Rescue Of F508del-CFTR Research Articles

Identification of 6,9-dihydro-5H-pyrrolo[3,2-h]quinazolines as a new class of F508del-CFTR correctors for the treatment of cystic fibrosis

Although substantial advances have been obtained in the pharmacological treatment of cystic fibrosis (CF) with the approval of Kaftrio, a combination of two correctors (VX-661, VX-445) and one potentiator (VX-770), new modulators are still needed to rescue F508del and other CFTR mutants with trafficking defects. We have previously identified PP compounds based on a tricyclic core as correctors with high efficacy in the rescue of F508del-CFTR on native epithelial cells of CF patients, particularly in combination with class 1 correctors (VX-809, VX-661). Compound PP028 was found as a lead candidate for the high rescue of F508del-CFTR and used for mechanistic insight indicating that PP028 behaves as a class 3 corrector, similarly to VX-445.From the exploration of the chemical space around the hit structure, based on iterative cycles of chemical synthesis and functional testing, the class of 6,9-dihydro-5H-pyrrolo [3,2-h]quinazolines with corrector activity was discovered. Within a series of 38 analogues, two derivatives emerged as promising candidates and used for further insight to assess the mechanism of action. Both compounds, decorated with a benzensulfonylamino group at the pyrimidine moiety, were able to generate a dose-dependent increase in CFTR function, particularly in the presence of VX-809. Half-effective concentrations (EC50) were in the single digit micromolar range and decreased in the presence of VX-809 thus indicating a synergistic interaction with class 1 correctors. Synergy was also observed with corr-4a (class 2 corrector) but not with VX-445 and PP028 (class 3 correctors) indicating that the new compounds behave as class 3 correctors. These results suggest that tricyclic pyrrolo-quinazolines interact with CFTR at a site different from that of VX-809 and represent a novel class of CFTR correctors suitable for combinatorial pharmacological treatments for the basic defect in CF.

P035 Partial correction of F508del-CFTR trafficking and stability defects by the combination of PTI-801 with ABBV-2222 or FDL-169

Objectives: Despite the significant progress in developing small-molecule correctors, the mechanism of action (MoA) of these compounds needs to be further elucidated to achieve a more robust rescue of F508del-CFTR. Here, we investigated the efficacy and MoA of PTI-801, a newly developed F508del-CFTR corrector.

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.

Targeting the E1 ubiquitin-activating enzyme (UBA1) improves elexacaftor/tezacaftor/ivacaftor efficacy towards F508del and rare misfolded CFTR mutants

The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors—elexacaftor/tezacaftor—and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients’ responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.

Probing Allosteric Hsp70 Inhibitors by Molecular Modelling Studies to Expedite the Development of Novel Combined F508del CFTR Modulators.

Cystic fibrosis (CF) is caused by different mutations related to the cystic fibrosis transmembrane regulator protein (CFTR), with F508del being the most common. Pioneering the development of CFTR modulators, thanks to the development of effective correctors or potentiators, more recent studies deeply encouraged the administration of triple combination therapeutics. However, combinations of molecules interacting with other proteins involved in functionality of the CFTR channel recently arose as a promising approach to address a large rescue of F508del-CFTR. In this context, the design of compounds properly targeting the molecular chaperone Hsp70, such as the allosteric inhibitor MKT-077, proved to be effective for the development of indirect CFTR modulators, endowed with ability to amplify the accumulation of the rescued protein. Herein we performed structure-based studies of a number of allosteric HSP70 inhibitors, considering the recent X-ray crystallographic structure of the human enzyme. This allowed us to point out the main interaction supporting the binding mode of MKT-077, as well as of the related analogues. In particular, cation-π and π–π stacking with the conserve residue Tyr175 deeply stabilized inhibitor binding at the HSP70 cavity. Molecular docking studies had been followed by QSAR analysis and then by virtual screening of aminoaryl thiazoles (I–IIIa) as putative HSP70 inhibitors. Their effectiveness as CFTR modulators has been verified by biological assays, in combination with VX-809, whose positive results confirmed the reliability of the whole applied computational method. Along with this, the “in-silico” prediction of absorption, distribution, metabolism, and excretion (ADME) properties highlighted, once more, that AATs may represent a chemical class to be further investigated for the rational design of novel combination of compounds for CF treatment.

The rescue of F508del-CFTR by elexacaftor/tezacaftor/ivacaftor (Trikafta) in human airway epithelial cells is underestimated due to the presence of ivacaftor

Trikafta, currently the leading therapeutic in cystic fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch-clamp recordings, Western blot and immunolocalisation experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and nontreated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor, unlike the CFTR (cystic fibrosis transmembrane conductance regulator) potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.

