lung disease characterized by hyperinflammation and endothelial barrier failure. Infectious and inflammatory stimuli can cause rapid downregulation of cystic fibrosis transmembrane conductance regulator (CFTR), and inhibition of CFTR has been proposed to increase lung microvascular endothelial permeability in vitro. Here, we identify loss of lung microvascular endothelial CFTR as important pathomechanism in lung barrier failure in pneumonia‐induced ARDS, delineate the molecular signaling pathway underlying this effect and identify CFTR potentiation as novel therapeutic strategy in ARDS.CFTR was downregulated in endothelial cells following Streptococcus pneumoniae(S.pn.) infection in human and murine lung tissue. Isolated perfused lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular Cl‐ and Ca2+concentrations ([Cl‐]i, [Ca2+]i). Inhibition of the Cl‐ sensitive with‐no‐lysine kinase 1 (WNK1) replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i while WNK1 activation attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4‐/‐) developed less lung edema and protein leak than their wild‐type littermates following infection with S. pn. Conversely, lungs of heterozygous Wnk1‐deficient mice (Wnk1+/‑) showed spontaneous leak. The CFTR potentiator ivacaftor prevented CFTR loss and reduced endothelial leak in response to pneumolysin (PLY), a key S.pn. virulence factor, or plasma from COVID‐19 patients in vitro, and prevented lung edema and protein leak after S. pn.infection in vivo.Lung infection causes rapid loss of CFTR that promotes lung edema formation through intracellular Cl‐accumulation, inhibition of WNK1 and subsequent disinhibition of TRPV4, resulting in endothelial Ca2+ influx and vascular barrier failure. Ivacaftor prevents CFTR loss and may thus present a promising therapeutic strategy in ARDS due to severe pneumonia including COVID‐19.

Alveolar Responses to Influenza Lung Infection Determined by Confocal Imaging of Live Lungs

One‐third of patients with severe lung infection by influenza A virus (IAV) develop secondary lung infection by inhaled S. aureus(SA), leading to mortality in 30% of cases. It is unclear how IAV promotes secondary SA infection in lung alveoli, where fluid barrier dysfunction causes fatal acute lung injury (ALI). Our objective is to define alveolar responses to IAV that promote SA infection, leading to SA‐induced alveolar damage and mortality. Our hypothesis is IAV lung infection blocks alveolar wall liquid (AWL) secretion, a homeostatic alveolar function that defends against alveolar stabilization of inhaled particles. AWL secretion depends on function of the alveolar epithelial cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. Our methods include optical imaging of live, intact mouse lungs and mouse survival determinations. Mice were untreated or intranasally instilled with IAV (2000 PFU, A/PR/8/34). We excised, inflated, and blood‐perfused the lungs at 24h, then viewed live alveoli by confocal microscopy. Alveolar airspaces were microinstilled separately with: (A) the fluorescent cytosolic dye, calcein‐AM; (B) the fluorescent AWL tracer, tetramethylrhodamine‐conjugated dextran (70 kD); (C) the CFTR potentiator, ivacaftor; and (D) the SA toxin, alpha hemolysin (Hla). For survival studies, IAV‐infected mice were intranasally instilled with SA (USA300, 1x10^8 CFU) at 24h, then observed for 72h. Mice were given intraperitoneal injections of ivacaftor or vehicle at 4, 24, and 48h after SA instillation. Analysis was by t test (n=3) and log rank. Our results follow. Calcein fluorescence indicated the alveolar epithelium of IAV‐infected lungs was viable. In alveoli of untreated lungs, airspace dextran fluorescence decreased over 1h, indicating dextran was progressively diluted by AWL secretion. However, in IAV‐infected lungs, dextran fluorescence was steady (P<0.05), indicating IAV blocked AWL secretion. Since alveolar ivacaftor pretreatment restored dextran loss (P=NS versus untreated), we interpret IAV blocked AWL secretion in a CFTR‐dependent manner, and CFTR potentiation rescued AWL secretion. Hla microinstillation into alveoli of IAV‐infected lungs caused loss of epithelial calcein fluorescence, but alveolar pretreatment with ivacaftor blocked the loss (P<0.05). We interpret that rescue of AWL secretion protected against Hla‐induced alveolar damage, probably by promoting Hla clearance and disrupting Hla‐epithelial interactions. IAV‐infected mice treated with ivacaftor were protected from SA‐induced mortality (60% and 0% in vehicle‐ versus ivacaftor‐treated mice, respectively; P<0.05; N=10 each). We conclude that IAV disrupted AWL secretion, causing retention of airspace contents and augmenting Hla‐induced alveolar damage. Rescue of AWL secretion blocked Hla‐induced alveolar damage and increased survival in IAV‐SA coinfected mice. We propose strategies that rescue AWL secretion may represent a new therapeutic approach for ALI caused by IAV‐SA coinfection.

