Determining appropriate substrate conversion for enzymatic assays in high-throughput screening
Determining appropriate substrate conversion for enzymatic assays in high-throughput screening
复制标题
DOI：
10.1177/1087057103260050
复制
发表时间：
2003-12-01
影响因子：
--
通讯作者：
Hodge, CN
中科院分区：
文献类型：
--
作者：
Wu, G;Yuan, Y;Hodge, CN
一般认为，在研究酶动力学时，底物的转化率应保持在所用总底物的10%以下。然而，10%或更少的底物转化通常不会产生稳健的高通量筛选（HTS）所需的足够的信号变化。为了增加信号背景比，HTS通常在高于10%的底物转化率下进行。由于对高底物转化率的后果知之甚少，筛选结果有时受到酶学家的质疑。通过在HTS条件下检测抑制剂的能力来判断测定的质量，这取决于主要检测信号（Z因子）的耐用性和对抑制剂的灵敏度。当收集单点数据时，在固定化合物浓度下观察到的IC 50值或抑制百分比反映了对抑制剂的测定灵敏度。在高底物转化率下的酶测定的主要问题是筛选的灵敏度可能会受到损害。在这里，我们推导出一个给定的抑制剂的IC 50值和底物转化的百分比之间的关系，使用一阶动力学模型的条件下，服从亨利-米氏-门滕动力学。推导出的理论进一步验证了实验与cAMP依赖性蛋白激酶。该模型为检测开发人员提供了指导，以在设计酶检测时选择适当的底物转化，平衡对稳健信号和对抑制剂敏感性的需求。
It is generally accepted that the conversion of substrate should be kept at less than 10% of the total substrate used when studying enzyme kinetics. However, 10% or less substrate conversion often will not produce sufficient signal changes required for robust high-throughput screening (HTS). To increase the signal-to-background ratio, HTS is often performed at higher than 10% substrate conversion. Because the consequences of high substrate conversion are poorly understood, the screening results are sometimes questioned by enzymologists. The quality of an assay is judged by the ability to detect an inhibitor under HTS conditions, which depends on the robustness of the primary detection signal (Z factor) and the sensitivity to an inhibitor. The assay sensitivity to an inhibitor is reflected in the observed IC50 value or percent inhibition at a fixed compound concentration when single-point data are collected. The major concern for an enzymatic assay under high substrate conversion is that the sensitivity of the screen may be compromised. Here we derive the relationship between the IC50 value for a given inhibitor and the percentage of substrate conversion using a first-order kinetic model under conditions that obey Henri-Michaelis-Menten kinetics. The derived theory was further verified experimentally with a cAMP-dependent protein kinase. This model provides guidance for assay developers to choose an appropriate substrate conversion in designing an enzymatic assay, balancing the needs for robust signal and sensitivity to inhibitors.