Characterization of the mechanism of action of RDR01752, a novel corrector of F508del-CFTR

Despite progress in developing pharmacotherapies to rescue F508del-CFTR, the most prevalent Cystic Fibrosis (CF)-causing mutation, individuals homozygous for this mutation still face several disease-related symptoms. Thus, more potent compound combinations are still needed. Here, we investigated the mechanism of action (MoA) of RDR01752, a novel F508del-CFTR trafficking corrector. F508del-CFTR correction by RDR01752 was assessed by biochemical, immunofluorescence microscopy and functional assays in cell lines and in intestinal organoids. To determine the MoA of RDR01752, we assessed its additive effects to those of genetic revertants of F508del-CFTR, the FDA-approved corrector drugs VX-809 and VX-661, and low temperature. Our data demonstrated that RDR01752 rescues F508del-CFTR processing and plasma membrane (PM) expression to similar levels of VX-809 in cell lines, although RDR01752 produced lower functional rescue. However, in functional assays using intestinal organoids (F508del/F508del), RDR01752, VX-809 and VX-661 had similar efficacy. RDR01752 demonstrated additivity to revertants 4RK and G550E, but not to R1070W, as previously shown for VX-809. RDR01752 was also additive to low temperature. Co-treatment of RDR01752 and VX-809 did not increase F508del-CFTR PM expression and function compared to each corrector alone. The lack of additivity of RDR01752 with the genetic revertant R1070W suggests that this compound has the same effect as the insertion of tryptophan at 1070, i.e., filling the pocket at the NBD1:ICL4 interface in F508del-CFTR, similarly to VX-809. Combination of RDR01752 with correctors mimicking the rescue by revertants G550E or 4RK could thus maximize rescue of F508del-CFTR.

Full Rescue of F508del-CFTR Processing and Function by CFTR Modulators Can Be Achieved by Removal of Two Regulatory Regions.

Cystic Fibrosis (CF) is caused by mutations in the CF Transmembrane conductance Regulator (CFTR), the only ATP-binding cassette (ABC) transporter functioning as a channel. Unique to CFTR is a regulatory domain which includes a highly conformationally dynamic region—the regulatory extension (RE). The first nucleotide-binding domain of CFTR contains another dynamic region—regulatory insertion (RI). Removal of RI rescues the trafficking defect of CFTR with F508del, the most common CF-causing mutation. Here we aimed to assess the impact of RE removal (with/without RI or genetic revertants) on F508del-CFTR trafficking and how CFTR modulator drugs VX-809/lumacaftor and VX-770/ivacaftor rescue these variants. We generated cell lines expressing ΔRE and ΔRI CFTR (with/without genetic revertants) and assessed CFTR expression, stability, plasma membrane levels, and channel activity. Our data demonstrated that ΔRI significantly enhanced rescue of F508del-CFTR by VX-809. While the presence of the RI seems to be precluding full rescue of F508del-CFTR processing by VX-809, this region appears essential to rescue its function by VX-770, suggesting some contradictory role in rescue of F508del-CFTR by these two modulators. This negative impact of RI removal on VX-770-stimulated currents on F508del-CFTR can be compensated by deletion of the RE which also leads to the stabilization of this mutant. Despite both regions being conformationally dynamic, RI precludes F508del-CFTR processing while RE affects mostly its stability and channel opening.

TGF-β1 Augments the Apical Membrane Abundance of Lemur Tyrosine Kinase 2 to Inhibit CFTR-Mediated Chloride Transport in Human Bronchial Epithelia.

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl– channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-β1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl– transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-β1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-β1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.

Augmentation of Cystic Fibrosis Transmembrane Conductance Regulator Function in Human Bronchial Epithelial Cells via SLC6A14-Dependent Amino Acid Uptake. Implications for Treatment of Cystic Fibrosis.

SLC6A14-mediated l-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia. We sought to determine the role of l-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from patients with cystic fibrosis (CF). Human bronchial epithelial (HBE) cells from lung explants of patients without CF (HBE) and those with CF (CF-HBE) were used for H3-flux, airway surface liquid, and Ussing chamber studies. We used α-methyltryptophan as a specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR. SLC6A14 is functionally expressed in CF-HBE cells. l-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14-mediated l-arginine uptake also increased the airway surface liquid in CF-HBE cells. Using CFBE41o cells, we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide (NO) synthase activity, nitrogen oxides, including NO, and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE, as inducible NO synthase inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR. In summary, SLC6A14-mediated l-arginine transport augments residual F508del-CFTR channel function via a noncanonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be used for the development of companion therapy in CF.

