Real-time bioluminescent assay for inhibitors of RNA and DNA polymerases and other ATP-dependent enzymes

Kalvin J. Gregory*, Ye Sun, G. Chen, Valeri Golovlev

*Corresponding author for this work

Research output: Contribution to journalArticle

3 Scopus citations

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 Km values that compared well with literature values. The assay could identify the mechanism of inhibition and determine inhibition constants (Ki) 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 Ki>100μM, making it a viable technique for fragment-based drug discovery.

Original languageEnglish
Pages (from-to)226-234
Number of pages9
JournalAnalytical Biochemistry
Volume408
Issue number2
DOIs
StatePublished - 15 Jan 2011

Keywords

  • Bioluminescence
  • Fragment-based drug discovery (FBDD)
  • Polymerase assay
  • Real-time
  • Viral polymerase inhibitors

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