A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5′-diphosphate accumulation
A generic, homogenous method for measuring kinase and inhibitor activity via adenosine 5′-diphosphate accumulation
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DOI：
10.1177/1087057106286829
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发表时间：
2006-06-01
影响因子：
--
通讯作者：
Eglen, Richard M.
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文献类型：
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作者：
Charter, Neil W.;Kauffman, Lindy;Eglen, Richard M.
作者描述了一种测定方法来测量由激酶磷酸化底物产生的5'-二磷酸腺苷（ADP）的产生。ADP积累是通过一个耦合酶系统转化为荧光信号来检测的。该技术在抑制剂效价和作用方式的评估以及酶活性的动力学分析方面具有潜在的应用前景。该分析具有宽动态范围（0.25-75 μ M），并已通过几种激酶验证，包括高活性环腺苷单磷酸依赖蛋白激酶（PKA α），酪蛋白激酶1 （CK1）和弱活性Jun n -末端激酶2 （Jnk2 α 2）。激酶活性可以以终点或连续模式测量。终点模式下的分析性能与腺苷5'-三磷酸（ATP）耗尽试验和连续模式下的丙酮酸激酶/乳酸脱氢酶偶联试验进行了比较。通过对PKA α、CK1和Jnk2 α 2的ATP/底物亲和力（Michaelis-Menten常数；K-m）值的推导，证明了表征激酶动力学的能力。该试验很容易用蛋白底物测量激酶反应的活性，表明适合与大分子一起使用。即使在存在高ATP浓度的情况下，也可以确定广泛的抑制剂活性，这使得该分析非常适合表征所讨论的抑制剂的作用模式。总的来说，该分析为激酶药物发现中的许多应用提供了一种均匀的通用方法。
The authors describe an assay to measure the generation of adenosine 5'-diphosphate (ADP) resulting from phosphorylation of a substrate by a kinase. ADP accumulation is detected by conversion to a fluorescent signal via a coupled enzyme system. The technology has potential applications for the assessment of inhibitor potency and mode of action as well as kinetic analysis of enzyme activity. The assay has a wide dynamic range (0.25-75 mu M) and has been validated with several kinases including the highly active cyclic adenosine monophosphate-dependent protein kinase (PKA alpha), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk2 alpha 2). Kinase activity can be measured either in an end point or continuous mode. Assay performance in end point mode was compared with an adenosine 5'-triphosphate (ATP) depletion assay and in continuous mode with a pyruvate kinase/lactate dehydrogenase coupled assay. The ability to characterize kinase kinetics was demonstrated by deriving ATP/substrate affinity (Michaelis-Menten constant; K-m) values for PKA alpha, CK1, and Jnk2 alpha 2. The assay readily measured activity with kinase reactions using protein substrates, indicating the suitability for use with large macromolecules. A wide range of inhibitor activities could be determined even in the presence of high ATP concentrations, making the assay highly suitable to characterize the mode of action of the inhibitor in question. Collectively, this assay provides a homogenous, generic method for a number of applications in kinase drug discovery.