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.     

Proximity Profiling of the CFTR Interaction Landscape in Response to Orkambi

Deletion of phenylalanine 508 (∆F508) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel protein is the leading cause of Cystic Fibrosis (CF). Here, we report the analysis of CFTR and ∆F508-CFTR interactomes using BioID (proximity-dependent biotin identification), a technique that can also detect transient associations. We identified 474 high-confidence CFTR proximity-interactors, 57 of which have been previously validated, with the remainder representing novel interaction space. The ∆F508 interactome, comprising 626 proximity-interactors was markedly different from its wild type counterpart, with numerous alterations in protein associations categorized in membrane trafficking and cellular stress functions. Furthermore, analysis of the ∆F508 interactome in cells treated with Orkambi identified several interactions that were altered as a result of this drug therapy. We examined two candidate CFTR proximity interactors, VAPB and NOS1AP, in functional assays designed to assess surface delivery and overall chloride efflux. VAPB depletion impacted both CFTR surface delivery and chloride efflux, whereas NOS1AP depletion only affected the latter. The wild type and ∆F508-CFTR interactomes represent rich datasets that could be further mined to reveal additional candidates for the functional rescue of ∆F508-CFTR.     

Molecular Motors and Apical CFTR Traffic in Epithelia

Intracellular protein traffic plays an important role in the regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channels. Microtubule and actin-based motor proteins direct CFTR movement along trafficking pathways. As shown for other regulatory proteins such as adaptors, the involvement of protein motors in CFTR traffic is cell-type specific. Understanding motor specificity provides insight into the biology of the channel and opens opportunity for discovery of organ-specific drug targets for treating CFTR-mediated diseases.     

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.     

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.     

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.     

Assays of CFTR Function In Vitro,Ex Vivo and In Vivo

Cystic fibrosis, a multi-organ genetic disease, is characterized by abnormal function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel at the apical membrane of several epithelia. In recent years, therapeutic strategies have been developed to correct the CFTR defect. To evaluate CFTR function at baseline for diagnosis, or the efficacy of CFTR-restoring therapy, reliable tests are needed to measure CFTR function, in vitro, ex vivo and in vivo. In vitro techniques either directly or indirectly measure ion fluxes; direct measurement of ion fluxes and quenching of fluorescence in cell-based assays, change in transmembrane voltage or current in patch clamp or Ussing chamber, swelling of CFTR-containing organoids by secondary water influx upon CFTR activation. Several cell or tissue types can be used. Ex vivo and in vivo assays similarly evaluate current (intestinal current measurement) and membrane potential differences (nasal potential difference), on tissues from individual patients. In the sweat test, the most frequently used in vivo evaluation of CFTR function, chloride concentration or stimulated sweat rate can be directly measured. Here, we will describe the currently available bio-assays for quantitative evaluation of CFTR function, their indications, advantages and disadvantages, and correlation with clinical outcome measures.     

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.     

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.     

Correctors Rescue CFTR Mutations in Nucleotide‐Binding Domain 1 (NBD1) by Modulating Proteostasis

We evaluated whether small molecule correctors could rescue four nucleotide‐binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos‐7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK‐293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT‐002, CFFT‐003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.     

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.     

Differential CFTR-Interactome Proximity Labeling Procedures Identify Enrichment in Multiple SLC Transporters

Proteins interacting with CFTR and its mutants have been intensively studied using different experimental approaches. These studies provided information on the cellular processes leading to proper protein folding, routing to the plasma membrane, recycling, activation and degradation. Recently, new approaches have been developed based on the proximity labeling of protein partners or proteins in close vicinity and their subsequent identification by mass spectrometry. In this study, we evaluated TurboID- and APEX2-based proximity labeling of WT CFTR and compared the obtained data to those reported in databases. The CFTR-WT interactome was then compared to that of two CFTR (G551D and W1282X) mutants and the structurally unrelated potassium channel KCNK3. The two proximity labeling approaches identified both known and additional CFTR protein partners, including multiple SLC transporters. Proximity labeling approaches provided a more comprehensive picture of the CFTR interactome and improved our knowledge of the CFTR environment.     

