Real-time bioluminescent assay for inhibitors of RNA and DNA polymerases and other ATP-dependent enzymes.
Real-time bioluminescent assay for inhibitors of RNA and DNA polymerases and other ATP-dependent enzymes.
复制标题
RNA 和 DNA 聚合酶以及其他 ATP 依赖性酶抑制剂的实时生物发光测定。
DOI：
10.1016/j.ab.2010.08.016
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发表时间：
2011
影响因子：
2.9
通讯作者：
Golovlev,Valeri
中科院分区：
文献类型：
--
作者：
Gregory,KalvinJ;Sun,Ye;Chen,NelsonG;Golovlev,Valeri
病毒聚合酶是药物开发的重要靶标。然而，目前用于鉴定和表征聚合酶抑制剂的方法是耗时的，使用放射性标记的试剂，并且成本低。在这里，我们提出了一种生物发光检测聚合酶抑制剂的鉴定和表征，以及其他ATP依赖性酶，监测ATP或dATP的减少在真实的时间，允许检测酶抑制的基础上的ATP/dATP消耗的差异。该测定使用RNA和DNA模板与多种RNA和DNA聚合酶一起工作。该检测方法可在真实的时间内测量底物浓度的变化，并为抑制的动力学研究提供了更快的替代方法。Michaelis-Menten图从一个单一的反应，得到Km值，以及与文献值相比。该试验可以确定抑制机制，并确定抑制常数（Ki）的Klenow片段的弱竞争性抑制剂和两个强的非竞争性抑制剂的HIV-1逆转录酶与一系列的抑制剂浓度，减少了实验的总数，通常需要。该检测方法也足够灵敏，可以检测Ki>100μM的弱抑制剂，使其成为基于片段的药物发现的可行技术。
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 Kmvalues 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.