Construction and application of a high-content analysis for identifying human carboxylesterase 2 inhibitors in living cell system.

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.