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Prolonged incubation with VX-770 reduces the maximal CFTR-mediated anion current in CFBE and primary cultures of D F508-CFTR but not WT-CFTR HBE. ( A and B ) Representative I sc recordings (A) and quantification of the changes in I sc ( n = 3) (B) in CFBE monolayer expressing
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Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit....
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Context 1
... that VX-770 and some, but not all, other potentiators cause D F508-CFTR destabilization at multiple cellular sites in model sys- tems and primary CF HBE, with consequent reduced functional expression of D F508-CFTR at the cell surface. To investigate the effect of prolonged exposure to VX-770 on F508- CFTR PM expression, we first used the human CF bronchial epithelial cell line CFBE41o − (referred to as CFBE), a widely validated model system with CFTR D F508/ D F508 genetic background but no detectable CFTR protein expression ( 32 ). CFBE cells were engineered for inducible expression of CFTR variants as described ( 10 , 33 ). To facilitate the PM detection of D F508-CFTR, horseradish peroxidase isoenzyme C (HRP-C) was genetically engineered into its fourth extracellular loop. The functional and biochemical properties of D F508-CFTR-HRP are similar to those of the 3HA-tagged variant ( 13 , 34 ) (fig. S1, A to D). Acute addition of VX-770 to low temperature – rescued D F508-CFTR (r D F508) in CFBE cells increased the cAMP-dependent protein kinase (PKA) – activated current by up to sixfold with EC 50 of 12.8 ± 1.0 nM (fig. S2, A and B), similar to that reported in D F508/ D F508 HBE cells (22 ± 10 nM) ( 24 ). Prolonged exposure (24 hours) to VX-770, however, caused a concentration-dependent decrease in the PM density of D F508- CFTR, regardless of whether the preincubation with VX-770 was done at physiological temperature or at 26 to 30°C, which facilitated D F508 CFTR biosynthetic processing (Fig. 1, A and B). The maximal reduction in D F508-CFTR PM density was attained at ~30 nM VX-770, well below the plasma concentration of ~3.5 m M in VX-770 – treated CF patients ( ). Although increasing the concentration of human serum (0 to 100%) raised the EC 50 of VX-770 from 2.5 ± 0.2 nM to 23.1 ± 4.6 nM in the presence of VX-809, it did not affect the reduced PM density achieved by long-term treatment with ≥ 100 nM VX-770 (fig. S2, G and H). In contrast, the PM density of wild-type CFTR or G551D-CFTR was not reduced by prolonged VX-770 exposure (Fig. 1, A and C). VX-809 partially restored D F508-CFTR biogenesis, function, and PM expression by about three- to fourfold in CFBE and primary HBE monolayers (fig. S2C) ( 10 , 13 , 28 ). VX-809 alone or in combination with low-temperature rescue, however, failed to prevent the VX-770 – dependent reduction in D F508-CFTR PM density (Fig. 1, A and B, and fig. S2C). Similar results were obtained for D F508-CFTR rescued with the corrector VX-661 (fig. S2D). The VX-770 – induced reduction in D F508-CFTR PM density was independent of channel gating because neither activation of adenyl cyclase by forskolin nor blocking the channel with BPO-27 ( 36 ) influenced the VX-770 effect (Fig. 1B and fig. S2C). Extended exposure to VX-770 did not affect cell viability (fig. S2E). PM down-regulation of 3HA-tagged D F508-CFTR by VX-770 in low temperature – rescued CFBE, NCI-H441 (a lung adeno- carcinoma cell line exhibiting some Clara cell features), and MDCK II (Madin-Darby canine kidney) epithelial cells suggested that the VX-770 effect is not CFBE-specific or related to the HRP-tag insertion (Fig. 1D and fig. S2F). To evaluate whether VX-770 causes the redistribution of PM resi- dent D F508-CFTR to intracellular pools or exerts a global down- regulation of mature D F508-CFTR in post-ER compartments, we determined the cellular expression of D F508-CFTR by immunoblot analysis. VX-770 treatment for 24 hours decreased the amount of the