Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population. It is caused by mutations in the CF gene, encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most prevalent CF mutation, deletion of phenylalanine 508 (?F508) impairs the posttranslational folding, gating characteristics, and the biosynthetic and endocytic processing of CFTR. The functional expression defect of CFTR at the plasma membrane leads to impaired chloride, bicarbonate and fluid transport in secretory epithelia, manifesting in recurrent lung infection, the primary cause of mortality in CF. A major focus of CF research is the identification of small-molecule correctors of defective CFTR processing and potentiators of defective gating that target that the ?F508 and other CFTR mutations. The efficacy of the best correctors available (VX-809) is low, such that treated cells show only ?15% of chloride conductance compared to non-CF cells. We and others have demonstrated that chronic exposure to gating potentiators (e.g. VX-770) destabilizes ?F508-CFTR and reduces correction efficacy. Phase III clinical data, indeed, showed only modest but significant clinical efficacy of combination VX-809 and VX-770 therapy. This competitive renewal builds on our recent discoveries of: a) corrector and potentiator molecules by high throughput screening (HTS) assays; b) the correction requirement of both primary folding defects of ?F508-CFTR, including destabilization of the NBD1 conformation and the NBD1-MSD2 interface in the context of cooperative domain folding; c) preliminary identification of novel potentiator molecules that do not destabilize ?F508-CFTR; and d) correction of the functional expression defect of the W128X-CFTR nonsense mutation synergistically by corrector and potentiator combination. To identify corrector-potentiator combinations that restore ?F508-CFTR folding and chloride channel function to >50% of its wild-type counterpart, we propose to identify and validate efficacious potentiators that lack destabilizing effect of the ?F508-CFTR in Aim 1.
In Aim 2 we will utilize novel localized structure defect-targeted (LSDT) screening approaches to identify distinct, structure-specific correctors as pharmacological chaperones that stabilize NBD1 and act synergistically with interface stabilizing drugs (e.g. VX-809). Engineered primary airways epithelial cells will increase the success rate of these screens. The mechanism of action of novel correctors will be established by biophysical, biochemical and cell biological assays. Based on the results of Aim 1-2, in Aim 3 novel mechanistic studies on the W1282X-CFTR functional expression defect, mutation-specific biochemical and functional HTS assays will be implemented to identify small-molecule correctors and potentiator and establish their mechanism of action.

Public Health Relevance

The presently available therapies for cystic fibrosis (CF) patients with deletion of F508 residue and most other rare mutations do not correct the underlying CFTR defect, and have not improved median life expectancy beyond 40 years partly due to the inefficiency or lack of drugs that can correct the basic defect. This proposal will address these deficiencies by a) identifying novel gating potentiator molecules lacking CFTR destabilizing effect, b) utilizing novel high throughput screening techniques and c) gaining more knowledge about the biochemical/structural defects caused by CF mutations. The outcome of this research will include improved understanding of the CF protein and the isolation of potentially new, more efficient therapies for cystic fibrosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK075302-10A1
Application #
9102561
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Eggerman, Thomas L
Project Start
2006-08-15
Project End
2021-03-31
Budget Start
2016-04-15
Budget End
2017-03-31
Support Year
10
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Mcgill University
Department
Type
DUNS #
205667090
City
Montreal
State
QC
Country
Canada
Zip Code
H3 0G4
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Phuan, Puay-Wah; Veit, Guido; Tan, Joseph-Anthony et al. (2018) ?F508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter. SLAS Discov 23:823-831
Bagdany, Miklos; Veit, Guido; Fukuda, Ryosuke et al. (2017) Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell. Nat Commun 8:398
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Veit, Gudio; Avramescu, Radu G; Chiang, Annette N et al. (2016) From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations. Mol Biol Cell 27:424-33
Schnúr, Andrea; Hegyi, Péter; Rousseau, Simon et al. (2016) Epithelial Anion Transport as Modulator of Chemokine Signaling. Mediators Inflamm 2016:7596531

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