Therapeutic CFTR Correction Normalizes Systemic and Lung-Specific S1P Level Alterations Associated with Heart Failure

Heart failure (HF) is among the main causes of death worldwide. Alterations of sphingosine-1-phosphate (S1P) signaling have been linked to HF as well as to target organ damage that is often associated with HF. S1P’s availability is controlled by the cystic fibrosis transmembrane regulator (CFTR), which acts as a critical bottleneck for intracellular S1P degradation. HF induces CFTR downregulation in cells, tissues and organs, including the lung. Whether CFTR alterations during HF also affect systemic and tissue-specific S1P concentrations has not been investigated. Here, we set out to study the relationship between S1P and CFTR expression in the HF lung. Mice with HF, induced by myocardial infarction, were treated with the CFTR corrector compound C18 starting ten weeks post-myocardial infarction for two consecutive weeks. CFTR expression, S1P concentrations, and immune cell frequencies were determined in vehicle- and C18-treated HF mice and sham controls using Western blotting, flow cytometry, mass spectrometry, and qPCR. HF led to decreased pulmonary CFTR expression, which was accompanied by elevated S1P concentrations and a pro-inflammatory state in the lungs. Systemically, HF associated with higher S1P plasma levels compared to sham-operated controls and presented with higher S1P receptor 1-positive immune cells in the spleen. CFTR correction with C18 attenuated the HF-associated alterations in pulmonary CFTR expression and, hence, led to lower pulmonary S1P levels, which was accompanied by reduced lung inflammation. Collectively, these data suggest an important role for the CFTR-S1P axis in HF-mediated systemic and pulmonary inflammation.     

Purification of CFTR for mass spectrometry analysis: identification of palmitoylation and other post-translational modifications

Post-translational modifications (PTMs) play a crucial role during biogenesis of many transmembrane proteins. Previously, it had not been possible to evaluate PTMs in cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial ion channel responsible for cystic fibrosis, because of difficulty obtaining sufficient amounts of purified protein. We recently used an inducible overexpression strategy to generate recombinant CFTR protein at levels suitable for purification and detailed analysis. Using liquid chromatography (LC) tandem and multiple reaction ion monitoring (MRM) mass spectrometry, we identified specific sites of PTMs, including palmitoylation, phosphorylation, methylation and possible ubiquitination. Many of these covalent CFTR modifications have not been described previously, but are likely to influence key and clinically important molecular processes including protein maturation, gating and the mechanisms underlying certain mutations associated with disease.     

Identification of Potential Leukocyte Biomarkers Related to Drug Recovery of CFTR: Clinical Applications in Cystic Fibrosis

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC–MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl⁻ transport in CF.     

Isorhamnetin Ameliorates Dry Eye Disease via CFTR Activation in Mice

Dry eye disease is one of the most common diseases, with increasing prevalence in many countries, but treatment options are limited. Cystic fibrosis transmembrane conductance regulator (CFTR) is a major ion channel that facilitates fluid secretion in ocular surface epithelium and is a potential target of therapeutic agent for the treatment of dry eye disease. In this study, we performed a cell-based, high-throughput screening for the identification of novel natural products that activate CFTR and restore the aqueous deficiency in dry eye. Screening of 1000 natural products revealed isorhamnetin, a flavonol aglycone, as a novel CFTR activator. Electrophysiological studies showed that isorhamnetin significantly increased CFTR chloride current, both wild type and ∆F508-CFTR. Isorhamnetin did not alter intracellular cAMP levels and the activity of other ion channels, including ANO1, ENaC, and hERG. Notably, application of isorhamnetin on mouse ocular surface induced CFTR activation and increased tear volume. In addition, isorhamnetin significantly reduced ocular surface damage and expression of interleukin (IL)-1β, IL-8, and tumor necrosis factor (TNF)-α in an experimental mouse model of dry eye. These data suggest that isorhamnetin may be used to treat dry eye disease.     

Dysfunctional Inflammation in Cystic Fibrosis Airways: From Mechanisms to Novel Therapeutic Approaches

Cystic fibrosis (CF) is an inherited disorder caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, an ATP-gated chloride channel expressed on the apical surface of airway epithelial cells. CFTR absence/dysfunction results in defective ion transport and subsequent airway surface liquid dehydration that severely compromise the airway microenvironment. Noxious agents and pathogens are entrapped inside the abnormally thick mucus layer and establish a highly inflammatory environment, ultimately leading to lung damage. Since chronic airway inflammation plays a crucial role in CF pathophysiology, several studies have investigated the mechanisms responsible for the altered inflammatory/immune response that, in turn, exacerbates the epithelial dysfunction and infection susceptibility in CF patients. In this review, we address the evidence for a critical role of dysfunctional inflammation in lung damage in CF and discuss current therapeutic approaches targeting this condition, as well as potential new treatments that have been developed recently. Traditional therapeutic strategies have shown several limitations and limited clinical benefits. Therefore, many efforts have been made to develop alternative treatments and novel therapeutic approaches, and recent findings have identified new molecules as potential anti-inflammatory agents that may exert beneficial effects in CF patients. Furthermore, the potential anti-inflammatory properties of CFTR modulators, a class of drugs that directly target the molecular defect of CF, also will be critically reviewed. Finally, we also will discuss the possible impact of SARS-CoV-2 infection on CF patients, with a major focus on the consequences that the viral infection could have on the persistent inflammation in these patients.