title: Real-time bioluminescent assay for inhibitors of RNA and DNA polymerases and other ATP-dependent enzymes.
abstract: Viral polymerases are important targets for drug development. However, current methods used to identify and characterize inhibitors of polymerases are time-consuming, use radiolabeled reagents, and are cost-inefficient. Here we present a bioluminescent assay for the identification and characterization of inhibitors of polymerases, as well as other ATP-dependent enzymes, that monitors the decrease of ATP or dATP in real time, allowing detection of enzyme inhibition based on differences in ATP/dATP consumption. The assay works with a variety of RNA and DNA polymerases, using both RNA and DNA templates. The assay measures changes in substrate concentration in real time and provides a faster alternative for kinetic studies of inhibition. Michaelis-Menten plots were obtained from a single reaction, yielding K(m) values that compared well with literature values. The assay could identify the mechanism of inhibition and determine inhibition constants (K(i)) for a weak competitive inhibitor of Klenow fragment and two strong noncompetitive inhibitors of HIV-1 reverse transcriptase with one series of inhibitor concentrations, reducing the total number of experiments that would normally be needed. The assay is also sensitive enough to detect a weak inhibitor with K(i)>100 μM, making it a viable technique for fragment-based drug discovery.
authors: Sci-Tec, Knoxville, TN 37932, USA. kjgreg@aurogene.com
authors: Kalvin J
authors: KJ
authors: Gregory
authors: Ye
authors: Y
authors: Sun
authors: Nelson G
authors: NG
authors: Chen
authors: Valeri
authors: V
authors: Golovlev
mesh_terms: D001667:Binding, Competitive; D004247:DNA / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D019384:Nucleic Acid Synthesis Inhibitors / Q000737:chemistry*; D012313:RNA / Q000096:biosynthesis; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry*
chemical_list: D003838:Deoxyadenine Nucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D018894:Reverse Transcriptase Inhibitors; D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate