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

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• 作者：Kalvin J. Gregory ; Ye Sun ; Nelson G. Chen ; Valeri Golovlev
• 关键词：Polymerase assay ; Bioluminescence ; Real-time ; Fragment-based drug discovery (FBDD) ; Viral polymerase inhibitors
• 刊名：Analytical Biochemistry
• 出版年：2011
• 出版时间：15 January 2011
• 年：2011
• 卷：408
• 期：2
• 页码：226-234
• 全文大小：1081 K

文摘

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.