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
    • "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 · 4.12 Impact Factor
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    • "IBMX (3-isobutyl-1-methylxanthine) an inhibitor of phosphodiesterase, seems to enhance F508del-CFTR function both by direct binding and by increasing cAMP cellular levels [112]. HTS has also identified several different potentiators such as VRT- 532 (described above) and VX-770 [113]. The latter (generic drug "
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    ABSTRACT: Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients - F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance , assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.
    Current pharmaceutical design 01/2013; 19. DOI:10.2174/13816128113199990318 · 3.29 Impact Factor
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