Graeme W. Carlile

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.

Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

Significance It has been shown that 14-3-3 proteins increase trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) to the plasma membrane by binding to its regulatory (R) domain. This paper contains a detailed characterization of the 14-3-3/CFTR interaction, showing that multiple phosphorylated binding sites in the CFTR R-domain are necessary for significant binding with 14-3-3. We find that one of these binding sites serves as an anchor, while surrounding weaker sites enhance the interaction. Furthermore, we show the druggability of this interaction using natural-product fusicoccin-A, which stabilizes the 14-3-3/CFTR interaction by selectively modifying a weaker binding site. This mechanism of action can serve as a model for the development of new trafficking corrector molecules to treat cystic fibrosis. Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics.

Experimental Design and Statistical Methods for Improved Hit Detection in High-Throughput Screening

Identification of active compounds in high-throughput screening (HTS) contexts can be substantially improved by applying classical experimental design and statistical inference principles to all phases of HTS studies. The authors present both experimental and simulated data to illustrate how true-positive rates can be maximized without increasing false-positive rates by the following analytical process. First, the use of robust data preprocessing methods reduces unwanted variation by removing row, column, and plate biases. Second, replicate measurements allow estimation of the magnitude of the remaining random error and the use of formal statistical models to benchmark putative hits relative to what is expected by chance. Receiver Operating Characteristic (ROC) analyses revealed superior power for data preprocessed by a trimmed-mean polish method combined with the RVM t-test, particularly for small- to moderate-sized biological hits.

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.

Ibuprofen rescues mutant cystic fibrosis transmembrane conductance regulator trafficking

BACKGROUND Small molecules as shown by VX809 can rescue the mislocalization of F508del-CFTR. The aim of this study was to identify correctors with a clinical history and their targets of action. METHODS CFTR correctors were screened using two F508del-CFTR expressing cell based HTS assays. Electrophysiological studies using CFBE41o(-) and HBE cells and in-vivo mouse assays confirmed CFTR rescue. The target of action was attained using pharmacological inhibitors and siRNA to specific genes. RESULTS Ibuprofen was identified as a CFTR corrector. Ibuprofen treatment of polarized CFBE41o(-) monolayers increased the short-circuit current (Isc) response to stimulation. In vivo CF mice treatment with ibuprofen restored the CFTR trafficking. SiRNA knock down of cyclooxygenase expression caused partial F508del-CFTR correction. CONCLUSION These studies show that ibuprofen is a CFTR corrector and that it causes correction by COX-1 inhibition. Hence ibuprofen may be suitable to be part of a future CF combination therapy.

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.

Unravelling druggable signalling networks that control F508del-CFTR proteostasis

Cystic fibrosis (CF) is caused by mutations in CF transmembrane conductance regulator (CFTR). The most frequent mutation (F508del-CFTR) results in altered proteostasis, that is, in the misfolding and intracellular degradation of the protein. The F508del-CFTR proteostasis machinery and its homeostatic regulation are well studied, while the question whether ‘classical’ signalling pathways and phosphorylation cascades might control proteostasis remains barely explored. Here, we have unravelled signalling cascades acting selectively on the F508del-CFTR folding-trafficking defects by analysing the mechanisms of action of F508del-CFTR proteostasis regulator drugs through an approach based on transcriptional profiling followed by deconvolution of their gene signatures. Targeting multiple components of these signalling pathways resulted in potent and specific correction of F508del-CFTR proteostasis and in synergy with pharmacochaperones. These results provide new insights into the physiology of cellular proteostasis and a rational basis for developing effective pharmacological correctors of the F508del-CFTR defect. DOI: http://dx.doi.org/10.7554/eLife.10365.001