Human carboxylesterase 2 (hCE2), one of the most principal drug-metabolizing enzymes, catalyzes the hydrolysis of a variety of endogenous esters, anticancer agents, and environmental toxicants. The significant roles of hCE2 in both endobiotic and xenobiotic metabolism sparked great interest in the discovery and development of efficacious and selective inhibitors. However, the safe and effective inhibitors of hCE2 are scarce, due to the lack of efficient screening and evaluation systems for complex biological systems. To offer a solution to this problem, a high-content analysis (HCA)-based cell imaging and multiparametric assay method was constructed for evaluating the inhibitory effect and safety of hCE2 inhibitors in living cell system. In this study, we first established a cell imaging-based method for identifying hCE2 inhibitors at the living cell level with hCE2 fluorescent probe NCEN. Meanwhile, two nuclear probes, Hoechst 33342 and PI, were integrated to evaluate the potential cytotoxicity of compounds simultaneously. Then, the accuracy of the HCA-based method was verified by the LC-FD-based method with a positive inhibitor BNPP, and the results showed that the HCA-based method exhibited excellent precision, robustness, and reliability. Finally, the newly established HCA-based multiparametric assay panel was successfully applied to re-evaluate a series of reported hCE2 inhibitors in living cells. In summary, the HCA-based multiparametric method could serve as an efficient tool for the accuracy measurement inhibitory effect and cytotoxicity of compounds against hCE2 in living cell system. Graphical abstract.

中文翻译：

---

高含量分析在活细胞系统中鉴定人羧酸酯酶2抑制剂的构建和应用。

人羧酸酯酶2（hCE2）是最主要的药物代谢酶之一，催化多种内源酯，抗癌剂和环境有毒物质的水解。hCE2在内源性和异源性代谢中的重要作用激发了人们对有效和选择性抑制剂的发现和开发的极大兴趣。但是，由于缺乏针对复杂生物系统的有效筛选和评估系统，因此缺乏安全有效的hCE2抑制剂。为了解决该问题，构建了基于高含量分析（HCA）的细胞成像和多参数分析方法，以评估hCE2抑制剂在活细胞系统中的抑制作用和安全性。在这个研究中，我们首先建立了一种基于细胞成像的方法，用hCE2荧光探针NCEN在活细胞水平上鉴定hCE2抑制剂。同时，整合了两个核探针，Hoechst 33342和PI，以同时评估化合物的潜在细胞毒性。然后，使用基于LC-FD的方法和阳性抑制剂BNPP验证了基于HCA的方法的准确性，结果表明基于HCA的方法显示出极好的精度，鲁棒性和可靠性。最后，新建立的基于HCA的多参数分析小组成功地用于重新评估活细胞中一系列报道的hCE2抑制剂。综上所述，基于HCA的多参数方法可作为一种有效工具，用于准确测量化合物对活细胞系统中hCE2的抑制作用和细胞毒性。