Cao_2026_Bioorg.Med.Chem_137_118624

Human carboxylesterase 2A (hCES2A) is a key mediator of drug metabolism and chemotherapeutic side effects, necessitating potent and selective inhibitors. Herein, 42 novel steroid derivatives were rationally designed via hCES2A homology modeling and synthesized. Compound 3g emerged as the lead inhibitor, exhibiting strong hCES2A inhibitory activity (IC50 = 1.257 microM) and excellent selectivity, with no cross-inhibition on hCES1A, hAchE, or hBchE. Structure-activity relationship (SAR) analysis and a validated 3D-QSAR model identified steric and hydrophobic effects as key activity determinants. Mechanistic studies confirmed 3g acts as a competitive inhibitor (K(i) = 1.503 microM) with favorable binding affinity (K(d) = 1.00 microM). Molecular docking revealed 3g occupies the hCES2A catalytic triad (Ser-228, Glu-345, His-457) pocket, stabilized by Pi-Pi stacking with Phe-106/Phe-469. Critical cellular validation was performed via high-content imaging, which confirmed specific hCES2A inhibition in HepG2 cells. Additionally, 3g exhibits low cytotoxicity and good metabolic stability. In conclusion, compound 3g was identified as a potent inhibitor of hCES2A.