Capturing the Direct Binding of CFTR Correctors to CFTR by Using Click Chemistry

Cystic fibrosis (CF) is a lethal genetic disease caused by the loss or dysfunction of the CF transmembrane conductance regulator (CFTR) channel. F508del is the most prevalent mutation of the CFTR gene and encodes a protein defective in folding and processing. VX‐809 has been reported to facilitate the folding and trafficking of F508del‐CFTR and augment its channel function. The mechanism of action of VX‐809 has been poorly understood. In this study, we sought to answer a fundamental question underlying the mechanism of VX‐809: does it bind CFTR directly in order to exert its action? We synthesized two VX‐809 derivatives, ALK‐809 and SUL‐809, that possess an alkyne group and retain the rescue capacity of VX‐809. By using Cu^I‐catalyzed click chemistry, we provide evidence that the VX‐809 derivatives bind CFTR directly in vitro and in cells. Our findings will contribute to the elucidation of the mechanism of action of CFTR correctors and the design of more potent therapeutics to combat CF.     

KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR

Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin–proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.     

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.     

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including β2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.     

CFTR,Cell Junctions and the Cytoskeleton

The multi-organ disease cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a cAMP regulated chloride (Cl⁻) and bicarbonate (HCO₃⁻) ion channel expressed at the apical plasma membrane (PM) of epithelial cells. Reduced CFTR protein results in decreased Cl⁻ secretion and excessive sodium reabsorption in epithelial cells, which consequently leads to epithelial dehydration and the accumulation of thick mucus within the affected organs, such as the lungs, pancreas, gastrointestinal (GI) tract, reproductive system and sweat glands. However, CFTR has been implicated in other functions besides transporting ions across epithelia. The rising number of references concerning its association to actin cytoskeleton organization, epithelial cell junctions and extracellular matrix (ECM) proteins suggests a role in the formation and maintenance of epithelial apical basolateral polarity. This review will focus on recent literature (the last 10 years) substantiating the role of CFTR in cell junction formation and actin cytoskeleton organization with its connection to the ECM.     

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.     

Emerging Concepts in Defective Macrophage Phagocytosis in Cystic Fibrosis

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Chronic inflammation and decline in lung function are major reasons for morbidity in CF. Mutant CFTR expressed in phagocytic cells such as macrophages contributes to persistent infection, inflammation, and lung disease in CF. Macrophages play a central role in innate immunity by eliminating pathogenic microbes by a process called phagocytosis. Phagocytosis is required for tissue homeostasis, balancing inflammation, and crosstalk with the adaptive immune system for antigen presentation. This review focused on (1) current understandings of the signaling underlying phagocytic mechanisms; (2) existing evidence for phagocytic dysregulation in CF; and (3) the emerging role of CFTR modulators in influencing CF phagocytic function. Alterations in CF macrophages from receptor initiation to phagosome formation are linked to disease progression in CF. A deeper understanding of macrophages in the context of CFTR and phagocytosis proteins at each step of phagosome formation might contribute to the new therapeutic development of dysregulated innate immunity in CF. Therefore, the review also indicates future areas of research in the context of CFTR and macrophages.     

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.     

Synergy in Cystic Fibrosis Therapies: Targeting SLC26A9

SLC26A9, a constitutively active Cl⁻ transporter, has gained interest over the past years as a relevant disease modifier in several respiratory disorders including Cystic Fibrosis (CF), asthma, and non-CF bronchiectasis. SLC26A9 contributes to epithelial Cl⁻ secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.     

The Effect of CFTR Modulators on Airway Infection in Cystic Fibrosis

The advent of Cystic fibrosis transmembrane receptor (CFTR) modulators in 2012 was a critical event in the history of cystic fibrosis (CF) treatment. Unlike traditional therapies that target downstream effects of CFTR dysfunction, CFTR modulators aim to correct the underlying defect at the protein level. These genotype-specific therapies are now available for an increasing number of CF patients, transforming the way we view the condition from a life-limiting disease to one that can be effectively managed. Several studies have demonstrated the vast improvement CFTR modulators have on normalization of sweat chloride, CFTR function, clinical endpoints, and frequency of pulmonary exacerbation. However, their impact on other aspects of the disease, such as pathogenic burden and airway infection, remain under explored. Frequent airway infections as a result of increased susceptibility and impaired innate immune response are a serious problem within CF, often leading to accelerated decline in lung function and disease progression. Current evidence suggests that CFTR modulators are unable to eradicate pathogenic organisms in those with already established lung disease. However, this may not be the case for those with relatively low levels of disease progression and conserved microbial diversity, such as young patients. Furthermore, it remains unknown whether the restorative effects exerted by CFTR modulators extend to immune cells, such as phagocytes, which have the potential to modulate the response of people with CF (pwCF) to infection. Throughout this review, we look at the potential impact of CFTR modulators on airway infection in CF and their ability to shape impaired pulmonary defences to pathogens.     

Anti-Inflammatory Influences of Cystic Fibrosis Transmembrane Conductance Regulator Drugs on Lung Inflammation in Cystic Fibrosis

Cystic fibrosis (CF) is caused by a defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) which instigates a myriad of respiratory complications including increased vulnerability to lung infections and lung inflammation. The extensive influx of pro-inflammatory cells and production of mediators into the CF lung leading to lung tissue damage and increased susceptibility to microbial infections, creates a highly inflammatory environment. The CF inflammation is particularly driven by neutrophil infiltration, through the IL-23/17 pathway, and function, through NE, NETosis, and NLRP3-inflammasome formation. Better understanding of these pathways may uncover untapped therapeutic targets, potentially reducing disease burden experienced by CF patients. This review outlines the dysregulated lung inflammatory response in CF, explores the current understanding of CFTR modulators on lung inflammation, and provides context for their potential use as therapeutics for CF. Finally, we discuss the determinants that need to be taken into consideration to understand the exaggerated inflammatory response in the CF lung.     

Targeting of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein with a Technetium‐99m Imaging Probe

Cystic fibrosis (CF) is caused by mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to almost total absence of CFTR at the plasma membrane, a defect potentially corrected via drug‐based therapies. Herein, we report the first proof‐of‐principle study of a noninvasive imaging probe able to detect CFTR at the plasma membrane. We radiolabeled the CFTR inhibitor, CFTR_inh‐172a, with technetium‐99m via a pyrazolyl‐diamine chelating unit, yielding a novel ^99mTc(CO)₃ complex. A non‐radioactive surrogate showed that the structural modifications introduced in the inhibitor did not affect its activity. The radioactive complex was able to detect plasma membrane CFTR, shown by its significantly higher uptake in wild‐type versus mutated cells. Furthermore, assessment of F508del CFTR pharmacological correction in human cells using the radioactive complex revealed differences in corrector versus control uptake, recapitulating the biochemical correction observed for the protein.     

The NHE3 Inhibitor Tenapanor Prevents Intestinal Obstructions in CFTR-Deleted Mice

Mutations in the CFTR chloride channel result in intestinal obstructive episodes in cystic fibrosis (CF) patients and in CF animal models. In this study, we explored the possibility of reducing the frequency of obstructive episodes in cftr^−/− mice through the oral application of a gut-selective NHE3 inhibitor tenapanor and searched for the underlying mechanisms involved. Sex- and age-matched cftr^+/+ and cftr^−/− mice were orally gavaged twice daily with 30 mg kg⁻¹ tenapanor or vehicle for a period of 21 days. Body weight and stool water content was assessed daily and gastrointestinal transit time (GTT) once weekly. The mice were sacrificed when an intestinal obstruction was suspected or after 21 days, and stool and tissues were collected for further analysis. Twenty-one day tenapanor application resulted in a significant increase in stool water content and stool alkalinity and a significant decrease in GTT in cftr^+/+ and cftr^−/− mice. Tenapanor significantly reduced obstructive episodes to 8% compared to 46% in vehicle-treated cftr^−/− mice and prevented mucosal inflammation. A decrease in cryptal hyperproliferation, mucus accumulation, and mucosal mast cell number was also observed in tenapanor- compared to vehicle-treated, unobstructed cftr^−/− mice. Overall, oral tenapanor application prevented obstructive episodes in CFTR-deficient mice and was safe in cftr^+/+ and cftr^−/− mice. These results suggest that tenapanor may be a safe and affordable adjunctive therapy in cystic fibrosis patients to alleviate constipation and prevent recurrent DIOS.     

