Stereo- and Regioselective Metabolism of the Omeprazole Enantiomers by CYP2C19: Insights from Binding Free Energy and QM/MM Calculations in a Curtin–Hammett Framework

Omeprazole, a widely used drug for acid-related disorders, is administered as a racemic mixture of its R- and S-enantiomers. The therapeutic efficacy of omeprazole is influenced by the differential metabolic stability of these enantiomers. Two human cytochrome P450 isoforms play critical roles in its metabolism: CYP2C19 preferentially hydroxylates the R-enantiomer to produce 5-hydroxyomeprazole, while CYP3A4 predominantly metabolizes the S-enantiomer (esomeprazole) to generate omeprazole sulfone. Additionally, CYP2C19 metabolizes esomeprazole at the 5′-methoxy position, yielding 5′-O-hydroxyomeprazole, which subsequently hydrolyzes to 5′-O-desmethylomeprazole. Despite the clinical significance of these metabolic pathways, the mechanisms and determinants underlying metabolic selectivity remain poorly understood. In this study, we introduced a computational protocol that integrates binding free energy calculations based on thermodynamic integration and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to investigate the stereo- and regioselective metabolism of the two omeprazole enantiomers by CYP2C19. This integrated protocol incorporates the Curtin–Hammett principle in simulations, enabling efficient and accurate modeling of metabolic selectivity. Water-mediated H-bond networks were found to play a significant role in determining substrate binding affinity. Furthermore, the activation energy barriers for the four metabolic pathways differ, highlighting the pivotal role of the active-site environment in modulating substrate hydroxylation reactivity.