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Primary Human Nasal Epithelial Cells: Biobanking in the Context of Precision Medicine.

Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl-) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells' electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.

Novel CFTR modulator combinations maximise rescue of G85E and N1303K in rectal organoids

IntroductionCystic fibrosis (CF) is a severe monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Several types of CFTR modulators (correctors/potentiators) have been developed to overcome protein dysfunction associated with these mutations. CFTR modulator therapy is now available for the major CF-causing mutations; however, 10% of people with CF remain without causal treatments. By combining investigational and market-approved CFTR modulators, we aimed to maximise functional rescue of iva-, luma- and tezacaftor refractory mutants G85E and N1303K.MethodsWe used the well-established forskolin-induced swelling (FIS) in primary rectal organoids to assess responses to different CFTR corrector and potentiator types. The FIS analysis was performed with brightfield microscopy, allowing both 1-h and 24-h follow-up. Corrector and potentiator activity of elexacaftor was investigated.ResultsFor G85E, maximal rescue was observed by a combination of elexacaftor and corr4a. For N1303K, the quadruple combination teza-elexa-ivacaftor with apigenin was required to obtain a rescue similar to that of luma-ivacaftor rescued F508del. Elexacaftor rescued G85E and N1303K by different mechanisms, with chronic corrector effects on G85E and acute potentiation of N1303K only in the presence of ivacaftor. Synergy in N1303K rescue for iva-elexacaftor and apigenin suggests at least three potentiator mechanisms for this mutant. 24-h FIS identified ivacaftor as the main CFTR modulator for N1303K and elexacaftor and apigenin as co-potentiators.ConclusionsNovel combinations of CFTR modulators can further improve functional rescue of G85E and N1303K in rectal organoids, although for N1303K, more effective CFTR modulators are still needed.

A small molecule CFTR potentiator restores ATP-dependent channel gating to the cystic fibrosis mutant G551D-CFTR.

Background and PurposeCystic fibrosis transmembrane conductance regulator (CFTR) potentiators are small molecules developed to treat the genetic disease cystic fibrosis (CF). They interact directly with CFTR Cl− channels at the plasma membrane to enhance channel gating. Here, we investigate the action of a new CFTR potentiator, CP‐628006 with a distinct chemical structure.Experimental ApproachUsing electrophysiological assays with CFTR‐expressing heterologous cells and CF patient‐derived human bronchial epithelial (hBE) cells, we compared the effects of CP‐628006 with the marketed CFTR potentiator ivacaftor.Key ResultsCP‐628006 efficaciously potentiated CFTR function in epithelia from cultured hBE cells. Its effects on the predominant CFTR variant F508del‐CFTR were larger than those with the gating variant G551D‐CFTR. In excised inside‐out membrane patches, CP‐628006 potentiated wild‐type, F508del‐CFTR, and G551D‐CFTR by increasing the frequency and duration of channel openings. CP‐628006 increased the affinity and efficacy of F508del‐CFTR gating by ATP. In these respects, CP‐628006 behaved like ivacaftor. CP‐628006 also demonstrated notable differences with ivacaftor. Its potency and efficacy were lower than those of ivacaftor. CP‐628006 conferred ATP‐dependent gating on G551D‐CFTR, whereas the action of ivacaftor was ATP‐independent. For G551D‐CFTR, but not F508del‐CFTR, the action of CP‐628006 plus ivacaftor was greater than ivacaftor alone. CP‐628006 delayed, but did not prevent, the deactivation of F508del‐CFTR at the plasma membrane, whereas ivacaftor accentuated F508del‐CFTR deactivation.Conclusions and ImplicationsCP‐628006 has distinct effects compared to ivacaftor, suggesting a different mechanism of CFTR potentiation. The emergence of CFTR potentiators with diverse modes of action makes therapy with combinations of potentiators a possibility.

Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.