complex-glycosylated D F508-CFTR (C-band) in CFBE lysates in a dose-dependent manner (Fig. 1E). The VX-770 effect was attenuated in VX-809 – or VX-661 – treated cells, probably due to partial stabilization of the mature D F508-CFTR pool by VX-809, as reported previously (Fig. 1, F and G) ( 10 , 28 , 37 ). In contrast, the complex-glycosylated form of wild-type CFTR and G551D-CFTR was not affected by prolonged VX-770 exposure (Fig. 1H). The modest, albeit significant ( P = 0.02), decrease in the steady-state level of core-glycosylated D F508- CFTR (B-band) may be due to reduced biogenesis and/or accelerated ER degradation upon exposure to 100 nM VX-770 (Fig. 1, E to G). These observations suggest that the VX-770 effect cannot be explained merely by accelerated internalization or impeded recycling of r D F508-CFTR. To assess the functional consequence of prolonged VX-770 exposure of CFBE and primary HBE cells, we performed short-circuit current ( I sc ) measurements after 24 hours of incubation with 100 nM VX-770. Forskolin-stimulated I sc was measured after inhibition of ENaC (epithelial sodium channel) by amiloride and maximal acute potentiation of cell surface D F508-CFTR function with 10 m M VX-770 (Fig. 2A). Forskolin-stimulated I sc (1.7 ± 0.3 m A/cm 2 ) was reduced to 1.1 ± 0.2 m A/cm after incubation of D F508-CFTR – expressing CFBE cells with VX-770 for 24 hours. A comparable reduction in I sc was observed in VX-809 – and VX-661 – corrected cells (Fig. 2, A and B). To confirm the relevance of these results to human tissues, we assessed the VX-770 effect in primary HBE cell cultures, isolated from the lungs of six CFTR D F508/ D F508 patients and four CFTR WT/WT donors. The HBE cells were differentiated on Snapwell filter inserts under ...
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... I sc in the D F508-CFTR HBE was augmented by treatment with the correctors VX-809 or VX-661 (3 m M, 24 hours) ( 13 ). To further increase CFTR- mediated I sc and isolate the apical anion conductance, some cells were differentiated in Ultroser G medium and analyzed after basolateral permeabilization and in the presence of a basolateral-to-apical Cl − gradient. Independent of the differentiation method and presence of a chloride gradient, exposure to VX-770 for 24 hours decreased the VX-809 – or VX-661 – corrected D F508-CFTR current by 33 ± 6% and 47 ± 8% (mean ± SEM, n = 6), respectively ( Fig. 2, C and D, and Table 1). In contrast, VX-770 pretreatment did not affect the PKA-acti- vated wild-type CFTR current in HBE (Fig. 2E and table S1), in line with the absence of changes in PM and C-band density in wild-type CFTR (Fig. 1H). To determine whether down-regulation of r F508-CFTR is a univer- sal phenomenon of long-term potentiator exposure, we tested a panel of CFTR potentiators with distinct chemical structures. These investigational small molecules, abbreviated as P1 to P10, were made available by the Cystic Fibrosis Foundation Therapeutics Inc. (CFFT) for the re- search community (fig. S3A). The potency and efficacy of P1 to P10 on the activity of low-temperature r D F508-CFTR were demonstrated in CFBE cells using the halide-sensitive yellow fluorescent protein (YFP) quenching assay. Acute addition of P1 to P8 confirmed the potentiation of the r D F508-CFTR activity, whereas P9 and P10 had only small effects (Fig. 3, A to H, and fig. S3, B to D). The dose-response curve of genistein (P6), a flavone widely used for acute potentiation of CFTR activity, did not reach saturation activity at 100 m M, suggesting that r D F508-CFTR has lower affinity for genistein than wild-type CFTR ( 40 , 41 ) (Fig. 3F). Prolonged treatment (24 hours) with most potentiators produced a concentration-dependent decrease in D F508-CFTR PM density in ...
Citations
... The inclusion of IVA, another component of Trikafta, might further decrease the stability of TEZ/ELX-rescued mature F508del-CFTR. Chronic treatment with IVA has been shown to destabilize the cell surface F508del-CFTR [93]. ...
