Rescue of ΔF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Vertex Pharmaceuticals, 11010 Torreyana Road, San Diego, CA 92121, USA.
AJP Lung Cellular and Molecular Physiology (Impact Factor: 4.04). 07/2006; 290(6):L1117-30. DOI: 10.1152/ajplung.00169.2005
Source: PubMed

ABSTRACT Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

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    • "To reverse the defective trafficking of F508del-CFTR, a number of methods have been employed including the use of proteostasis regulators, sarcoplasmic-reticulum Ca 2+ -ATPase inhibitors , such as thapsigargin (Egan et al., 2002; Norez et al., 2006a), nitric oxide donors (Zaman et al., 2001), the heat shock protein (HSP) modulator sodium 4-phenylbutyrate (Rubenstein and Zeitlin, 2000), the glucosidase inhibitor miglustat (Lubamba et al., 2009; Norez et al., 2009), the type V phosphodiesterase inhibitors sildenafil and vardenafil (Dormer et al., 2005) and histone deacetylase inhibitors (Hutt et al., 2010). Another strategy is to use pharmacological chaperones , that is small molecules directly interacting with the CFTR protein, that promote its folding (Pedemonte et al., 2005; Van et al., 2006; Norez et al., 2008). However, despite these advances, therapies for CF have not improved, possibly due to insufficient restoration of CFTR function and/or poor selectivity for the processing of CFTR compared with other proteins and off-target effects. "
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    ABSTRACT: Background and purposeThe most common mutation in Cystic Fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CFTR (CF Transmembrane conductance Regulator) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF.Experimental approachHuman CF airway epithelial cells were treated with roscovitine 100μM for 2h before examining CFTR maturation, expression and activity. Then determined the mechanism of action of roscovitine was explored recording ER Ca2+ depletion, impact of F508del-CFTR/calnexin interaction and measuring the proteasome activity.Key resultsHere, among several kinase inhibitors, we report on the discovery of a small molecule corrector, a cyclin-dependent kinase (CDK) inhibitor roscovitine that rescues the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. We found that neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, correct F508del-CFTR trafficking demonstrating a CDK inhibition-independent corrector effect of roscovitine. Competition studies with inhibitors of the Endoplasmic Reticulum Quality Control (ERQC) suggest that roscovitine acts on the calnexin pathway and on the degradation machinery. We demonstrate that roscovitine (1) induced a ER Ca2+ depletion disturbing F508del-CFTR/calnexin interaction and (2) inhibits directly the proteasome activity via a Ca2+ independent manner.Conclusions and ImplicationsWe conclude that roscovitine is a F508del-CFTR corrector preventing the ability of the ERQC to interact with and to degrade F508del-CFTR via two synergistic but CDK-independent mechanisms.
    British Journal of Pharmacology 07/2014; 171(21). DOI:10.1111/bph.12859 · 4.99 Impact Factor
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    • "Screening identified 13 structurally distinct scaffolds with corrector activity, six of which were also active on FRT cells with ∆F508-CFTR. The mechanistic data obtained on the quinazolinone class (i.e., VRT- 325) suggest that the compounds act primarily or initially at the level of the ER to facilitate the folding and export of ∆F508- CFTR (Van Goor et al., 2006). More important, the subsequent round of optimization of one of the hits from the primary screening led to the investigational drug VX-809 (Van Goor et al., 2011). "
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    ABSTRACT: The lack of phenylalanine 508 (ΔF508 mutation) in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel represents the most frequent cause of CF, a genetic disease affecting multiple organs such as lung, pancreas, and liver. ΔF508 causes instability and misfolding of CFTR protein leading to early degradation in the endoplasmic reticulum and accelerated removal from the plasma membrane. Pharmacological correctors of mutant CFTR protein have been identified by high-throughput screening of large chemical libraries, by in silico docking of virtual compounds on CFTR structure models, or by using compounds that affect the whole proteome (e.g., histone deacetylase inhibitors) or a single CFTR-interacting protein. The presence of multiple defects of the CFTR protein caused by the ΔF508 mutation and the redundancy of quality control mechanisms detecting ΔF508-CFTR as a defective protein impose a ceiling to the maximal effect that a single compound (corrector) may obtain. Therefore, treatment of patients with the most frequent CF mutation may require the optimized combination of two drugs having additive or synergic effects.
    Frontiers in Pharmacology 10/2012; 3:175. DOI:10.3389/fphar.2012.00175
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    • "Cell-based assays for " correctors " of F508del-CFTR trafficking have identified chemically diverse small molecules that correct Abbreviations: CF, cystic fibrosis; CFBE, cystic fibrosis bronchial epithelial; CFTR, cystic fibrosis transmembrane conductance regulator; CMAP, connectivity MAP; COPII, vesicle coat proteins; ER, endoplasmic reticulum; ES, enrichment score; FDR, false discovery rate; HA, hemagglutinin; HTS, high throughput screening; Na + /K + -ATPase, sodium-potassium adenosine triphosphatase; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells. trafficking with variable efficiency (Pedemonte et al., 2005; Van Goor et al., 2006; Carlile et al., 2007). Some of these correctors are thought to interact directly with CFTR by acting as stabilizing ligands or " pharmacological chaperones " (Loo et al., 2006; Sampson et al., 2011) or on other known cellular targets, e.g., phosphodiesterases (Dormer et al., 2005; Robert et al., 2008) and histone deacetylases (Hutt et al., 2010). "
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    ABSTRACT: 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.
    Frontiers in Pharmacology 10/2012; 3:176. DOI:10.3389/fphar.2012.00176
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