QM/MM Insights into Mechanism-Based Inactivation of Human CYP3A4: Formation of a Nitrosoalkane Intermediate from Erythromycin

Erythromycin, a naturally occurring 14-membered macrolide antibiotic, is widely used to treat various bacterial infections. CYP3A4 is the primary enzyme responsible for erythromycin metabolism in humans. However, this metabolic process can lead to the formation of a nitrosoalkane intermediate, ultimately resulting in mechanism-based inactivation (MBI) of the enzyme. Despite its clinical significance, the precise mechanistic pathway underlying the formation of this intermediate remains largely unclear. In this study, we employed molecular docking and molecular dynamics (MD) simulations to identify the most plausible catalytically competent binding mode of erythromycin in CYP3A4, which differs markedly from that observed in the available crystal structure. Based on the predicted binding mode, we carried out hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, which suggest that erythromycin undergoes N-demethylation at its tertiary amine as the initial metabolic step, yielding a secondary amine. Rather than undergoing further N-dealkylation, this intermediate is N-hydroxylated to form an N-hydroxy secondary amine. This species is subsequently dehydrogenated to produce a nitrone intermediate, which undergoes methylene cleavage to generate a nitrosoalkane that coordinates to the ferrous heme iron, thereby contributing to CYP3A4 inactivation.