title: A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin.
abstract: Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.
authors.affiliation: The Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Du
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Haian
authors.initials: H
authors.lastname: Fu
authors.forename: David G
authors.initials: DG
authors.lastname: Harrison
mesh_terms: D001708:Biopterins / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D010798:Phycocyanin / Q000032:analysis; D011485:Protein Binding; D054852:Small Molecule Libraries / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology
chemical_list: C101092:GCHFR protein, human; D047908:Intracellular Signaling Peptides and Proteins; D054852:Small Molecule Libraries; C002497:allophycocyanin; D010798:Phycocyanin; D001708:Biopterins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D006136:GTP Cyclohydrolase; C003402:sapropterin