Genetic Deletion of Mmp9 Does Not Reduce Airway Inflammation and Structural Lung Damage in Mice with Cystic Fibrosis-like Lung Disease

Elevated levels of matrix metalloprotease 9 (MMP-9) and neutrophil elastase (NE) are associated with bronchiectasis and lung function decline in patients with cystic fibrosis (CF). MMP-9 is a potent extracellular matrix-degrading enzyme which is activated by NE and has been implicated in structural lung damage in CF. However, the role of MMP-9 in the in vivo pathogenesis of CF lung disease is not well understood. Therefore, we used β-epithelial Na⁺ channel-overexpressing transgenic (βENaC-Tg) mice as a model of CF-like lung disease and determined the effect of genetic deletion of Mmp9 (Mmp9^-/-) on key aspects of the pulmonary phenotype. We found that MMP-9 levels were elevated in the lungs of βENaC-Tg mice compared with wild-type littermates. Deletion of Mmp9 had no effect on spontaneous mortality, inflammatory markers in bronchoalveolar lavage, goblet cell metaplasia, mucus hypersecretion and emphysema-like structural lung damage, while it partially reduced mucus obstruction in βENaC-Tg mice. Further, lack of Mmp9 had no effect on increased inspiratory capacity and increased lung compliance in βENaC-Tg mice, whereas both lung function parameters were improved with genetic deletion of NE. We conclude that MMP-9 does not play a major role in the in vivo pathogenesis of CF-like lung disease in mice.     

A monoclonal antibody prevents aggregation of the NBD1 domain of the cystic fibrosis transmembrane conductance regulator

The homozygous deletion of the phenylalanine at position 508 (DeltaPhe508) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF-causing genetic defect. It has been proposed that the propensity of NBD1 to aggregate may lead to a lower display of the CFTR chloride channel to the cell membrane and to the disease, thus opening an avenue for the pharmacological development of CFTR folding correctors. Here, we show that a human monoclonal antibody fragment specific to the folded conformation of NBD1 inhibits the aggregation of NBD1 in vitro. However, in contrast to the previously published observations, we proved experimentally that NBD1 of wild-type and DeltaPhe508 version of CFTR display comparable propensities to aggregate in vitro and that the corresponding full-length CFTR protein reaches the cell membrane with comparable efficiency in mammalian cell expression systems. On the basis of our results, the 'folding defect' hypothesis seems unlikely to represent the causal mechanism for the pathogenesis of CF. A solid understanding of how the DeltaPhe508 deletion leads to the disease represents an absolute requirement for the development of effective drugs against CF.     

Novel CFTR Activator Cact-3 Ameliorates Ocular Surface Dysfunctions in Scopolamine-Induced Dry Eye Mice

Cystic fibrosis transmembrane conductance regulator (CFTR) is highly expressed on the ocular epithelium and plays a pivotal role in the fluid secretion driven by chloride transport. Dry eye disease is one of the most common diseases with limited therapeutic options. In this study, a high-throughput screening was performed to identify novel CFTR activators capable of inducing chloride secretion on the ocular surface. The screening of 50,000 small molecules revealed three novel CFTR activators. Among them, the most potent CFTR activator, Cact-3 (7-(3,4-dimethoxyphenyl)-N-(4-ethoxyphenyl)pyrazolo [1,5-α]pyrimidine-2-carboxamide), produced large and sustained Cl⁻ currents in WT-CFTR-expressing FRT cells with no alterations of ANO1 and hERG channel activity. The application of Cact-3 strongly activated CFTR in the ocular epithelia of mice and it also significantly increased CFTR-mediated Cl⁻ transport in a primary cultured human conjunctival epithelium. Cact-3 strongly stimulated tear secretion in normal mice. In addition, Cact-3 significantly reduced ocular surface damage and the expression of proinflammatory factors, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in an experimental mouse model of dry eye disease. These results suggest that Cact-3, a novel CFTR activator, may be a potential development candidate for the treatment of dry eye disease.