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: 4.08). 07/2006; 290(6):L1117-30. DOI: 10.1152/ajplung.00169.2005
Source: PubMed


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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    • "Interestingly, ΔF508-CFTR can, at least partially, be rescued by incubation of cell cultures for 24 h at reduced temperature (<30°C) [6] or by incubation of cells with various small molecule correctors [7] [8] [9] leading to the appearance of ΔF508-CFTR in the plasma membrane. Recent FDA approval of a potentiator/corrector combination (Ivacaftor/Lumicaftor, marketed as Orkambi; Vertex Pharmaceuticals) represents the first pharmacological approach to treat patients with the F508 mutation. "
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    ABSTRACT: Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol. High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. Taken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound. Copyright © 2015. Published by Elsevier B.V.
    Biochimica et Biophysica Acta 09/2015; DOI:10.1016/j.bbagen.2015.08.020 · 4.66 Impact Factor
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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 purpose: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) 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 approach: Human CF airway epithelial cells were treated with roscovitine 100 μM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. Key results: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. Conclusions and implications: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.
    British Journal of Pharmacology 07/2014; 171(21). DOI:10.1111/bph.12859 · 4.84 Impact Factor
    • "The CFTR " potentiator " VX-770 [N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4- dihydroquinoline-3-carboxamide; Ivacaftor], which corrects defective channel gating of some CFTR mutants, has been approved for CF therapy caused by the defective channel gating but unimpaired cellular processing and plasma membrane targeting of the G551D-CFTR mutation (Van Goor et al., 2009; Accurso et al., 2010). Several small-molecule " correctors " of ΔF508-CFTR cellular processing have been identified (Pedemonte et al., 2005; Van Goor et al., 2006; Yu et al., 2008; Robert et al., 2010; Phuan et al., 2011). VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl] amino]-3-methyl-2-pyridinyl]-benzoic acid) is in clinical trials for CF caused by the ΔF508 mutation (Clancy et al., 2012). "
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    ABSTRACT: The most prevalent CFTR mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain 1 (NBD1) and stability of the interface between NBD1 and the membrane spanning domains (MSDs). The interfacial stability defect can be partially corrected by the investigational drug VX-809 or the R1070W mutation. 'Second-generation' ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing CF correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium- derived cell line (CFBE41o-) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop either in the presence or absence of R1070W. Using a luminescence read-out of HRP activity, screening of ~110,000 small molecules produced 9 novel corrector scaffolds that increased cell surface ΔF508-CFTR expression by up to 200 % in the presence vs. absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with EC50 <5 μM. 8 chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ΔF508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof-of-concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the 'therapeutic ceiling' of first-generation correctors.
    Molecular pharmacology 04/2014; 86(1). DOI:10.1124/mol.114.092478 · 4.13 Impact Factor
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