adenosine-5--(n-ethylcarboxamide) and Cystic-Fibrosis

Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.

Topics: Alleles; Benzoates; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endoplasmic Reticulum; Furans; Gene Deletion; HEK293 Cells; HeLa Cells; High-Throughput Screening Assays; Humans; Hydroxamic Acids; Microscopy, Fluorescence; Protein Folding; Protein Structure, Tertiary; Pyrazoles; RNA, Messenger; Small Molecule Libraries; Ubiquitination; Vorinostat

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cells, Cultured; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Egtazic Acid; Extracellular Space; Humans; Intracellular Membranes; Ionomycin; Membrane Proteins; Reference Values; Respiratory System