ABSTRACT
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause CF. The most common mutation, F508 deletion, causes CFTR misfolding and endoplasmic reticulum retention, preventing it from trafficking to the cell surface. One approach to CF treatment is to identify compounds that correct the trafficking defect. We screened a marine extract collection and, after extract, deconvolution identified the latonduines as F508del-CFTR trafficking correctors that give functional correction in vivo. Using a biotinylated azido derivative of latonduine, we identified the poly(ADP-ribose) polymerase (PARP) family as latonduine target proteins. We show that latonduine binds to PARPs 1, 2, 3, 4, 5a, and 5b and inhibits PARP activity, especially PARP-3. Thus, latonduine corrects F508del-CFTR trafficking by modulating PARP activity. Latonduines represent pharmacologic agents for F508del-CFTR correction, and PARP-3 is a pathway for the development of CF treatments.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Heterocyclic Compounds, 3-Ring/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Protein Transport/drug effects , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , Cell Line , Cricetinae , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Endoplasmic Reticulum/metabolism , Heterocyclic Compounds, 3-Ring/chemistry , Humans , Mice , Mutation , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , Porifera/chemistry , Protein Binding , Protein Folding/drug effects , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
High-throughput small-molecule screens hold great promise for identifying compounds with potential therapeutic value in the treatment of protein-trafficking diseases such as cystic fibrosis (CF) and nephrogenic diabetes insipidus (NDI). The approach usually involves expressing the mutant form of the gene in cells and assaying function in a multiwell format when cells are exposed to libraries of compounds. Although such functional assays are useful, they do not directly test the ability of a compound to correct defective trafficking of the protein. To address this we have developed a novel corrector-screening assay for CF, in which the appearance of the mutant protein at the cell surface is measured. We used this assay to screen a library of 2000 compounds and have isolated several classes of trafficking correctors that had not previously been identified. This novel screening approach to protein-trafficking diseases is robust and general, and could enable the selection of molecules that could be translated rapidly to a clinical setting.