Renaud Robert

Structural Analog of Sildenafil Identified as a Novel Corrector of the F508del-CFTR Trafficking Defect

The F508del mutation impairs trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane and results in a partially functional chloride channel that is retained in the endoplasmic reticulum and degraded. We recently used a novel high-throughput screening (HTS) assay to identify small-molecule correctors of F508del CFTR trafficking and found several classes of hits in a screen of 2000 compounds (Carlile et al., 2007). In the present study, we have extended the screen to 42,000 compounds and confirmed sildenafil as a corrector using this assay. We evaluated structural analogs of sildenafil and found that one such molecule called KM11060 (7-chloro-4-{4-[(4-chlorophenyl) sulfonyl] piperazino}quinoline) was surprisingly potent. It partially restored F508del trafficking and increased maturation significantly when baby hamster kidney (BHK) cells were treated with 10 nM for 24 h or 10 μM for 2 h. Partial correction was confirmed by the appearance of mature CFTR in Western blots and by using halide flux, patch-clamp, and short-circuit current measurements in unpolarized BHK cells, monolayers of human airway epithelial cells (CFBE41o-), and intestines isolated from F508del-CFTR mice (Cftrtm1Eur) treated ex vivo. Small-molecule correctors such as KM11060 may serve as useful pharmacological tools in studies of the F508del-CFTR processing defect and in the development of cystic fibrosis therapeutics.

Correctors of protein trafficking defects identified by a novel high-throughput screening assay

High‐throughput small‐molecule screens hold great promise for identifying compounds with potential therapeutic value in the treatment of protein‐trafficking diseases such as cystic fibrosis (CF) and nephrogenic diabetes insipidus (NDI). The approach usually involves expressing the mutant form of the gene in cells and assaying function in a multiwell format when cells are exposed to libraries of compounds. Although such functional assays are useful, they do not directly test the ability of a compound to correct defective trafficking of the protein. To address this we have developed a novel corrector‐screening assay for CF, in which the appearance of the mutant protein at the cell surface is measured. We used this assay to screen a library of 2000 compounds and have isolated several classes of trafficking correctors that had not previously been identified. This novel screening approach to protein‐trafficking diseases is robust and general, and could enable the selection of molecules that could be translated rapidly to a clinical setting.

Correction of the ΔPhe508 Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Defect by the Bioavailable Compound Glafenine

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-activated anion channel expressed in epithelial cells. The most common mutation ΔPhe508 leads to protein misfolding, retention by the endoplasmic reticulum, and degradation. One promising therapeutic approach is to identify drugs that have been developed for other indications but that also correct the CFTR trafficking defect, thereby exploiting their known safety and bioavailability in humans and reducing the time required for clinical development. We have screened approved, marketed, and off-patent drugs with known safety and bioavailability using a ΔPhe508-CFTR trafficking assay. Among the confirmed hits was glafenine, an anthranilic acid derivative with analgesic properties. Its ability to correct the misprocessing of CFTR was confirmed by in vitro and in vivo studies using a concentration that is achieved clinically in plasma (10 μM). Glafenine increased the surface expression of ΔPhe508-CFTR in baby hamster kidney (BHK) cells to ∼40% of that observed for wild-type CFTR, comparable with the known CFTR corrector 4-cyclohexyloxy-2-{1-[4-(4-methoxybenzensulfonyl)-piperazin-1-yl]-ethyl}-quinazoline (VRT-325). Partial correction was confirmed by the appearance of mature CFTR in Western blots and by two assays of halide permeability in unpolarized BHK and human embryonic kidney cells. Incubating polarized CFBE41o− monolayers and intestines isolated from ΔPhe508-CFTR mice (treated ex vivo) with glafenine increased the short-circuit current (Isc) response to forskolin + genistein, and this effect was abolished by 10 μM CFTRinh172. In vivo treatment with glafenine also partially restored total salivary secretion. We conclude that the discovery of glafenine as a CFTR corrector validates the approach of investigating existing drugs for the treatment of CF, although localized delivery or further medicinal chemistry may be needed to reduce side effects.

