Correction of defective deltaF508-CFTR processing in cystic fibrosis

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DESCRIPTION (provided by applicant): 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) disrupts the posttranslational folding, as well as the biosynthetic and endocytic processing of CFTR. The functional expression defect of CFTR at the plasma membrane leads to impaired chloride, bicarbonate and water 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 corrector. 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. In accord, initial data of phase II clinical trials indicate marginal clinical efficiency of VX-809. Identification of highly efficient corrector molecules is impeded by our incomplete understanding of the ?F508 CFTR misfolding. While recent results suggest that the ?F508 mutation energetically destabilizes the isolated nucleotide binding domain 1 (NBD1), the implication that ?F508-NBD1 conformational stabilization represents the ideal drug target remains to be validated in the context of full-length CFTR. Indeed, our preliminary data indicate that additional mechanism(s) play equally important role in the ?F508 CFTR misfolding. This competitive renewal builds on our discoveries of a) corrector and potentiator molecules by high throughput screening (HTS) assays, b) the CFTR cooperative domain folding mechanism, as well as c) preliminary data indicating that thermodynamic correction of the ?F508-NBD1 is necessary, but not sufficient to restore the ?F508 CFTR biosynthetic folding, processing and plasma membrane stability. To isolate correctors that restore the ?F508 CFTR folding and plasma membrane chloride channel function to >50% of its wild-type counterpart, we propose to identify distinct, structure-specific correctors as pharmaco- chaperones that act synergistically by using a novel, localized structure defect-targeted screening (LSDS) approach. Aim 1. Will elucidate the major structural defects responsible for ?F508-CFTR misfolding by quantifying the contribution of domain-domain interactions and the NBD1 energetics to the channel folding and function. The consequence of second site suppressor mutations of distinct structural defects in isolated NBD1 and full-length CFTR will be established by biophysical, biochemical and cell biological assays. Aim 2. will identify small-molecule correctors by multiple localized structure defect-targeted HTS and establish their mechanism of action in vitro and in vivo. Aim 3. Will determine the translational potential of LSDS-based combination ?F508-CFTR corrector therapy by using innovate surrogate human cellular and animal models of CF. PUBLIC HEALTH RELEVANCE: The presently available therapies for cystic fibrosis (CF) do not correct the underlying CFTR defect, and have not improved median life expectancy beyond 40 years. This proposal will establish a conceptually novel, structure-based high throughput screening techniques utilizing our knowledge about the biochemical and structural defects caused by the most common mutation in CF, deletion of F508 residue. 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.