Cystic Fibrosis (CF) is a genetic disease caused by mutations in CFTR gene expressing the anion selective channel CFTR located at the plasma membrane of different epithelial cells. The most commonly investigated variant causing CF is F508del. This mutation leads to structural defects in the CFTR protein, which are recognized by the endoplasmic reticulum (ER) quality control system. As a result, the protein is retained in the ER and degraded via the ubiquitin–proteasome pathway. Although blocking ubiquitination to stabilize the CFTR protein has long been considered a potential pharmacological approach in CF, progress in this area has been relatively slow. Currently, no compounds targeting this pathway have entered clinical trials for CF. On the other hand, the emergence of Orkambi initially, and notably the subsequent introduction of Trikafta/Kaftrio, have demonstrated the effectiveness of molecular chaperone-based therapies for patients carrying the F508del variant and even showed efficacy against other variants. These treatments directly target the CFTR variant protein without interfering with cell signaling pathways. This review discusses the limits and potential future of targeting protein ubiquitination in CF.
... Corrector therapy is a priority, and additional approaches to slowing the spread of the illness are being investigated (68). As a potential corrective method for people with F508del-CFTR, the direct and indirect regulation of the nitric oxide (NO) pathway has recently received attention (69). The most recent medication, Riociguat, is an oral NO-independent soluble guanylate cyclase (sGC) stimulator. ...
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). In 1949, it's been identified as a monogenic disease and was thought to primarily affect individuals of Northern European descent. It was the most prevalent autosomal recessive disease that shortens life. With the availability of multiple testing methodologies nowadays, there is a chance to create novel and enhanced treatment options. Even in the absence of a high sweat chloride test (SCT) result, the discovery of two causal mutations is diagnostic for cystic fibrosis (CF). For a CF diagnosis, however, at least two positive E sweat chloride tests are still required. In order to achieve early and active intervention to manage cystic fibrosis (CF) and its comorbidities, treatment regimens for pediatric patients should be evaluated, improved, and closely monitored. New developments in the treatment of cystic fibrosis (CF) have led to the development of medications derived from molecules that target the pathogenetic pathway of the illness. These options are very efficient and allow pediatric patients to receive individualized care. However, in order to better direct patient care and enhance patient outcomes, it is crucial to research uncommon CF mutations, which can provide crucial information about the prognosis of the disease and the relationships between genotype and phenotype. To ensure the success of creating novel, safer, and more efficient treatment approaches, a deeper understanding of the pathogeny of the illness is required. In the age of customized medicine, genetic research will be essential to improving patient care and quality of life for those with uncommon mutations.
... Exposure of CFTR-F508del intestinal organoids to LUM or ELX/TEZ enhanced processing to Band C, with the combination being more effective than LUM alone ( Figure 2D). Notably, ferret CFTR-F508del organoids exposed for 16 hours to the combination of LUM/IVA led to impaired accumulation of Band C, as compared with LUM alone (Figure 2D), similar to previous results in human CFTR-F508del epithelial cells (25). LUM treatment of airway ALI cultures also significantly enhanced ferret CFTR-F508del processing to Band C ( Figure 2, E-H). ...
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
... Additionally, small molecule compounds known as CFTR correctors play a pivotal role in rescuing the folding, processing, and trafficking of CFTR mutants that are retained within the ER due to misfolding (Pedemonte et al., 2005;Van Goor et al., 2011). These correctors, acting as pharmacological chaperones, have been identified through cell-based high-throughput screenings and in silico (virtual) screenings based on the CFTR structure (Pedemonte et al., 2005;Van Goor et al., 2011;Odolczyk et al., 2013;Phuan et al., 2014;Veit et al., 2018;Orro et al., 2021;Fossa et al., 2022). Pharmacological chaperones, including ligands and substrates, directly engage with their target proteins, often leading to an increase in thermodynamic stability (Bernier et al., 2004;Arakawa et al., 2006;Loo and Clarke, 2007). ...
... However, despite the success of Trikafta, there are persistent challenges. ΔF508-CFTR remains ubiquitinated even in the presence of Trikafta, and prolonged administration of IVA destabilizes ΔF508-CFTR on the cell surface (Cholon et al., 2014;Veit et al., 2014). Furthermore, the maturation achieved with Trikafta results in a considerably shorter half-life of ΔF508-CFTR compared to the wild-type CFTR (Capurro et al., 2021;Taniguchi et al., 2022). ...