A novel triple combination of pharmacological chaperones improves F508del-CFTR correction

Pharmacological chaperones (e.g. VX-809, lumacaftor) that bind directly to F508del-CFTR and correct its mislocalization are promising therapeutics for Cystic Fibrosis (CF). However to date, individual correctors provide only ~4% improvement in lung function measured as FEV1, suggesting that multiple drugs will be needed to achieve substantial clinical benefit. Here we examine if multiple sites for pharmacological chaperones exist and can be targeted to enhance the rescue of F508del-CFTR with the premise that additive or synergistic rescue by multiple pharmacological chaperones compared to single correctors indicates that they have different sites of action. First, we found that a combination of the pharmacological chaperones VX-809 and RDR1 provide additive correction of F508del-CFTR. Then using cellular thermal stability assays (CETSA) we demonstrated the possibility of a third pharmacologically important site using the novel pharmacological chaperone tool compound 4-methyl-N-[3-(morpholin-4-yl) quinoxalin-2-yl] benzenesulfonamide (MCG1516A). All three pharmacological chaperones appear to interact with the first nucleotide-binding domain (NBD1). The triple combination of MCG1516A, RDR1, and VX-809 restored CFTR function to >20% that of non-CF cells in well differentiated HBE cells and to much higher levels in other cell types. Thus the results suggest the presence of at least three distinct sites for pharmacological chaperones on F508del-CFTR NBD1, encouraging the development of triple corrector combinations.

Quorum Sensing Down-Regulation Counteracts the Negative Impact of Pseudomonas aeruginosa on CFTR Channel Expression, Function and Rescue in Human Airway Epithelial Cells.

The function of cystic fibrosis transmembrane conductance regulator (CFTR) channels is crucial in human airways. However unfortunately, chronic Pseudomonas aeruginosa infection has been shown to impair CFTR proteins in non-CF airway epithelial cells (AEC) and to alter the efficiency of new treatments with CFTR modulators designed to correct the basic CFTR default in AEC from cystic fibrosis (CF) patients carrying the F508del mutation. Our aim was first to compare the effect of laboratory strains, clinical isolates, engineered and natural mutants to determine the role of the LasR quorum sensing system in CFTR impairment, and second, to test the efficiency of a quorum sensing inhibitor to counteract the deleterious impact of P. aeruginosa both on wt-CFTR and on the rescue of F508del-CFTR by correctors. We first report that exoproducts from either the laboratory PAO1 strain or a clinical ≪Early≫ isolate (from an early stage of infection) altered CFTR expression, localization and function in AEC expressing wt-CFTR. Genetic inactivation of the quorum-sensing LasR in PAO1 (PAO1ΔlasR) or in a natural clinical mutant (≪Late≫ CF-adapted clinical isolate) abolished wt-CFTR impairment. PAO1 exoproducts also dampened F508del-CFTR rescue by VRT-325 or Vx-809 correctors in CF cells, whereas PAO1ΔlasR had no impact. Importantly, treatment of P. aeruginosa cultures with a quorum sensing inhibitor (HDMF) prevented the negative effect of P. aeruginosa exoproducts on wt-CFTR and preserved CFTR rescue by correctors in CF AEC. These findings indicate that LasR-interfering strategies could be of benefits to counteract the deleterious effect of P. aeruginosa in infected patients.

Evaluation of a systems biology approach to identify pharmacological correctors of the mutant CFTR chloride channel

BackgroundMistrafficking of CFTR protein caused by F508del, the most frequent mutation in cystic fibrosis (CF), can be corrected by cell incubation at low temperature, an effect that may be mediated by altered expression of proteostasis genes. MethodsTo identify small molecules mimicking low temperature, we compared gene expression profiles of cells kept at 27°C with those previously generated from more than 1300 compounds. The resulting candidates were tested with a functional assay on a bronchial epithelial cell line. ResultsWe found that anti-inflammatory glucocorticoids, such as mometasone, budesonide, and fluticasone, increased mutant CFTR function. However, this activity was not confirmed in primary bronchial epithelial cells. Actually, glucocorticoids enhanced Na+ absorption, an effect that could further impair mucociliary clearance in CF airways. ConclusionsOur results suggest that rescue of F508del-CFTR by low temperature cannot be easily mimicked by small molecules and that compounds with closer transcriptional and functional effects need to be found.

WS06.3 Evaluation of novel corrector–potentiator combinations for treating cystic fibrosis in various assays

Defects in CFTR that result in cystic fibrosis can be broadly categorized into three processes: translational, folding/maturation, and functional. To address the underlying causes affecting the folding/maturation and functional properties of CFTR, two biomolecular activities are required, namely correctors to increase CFTR levels at the cell surface, and potentiators to allow the effective opening of the CFTR channel. Combined, these activities allow chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. Previously, we reported the discovery of new corrector molecules via a HTS screen on CFBe41o-cells harboring HRP-tagged F508del CFTR and on U2OS cells ProLink-tagged F508del CFTR (NACFC 2014). Corrector compounds from two distinct series exhibited channel opening activity in primary cells derived from F508del CFTR homozygous patients as measured by trans-epithelial clamp circuit (TECC). Here, we report a series of assays developed to better understand the mechanism of action of the corrector molecules. More specifically, assays using differently tagged F508del CFTR were employed to evaluate the effect of correctors alone and in various combinations with a potentiator on the correction time-dependency and on the cell surface residence time of F508del CFTR. Other characterization endpoints will also be discussed. In summary, we developed several assays to evaluate the impact of corrector/potentiator combinations on the rescue of F508del CFTR, and to understand the contribution of each component in combination cocktails.

