Division for Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.
Division of Toxicology, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Laurel, MD, USA.
Toxicol In Vitro. 2019 Apr;56:93-100. doi: 10.1016/j.tiv.2019.01.002. Epub 2019 Jan 6.
The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.
乙酰胆碱酯酶（AChE）的抑制作用具有药物应用价值，同时也具有潜在的神经毒性。一些化学物质的体内代谢物，包括有机磷农药，与母体化合物相比，可能成为更有效的 AChE 抑制剂。为了说明生物转化的影响，我们开发并表征了一种高通量筛选方法，用于识别在异生物质代谢后变得活跃或更有效的 AChE 抑制剂。在这项研究中，使用重组人 AChE 结合人或大鼠肝微粒体，在 1536 孔板中开发了一种基于酶的测定法。通过两种具有不同读数的方法测量 AChE 活性：比色法和荧光法。该测定法具有出色的性能特征，包括大的测定信号窗口、低孔间变异性和高重现性。通过测试一组已知的 AChE 抑制剂，包括母体化合物及其代谢物，对添加微粒体的测定法的性能进行了表征。在测定法中添加微粒体后，观察到母体化合物和代谢物之间的效力差异很大。两种测定读数都需要最大的灵敏度。这些结果表明，该平台是一种有前途的方法，可以对需要代谢激活以抑制 AChE 活性的大量化学物质进行分析。