Cystic fibrosis (CF) is a monogenetic disease caused by the mutation of CFTR, a cAMP-regulated Cl ⁻ channel expressing at the apical plasma membrane (PM) of epithelia. ∆F508-CFTR, the most common mutant in CF, fails to reach the PM due to its misfolding and premature degradation at the endoplasmic reticulum (ER). Recently, CFTR modulators have been developed to correct CFTR abnormalities, with some being used as therapeutic agents for CF treatment. One notable example is Trikafta, a triple combination of CFTR modulators (TEZ/ELX/IVA), which significantly enhances the functionality of ΔF508-CFTR on the PM. However, there’s room for improvement in its therapeutic effectiveness since TEZ/ELX/IVA doesn't fully stabilize ΔF508-CFTR on the PM. To discover new CFTR modulators, we conducted a virtual screening of approximately 4.3 million compounds based on the chemical structures of existing CFTR modulators. This effort led us to identify a novel CFTR ligand named FR3. Unlike clinically available CFTR modulators, FR3 appears to operate through a distinct mechanism of action. FR3 enhances the functional expression of ΔF508-CFTR on the apical PM in airway epithelial cell lines by stabilizing NBD1. Notably, FR3 counteracted the degradation of mature ΔF508-CFTR, which still occurs despite the presence of TEZ/ELX/IVA. Furthermore, FR3 corrected the defective PM expression of a misfolded ABCB1 mutant. Therefore, FR3 may be a potential lead compound for addressing diseases resulting from the misfolding of ABC transporters.
... Previous studies demonstrate that micromolar concentrations of ivacaftor attenuate the plasma membrane expression and function of some CFTR variants (Avramescu et al., 2017;Cholon et al., 2014;Veit et al., 2014;Wang et al., 2014). For example, micromolar concentrations of ivacaftor inhibit F508del-CFTR both by destabilising its structure (Cholon et al., 2014;Veit et al., 2014) and disrupting the integrity of the ...
... Previous studies demonstrate that micromolar concentrations of ivacaftor attenuate the plasma membrane expression and function of some CFTR variants (Avramescu et al., 2017;Cholon et al., 2014;Veit et al., 2014;Wang et al., 2014). For example, micromolar concentrations of ivacaftor inhibit F508del-CFTR both by destabilising its structure (Cholon et al., 2014;Veit et al., 2014) and disrupting the integrity of the ...
Some residues in the cystic fibrosis transmembrane conductance regulator (CFTR) channel are the site of more than one CFTR variant that cause cystic fibrosis. Here, we investigated the function of S1159F and S1159P, two variants associated with different clinical phenotypes, which affect the same pore‐lining residue in transmembrane segment 12 that are both strongly potentiated by ivacaftor when expressed in CFBE41o⁻ bronchial epithelial cells. To study the single‐channel behaviour of CFTR, we applied the patch‐clamp technique to Chinese hamster ovary cells heterologously expressing CFTR variants incubated at 27°C to enhance channel residence at the plasma membrane. S1159F‐ and S1159P‐CFTR formed Cl⁻ channels activated by cAMP‐dependent phosphorylation and gated by ATP that exhibited thermostability at 37°C. Both variants modestly reduced the single‐channel conductance of CFTR. By severely attenuating channel gating, S1159F‐ and S1159P‐CFTR reduced the open probability (Po) of wild‐type CFTR by ≥75% at ATP (1 mM); S1159F‐CFTR caused the greater decrease in Po consistent with its more severe clinical phenotype. Ivacaftor (10–100 nM) doubled the Po of both CFTR variants without restoring Po values to wild‐type levels, but concomitantly, ivacaftor decreased current flow through open channels. For S1159F‐CFTR, the reduction of current flow was marked at high (supersaturated) ivacaftor concentrations (0.5–1 μM) and voltage‐independent, identifying an additional detrimental action of elevated ivacaftor concentrations. In conclusion, S1159F and S1159P are gating variants, which also affect CFTR processing and conduction, but not stability, necessitating the use of combinations of CFTR modulators to optimally restore their channel activity. image
Key points
Dysfunction of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes the genetic disease cystic fibrosis (CF).
This study investigated two rare pathogenic CFTR variants, S1159F and S1159P, which affect the same amino acid in CFTR, to understand the molecular basis of disease and response to the CFTR‐targeted therapy ivacaftor.
Both rare variants diminished CFTR function by modestly reducing current flow through the channel and severely inhibiting ATP‐dependent channel gating with S1159F exerting the stronger adverse effect, which correlates with its association with more severe disease.
Ivacaftor potentiated channel gating by both rare variants without restoring their activity to wild‐type levels, but concurrently reduced current flow through open channels, particularly those of S1159F‐CFTR.