Calpain Inhibition Promotes the Rescue of F508del-CFTR in PBMC from Cystic Fibrosis Patients

A basal calpain activity promotes the limited proteolysis of wild type (WT) cystic fibrosis conductance regulator (CFTR), inducing the internalization of the split channel. This process contributes to the regulation in the level of the active CFTR at the plasma membranes. In peripheral blood mononuclear cells (PBMC) from 16 healthy donors, the inhibition of calpain activity induces a 3-fold increase in the amount of active WT CFTR at the plasma membranes. Instead, in PBMC from cystic fibrosis (CF) patients, calpain activity is expressed at aberrant levels causing the massive removal of F508del-CFTR from the cell surface. In these patients, the inhibition of such abnormal proteolysis rescues physiological amounts of active mutated CFTR in 90% of the patients (25 over 28). The recovery of functional F508del-CFTR at the physiological location, in cells treated with a synthetic calpain inhibitor, indicates that F508del-CFTR folding, maturation, and trafficking operate in CF-PBMC at significant rate. Thus, an increase in the basal calpain activity seems primarily involved in the CFTR defect observed in various CF cells. Furthermore, in CF-PBMC the recovery of the scaffolding protein Na+/H+ exchanger regulatory factor 1 (NHERF-1), occurring following inhibition of the aberrant calpain activity, can contribute to rescue CFTR-functional clusters.

WS4.3 Rescue of F508del-CFTR without the regulatory insertion (ΔRI) and regulatory extension (ΔRE) in combination with genetic revertants and small molecules

WS4.3 Rescue of F508del-CFTR without the regulatory insertion (ΔRI) and regulatory extension (ΔRE) in combination with genetic revertants and small molecules

Ouabain Mimics Low Temperature Rescue of F508del-CFTR in Cystic Fibrosis Epithelial Cells

Most cases of cystic fibrosis (CF) are caused by the deletion of a single phenylalanine residue at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutant F508del-CFTR is retained in the endoplasmic reticulum and degraded, but can be induced by low temperature incubation (29°C) to traffic to the plasma membrane where it functions as a chloride channel. Here we show that, cardiac glycosides, at nanomolar concentrations, can partially correct the trafficking of F508del-CFTR in human CF bronchial epithelial cells (CFBE41o-) and in an F508del-CFTR mouse model. Comparison of the transcriptional profiles obtained with polarized CFBE41o-cells after treatment with ouabain and by low temperature has revealed a striking similarity between the two corrector treatments that is not shared with other correctors. In summary, our study shows a novel function of ouabain and its analogs in the regulation of F508del-CFTR trafficking and suggests that compounds that mimic this low temperature correction of trafficking will provide new avenues for the development of therapeutics for CF.

Disruption of cytokeratin-8 interaction with F508del-CFTR corrects its functional defect

We have previously reported an increased expression of cytokeratins 8/18 (K8/K18) in cells expressing the F508del mutation of cystic fibrosis transmembrane conductance regulator (CFTR). This is associated with increased colocalization of CFTR and K18 in the vicinity of the endoplasmic reticulum, although this is reversed by treating cells with curcumin, resulting in the rescue of F508del-CFTR. In the present work, we hypothesized that (i) the K8/K18 network may interact physically with CFTR, and that (ii) this interaction may modify CFTR function. CFTR was immunoprecipitated from HeLa cells transfected with either wild-type (WT) CFTR or F508del-CFTR. Precipitates were subjected to 2D-gel electrophoresis and differential spots identified by mass spectrometry. K8 and K18 were found significantly increased in F508del-CFTR precipitates. Using surface plasmon resonance, we demonstrate that K8, but not K18, binds directly and preferentially to the F508del over the WT human NBD1 (nucleotide-binding domain-1). In vivo K8 interaction with F508del-CFTR was confirmed by proximity ligation assay in HeLa cells and in primary cultures of human respiratory epithelial cells. Ablation of K8 expression by siRNA in F508del-expressing HeLa cells led to the recovery of CFTR-dependent iodide efflux. Moreover, F508del-expressing mice topically treated with K8-siRNA showed restored nasal potential difference, equivalent to that of WT mice. These results show that disruption of F508del-CFTR and K8 interaction leads to the correction of the F508del-CFTR processing defect, suggesting a novel potential therapeutic target in CF.

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca 2+-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

Proteasome-Dependent Pharmacological Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Revealed by Mutation of Glycine 622

The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.