A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

• Type of Material: Article
• Authors: Li Li, Emory University; Yuhong Du, Emory University; Haian Fu, Emory University; Wei Chen, Vanderbilt University; David G. Harrison, Vanderbilt University
• Language: English
• Date: 2011-09-01
• Publisher: SAGE PUBLICATIONS INC
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2011 Society for Laboratory Automation and Screening.
• Final Published Version (URL): http://dx.doi.org/10.1177/1087057111411088
• Title of Journal or Parent Work: Journal of Biomolecular Screening
• Volume: 16
• Issue: 8
• Start Page: 836
• End Page: 844
• Grant/Funding Information: This work was supported by National Institutes of Health (NIH) grants P01HL58000 and R01HL39006 (to D.G.H.) and an American Heart Association Predoctoral Grant (to L.L.), Emory URC (to Y.D.), Georgia Cancer Coalition, Georgia Research Alliance, and Emory Faculty Distinction Fund (to H.F.).
• Keywords: ATHEROSCLEROSIS; Biochemistry & Molecular Biology; Chemistry; transcription factors; Science & Technology; Biochemical Research Methods; protein-protein interactions; OXIDATION; NITRIC-OXIDE SYNTHASE; GTP CYCLOHYDROLASE-I; BIOCHEMICAL RESEARCH METHODS; signal transduction; BIOTECHNOLOGY & APPLIED MICROBIOLOGY; CHEMISTRY, ANALYTICAL; Physical Sciences; FEEDBACK REGULATORY PROTEIN; ENOS; PROGRESSION; fluorescence methods; Biotechnology & Applied Microbiology; Chemistry, Analytical; cell-based assays; INHIBITION; Life Sciences & Biomedicine; cardiac diseases
• Research Categories: Biology, Microbiology; Chemistry, Biochemistry; Health Sciences, Pharmacology

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 signalto-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.