Our data demonstrate that S1159F and S1159P cause CFTR dysfunction by multiple mechanisms that require combinations of CFTR‐targeted therapies to fully restore channel function.
... ∆F508-CFTR function assay by halide-sensitive YFP fluorescence quenching was performed as described [22,[24][25][26]. CFBE cells expressing both inducible ∆F508-CFTR-3HA and halide sensor YFP-F46L/H148Q/I152L were seeded onto black 96-well microplates and transfected with siRNA (50 nM each) and dsiRNA (25 nM). ...
The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.
... D Chronic exposure to 1 on lumacaftor-rescued ΔF508-CFTR protein inhibits the positive stabilizing effects of lumacaftor. 11,23,35 Even at high concentrations of the much less lipophilic 3, we did not observe attenuation of (lumacaftor and low temperature rescued) ΔF508-CFTR protein surface levels. Lumacaftor binds to a deep hydrophobic pocket spanning TM helices 1, 2, 3, and 6 on TMD1. ...
Mutations in the unique ATP-binding cassette anion channel, the cystic fibrosis conductance regulator (CFTR), lead to the inherited fatal disease known as cystic fibrosis (CF). Ivacaftor enhances channel gating of CFTR by stabilizing its open state and has been approved as monotherapy for CF patients with CFTR gating mutations (e.g., G551D) and as part of combination therapy with lumacaftor for CFTR folding mutations (e.g., ΔF508). However, in the latter context, ivacaftor may destabilize folding-rescued ΔF508-CFTR and membrane-associated proteins and attenuate lumacaftor pharmacotherapy. Here, we tested the hypothesis that the high lipophilicity of ivacaftor may contribute to this effect. We describe the synthesis of three glutamic acid ivacaftor derivatives with reduced lipophilicity that bear different charges at neutral pH (compounds 2, 3, 4). In a cellular ion flux assay, all three restored G551D-CFTR channel activity at comparable or better levels than ivacaftor. Furthermore, unlike ivacaftor, compound 3 did not attenuate levels of folding-rescued ΔF508 at the cell surface. Molecular modeling predicts that the increased polarity of compound 3 allows engagement with polar amino acids present in the binding pocket with hydrogen bonding and ionic interactions, which are collectively higher in strength as compared to hydrophobic interactions that stabilize ivacaftor. Overall, the data suggests that reduced lipophilicity may improve the efficacy of this class of CFTR potentiators when used for folding-rescued ΔF508-CFTR.
... Even though there are CFTR modulators on the market that can help relieve disease related symptoms, there is still no way to cure CF disease permanently. Primary human bronchial epithelial (HBE) cells from CF patients with F508del mutation (F508del/F508del CF HBEs) are widely recognized as the gold standard assay for preclinical CF studies most predictive of clinical benefit 37,38 . Because F508del HBEs are more commonly available than G542X HBEs, we had to focus on correcting the F508del mutation in the HBE assay. ...
Approximately 10% of Cystic Fibrosis (CF) patients, particularly those with CF transmembrane conductance regulator (CFTR) gene nonsense mutations, lack effective treatments. The potential of gene correction therapy through delivery of the CRISPR/Cas system to CF-relevant organs/cells is hindered by the lack of efficient genome editor delivery carriers. Herein, we report improved Lung Selective Organ Targeting Lipid Nanoparticles (SORT LNPs) for efficient delivery of Cas9 mRNA, sgRNA, and donor ssDNA templates, enabling precise homology-directed repair-mediated gene correction in CF models. Optimized Lung SORT LNPs deliver mRNA to lung basal cells in Ai9 reporter mice. SORT LNP treatment successfully corrected the CFTR mutations in homozygous G542X mice and in patient-derived human bronchial epithelial cells with homozygous F508del mutations, leading to the restoration of CFTR protein expression and chloride transport function. This proof-of-concept study will contribute to accelerating the clinical development of mRNA LNPs for CF treatment through CRISPR/Cas gene correction.
... We hypothesized that the measured proteostatic differences between P67L and L206W may be important for the pharmacological correction in these variants. To interrogate the role of proteostasis factors on CFTR trafficking, siRNA knockdown (KD) was performed in a Cystic Fibrosis Bronchial Epithelial cell-line with tetracyclin-inducible P67L CFTR expression (TetON P67L CFBE) (39). First, we selected a panel of targets in the pathways including translation, degradation, P67L CFTR interactome remodeling by VX-445 nuclear import/export, and RNA processing, from the waterfall plot (Fig. 4, A and D). ...