Identification of a NBD1-Binding Pharmacological Chaperone that Corrects the Trafficking Defect of F508del-CFTR

Most cases of cystic fibrosis (CF) are attributable to the F508del allele of CFTR, which causes the protein to be retained in the endoplasmic reticulum (ER) and subsequently degraded. One strategy for CF therapy is to identify corrector compounds that help traffic F508del-CFTR to the cell surface. Pharmacological chaperones, or correctors that bind specifically to F508del-CFTR and restore function, would be the most promising drug development candidates, but few pharmacological chaperones exist for F508del-CFTR. Using differential scanning fluorimetry (DSF), we have surveyed corrector compounds and identified one, RDR1, which binds directly to the first nucleotide binding domain (NBD1) of F508del-CFTR. We show that RDR1 treatment partially rescues F508del-CFTR function in both cells and in an F508del-CF mouse model. Thus, RDR1 is a pharmacological chaperone of F508del-CFTR and represents a novel scaffold for drug development.

Lack of CFTR in Skeletal Muscle Predisposes to Muscle Wasting and Diaphragm Muscle Pump Failure in Cystic Fibrosis Mice

Cystic fibrosis (CF) patients often have reduced mass and strength of skeletal muscles, including the diaphragm, the primary muscle of respiration. Here we show that lack of the CF transmembrane conductance regulator (CFTR) plays an intrinsic role in skeletal muscle atrophy and dysfunction. In normal murine and human skeletal muscle, CFTR is expressed and co-localized with sarcoplasmic reticulum-associated proteins. CFTR–deficient myotubes exhibit augmented levels of intracellular calcium after KCl-induced depolarization, and exposure to an inflammatory milieu induces excessive NF-kB translocation and cytokine/chemokine gene upregulation. To determine the effects of an inflammatory environment in vivo, sustained pulmonary infection with Pseudomonas aeruginosa was produced, and under these conditions diaphragmatic force-generating capacity is selectively reduced in Cftr −/− mice. This is associated with exaggerated pro-inflammatory cytokine expression as well as upregulation of the E3 ubiquitin ligases (MuRF1 and atrogin-1) involved in muscle atrophy. We conclude that an intrinsic alteration of function is linked to the absence of CFTR from skeletal muscle, leading to dysregulated calcium homeostasis, augmented inflammatory/atrophic gene expression signatures, and increased diaphragmatic weakness during pulmonary infection. These findings reveal a previously unrecognized role for CFTR in skeletal muscle function that may have major implications for the pathogenesis of cachexia and respiratory muscle pump failure in CF patients.

Cystic fibrosis transmembrane conductance regulator trafficking modulates the barrier function of airway epithelial cell monolayers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane glycoprotein which functions as an anion channel and influences diverse cellular processes. We studied its role in the development of epithelial tightness by expressing wild‐type (WT‐CFTR) or mutant (ΔF508‐CFTR) CFTR in human airway epithelial cell monolayers cultured at the air–liquid interface. Green fluorescent protein (GFP)‐tagged WT or ΔF508 constructs were expressed in the CF bronchial cell line CFBE41o− using adenoviruses, and the results were compared with those obtained using CFBE41o− lines stably complemented with wild‐type or mutant CFTR. As predicted, GFP‐WT‐CFTR reached the apical membrane whereas GFP‐ΔF508‐CFTR was only detected intracellularly. Although CFTR expression would be expected to reduce transepithelial resistance (TER), expressing GFP‐CFTR significantly increased the TER of CFBE41o− monolayers whilst GFP‐ΔF508‐CFTR had no effect. Similar results were obtained with cell lines stably overexpressing ΔF508‐CFTR or WT‐CFTR. Preincubating ΔF508‐CFTR monolayers at 29°C reduced mannitol permeability and restored TER, and the effect on TER was reversible during temperature oscillations. Expression of GFP‐ΔF508‐CFTR or GFP‐WT‐CFTR in a cell line already containing endogenous WT‐CFTR (Calu‐3) did not alter TER. The CFTR‐ and temperature‐dependence of TER were not affected by the CFTR inhibitor CFTRinh172 or low‐chloride medium; therefore the effect of CFTR on barrier function was unrelated to its ion channel activity. Modulation of TER was blunted but not eliminated by genistein, implying the involvement of tyrosine phosphorylation and other mechanisms. Modulation of CFTR trafficking was correlated with an increase in tight junction depth. The results suggest that CFTR trafficking is required for the normal organisation and function of tight junctions. A reduction in barrier function caused by endoplasmic reticulum retention of ΔF508‐CFTR may contribute to fluid hyperabsorption in CF airways.

Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle.

Patients with cystic fibrosis (CF) suffer from asthma-like symptoms and gastrointestinal cramps, attributed to a mutation in the CF transmembrane conductance regulator (CFTR) gene present in a variety of cells. Pulmonary manifestations of the disease include the production of thickened mucus and symptoms of asthma, such as cough and wheezing. A possible alteration in airway smooth muscle (ASM) cell function of patients with CF has not been investigated. The aim of this study was to determine whether the (CFTR) channel is present and affects function of human ASM cells. Cell cultures were obtained from the main or lobar bronchi of patients with and without CF, and the presence of the CFTR channel detected by immunofluorescence. Cytosolic Ca(2+) was measured using Fura-2 and dual-wavelength microfluorimetry. The results show that CFTR is expressed in airway bronchial tissue and in cultured ASM cells. Peak Ca(2+) release in response to histamine was significantly decreased in CF cells compared with non-CF ASM cells (357 +/- 53 nM versus 558 +/- 20 nM; P < 0.001). The CFTR pharmacological blockers, glibenclamide and N-phenyl anthranilic acid, significantly reduced histamine-induced Ca(2+) release in non-CF cells, and similar results were obtained when CFTR expression was varied using antisense oligonucleotides. In conclusion, these data show that the CFTR channel is present in ASM cells, and that it modulates the release of Ca(2+) in response to contractile agents. In patients with CF, a dysfunctional CFTR channel could contribute to the asthma diathesis and gastrointestinal problems experienced by these patients.

Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL-8 secretion by cystic fibrosis airways

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, causes retention of CFTR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca2+ homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store‐operated calcium entry (SOCE) through Orai1 is abnormal in CF. The significance of Orai1‐mediated SOCE for increased interleukin‐8 (IL‐8) expression in CF was also investigated. CF and non‐CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca2+ imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orai1 function in CF vs. non‐CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store‐operated Ca2+ entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ~1.8‐ and ~2.5‐fold for total Ca2+i increase and Ca2+ influx rate, respectively, and ~2‐fold increase in the ICRAC current) and is caused by increased exocytotic insertion (~2‐fold) of Orai1 channels into the plasma membrane, which is normalized by rescue of ΔF508‐CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL‐8 secretion (~2‐fold). CFTR normally down‐regulates the Orai1/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells.—Balghi, H., Robert, R., Rappaz, B., Zhang, X., Wohlhuter‐Haddad, A., Evagelidis, A., Luo, Y., Goepp, J., Ferraro, P., Roméo, P., Trebak, M., Wiseman, P. W., Thomas, D. Y., Hanrahan, J. W. Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL‐8 secretion by cystic fibrosis airways. FASEB J. 25, 4274–4291 (2011). www.fasebj.org

Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX‐770 (ivacaftor)

The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a ‘potentiator’ to increase channel open probability and a ‘corrector’ that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood.

Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

•  The mechanisms of anion and fluid transport by airway submucosal glands are not well understood and may differ from those in surface epithelium. •  The Calu‐3 cell line is often used as a model for submucosal gland serous cells and has cAMP‐stimulated fluid secretion; however, it does not actively transport chloride under short‐circuit conditions. •  In this study we show that fluid secretion requires chloride, bicarbonate and sodium, that chloride is the predominant anion in Calu‐3 secretions, and that a large fraction of the basolateral chloride loading during cAMP stimulation occurs by Cl−/HCO3− exchange. •  The results suggest a novel cellular model for anion and fluid secretion by Calu‐3 and submucosal gland acinar cells