Identification of soluble epoxide hydrolase inhibitors from natural products via structure-based virtual screening, in vitro assays, and molecular dynamics simulations.

Soluble epoxide hydrolase (sEH) is a key enzyme regulating the metabolism of epoxidized fatty acids and is involved in various pathological processes, including inflammation, cardiovascular disease, and metabolic disorders; therefore, the development of novel sEH inhibitors is of great significance. In this study, we screened 715,343 natural products using the COCONUT 2.0 natural products database, with the human sEH crystal structure (PDB ID: 3ANS) as the target. Eight representative compounds were selected for in vitro validation. The results showed that Compound 1 (CNP0403849.1) and Compound 3 (CNP0059487.0) exhibited good sEH inhibitory activity, with IC₅₀ values of 0.852 ± 0.014 µM and 2.811 ± 0.243 µM, respectively, while the IC₅₀ value for the positive control AUDA was 0.014 ± 0.003 µM. Enzyme kinetic analysis indicates that these two active compounds inhibit sEH through a non-competitive mechanism. Cell-based experiments indicated that, within the concentration range of 5-40 µM, Compound 1 and Compound 3 exhibited no significant cytotoxicity toward RAW264.7 cells and could inhibit LPS-induced NO production to some extent. 100 ns molecular dynamics simulations revealed that both compounds could maintain stable binding within the active pocket of sEH. This study identified two sEH inhibitors with micromolar activity from natural products, with Compound 1 exhibiting superior in vitro enzyme inhibitory activity. This research provides a new structural basis and research leads for the discovery of sEH inhibitors derived from natural products and the subsequent optimization of lead compounds.