... Tet-On inducible human bronchial epithelial cells were generously provided by Dr Guido Veit and Dr Gergely Lukacs, McGill University (39). F508del-CFTR (iF508del-CFBE), P67L-CFTR (iP67L-CFBE), WT-CFTR (WT-CFBE), and parental CFTR null (CFBE41o -) were cultured in minimum essential medium (Gibco) supplemented with 10% FBS (Gibco), 1% HEPES (1 M, Gibco), 1% L-glutamine (200 mM, Gibco). ...
Cystic fibrosis (CF) is one of the most prevalent lethal genetic diseases with over 2000 identified mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Pharmacological chaperones such as Lumacaftor (VX-809), Tezacaftor (VX-661) and Elexacaftor (VX-445) treat mutation-induced defects by stabilizing CFTR and are called correctors. These correctors improve proper folding and thus facilitate processing and trafficking to increase the amount of functional CFTR on the cell surface. Yet, CFTR variants display differential responses to each corrector. Here, we report variants P67L and L206W respond similarly to VX-809 but divergently to VX-445 with P67L exhibiting little rescue when treated with VX-445. We investigate the underlying cellular mechanisms of how CFTR biogenesis is altered by correctors in these variants. Affinity purification-mass spectrometry (AP-MS) multiplexed with isobaric Tandem Mass Tags (TMT) was used to quantify CFTR protein-protein interaction changes between variants P67L and L206W. VX-445 facilitates unique proteostasis factor interactions especially in translation, folding, and degradation pathways in a CFTR variant-dependent manner. A number of these interacting proteins knocked down by siRNA, such as ribosomal subunit proteins, moderately rescued fully glycosylated P67L. Importantly, these knockdowns sensitize P67L to VX-445 and further enhance the trafficking correction of this variant. Partial inhibition of protein translation also mildly sensitizes P67L CFTR to VX-445 correction, supporting a role for translational dynamics in the rescue mechanism of VX-445. Our results provide a better understanding of VX-445 biological mechanism of action and reveal cellular targets that may sensitize unresponsive CFTR variants to known and available correctors.
... This classification has been useful to identify corrector combinations with complementary mechanisms since F508del (and several class II variants) promotes pleiotropic defects on the CFTR structure and single correctors demonstrated to have limited efficacy in rescuing several of them [25]. VX-809 and its analogs VX-661 and C18 are prototypical examples of type I correctors [28][29][30] (Table 2). VX-809 and VX-661 were the first CFTR correctors clinically approved (in combination with VX-770), as they improved lung function, despite modestly, in F508del-homozygous PwCF [19,20]. ...
... QBW251 (icenticaftor) potentiated CFTR function for classes III and IV variants and was superior to VX-770 in potentiating F508del-CFTR function [74,75]. Interestingly, both ABBV-974 and QBW251 did not reduce the stability of rescued CFTR at the PM [69,75], in contrast to VX-770 [29,76]. ...
Introduction:
Cystic fibrosis (CF), a potentially fatal genetic disease, is caused by loss-of-function variants in the gene encoding for the CFTR chloride/bicarbonate channel. Modulator drugs rescuing mutant CFTR traffic and function are now in the clinic, providing unprecedented breakthrough therapies for people with CF (PwCF) carrying specific genotypes. However, several CFTR variants are unresponsive to these therapies.
Area covered:
We discussed several therapeutic approaches that are under development to tackle the fundamental cause of CF, including strategies targeting defective CFTR mRNA and/or protein expression and function. Alternatively, defective chloride secretion and dehydration in CF epithelia could be restored by exploiting pharmacological modulation of alternative targets, i.e. alternative ion channels/transporters that concur with the maintenance of airway surface liquid homeostasis (e.g. ENaC, TMEM16A, SLC26A4, SLC26A9, and ATP12A). Finally, we assessed progress and challenges in the development of gene-based therapies to replace or correct the mutant CFTR gene.
Expert opinion:
CFTR modulators are benefiting many PwCF responsive to these drugs, yielding substantial improvements in various clinical outcomes. Meanwhile, the CF therapy development pipeline continues to expand with the development of novel CFTR modulators and alternative therapeutic strategies with the ultimate goal to provide effective therapies for all PwCF in the foreseeable future.
