QM/MM Study of the Metabolic Oxidation of 6′,7′-Dihydroxybergamottin Catalyzed by Human CYP3A4: Preferential Formation of the γ-Ketoenal Product in Mechanism-Based Inactivation

6',7'-Dihydroxybergamottin (DHB), a natural furanocoumarin found in grapefruit, is known to cause mechanism-based inactivation (MBI) of several cytochrome P450 enzymes (P450s) in humans, including CYP3A4. Despite its pharmacological significance, the precise microscopic mechanisms underlying the P450 MBI induced by DHB remain unclear. To address this, we employed molecular docking and molecular dynamics simulations to identify a plausible catalytic binding pose of DHB within CYP3A4. Subsequent quantum mechanics/molecular mechanics (QM/MM) calculations explored two possible reaction pathways (A and B). Path A involves the attack by compound I (Cpd I) at the C5 position of the furan moiety, leading to γ-ketoenal formation, while Path B targets the C4 position, yielding an epoxide. Path A exhibits a much lower activation energy barrier, indicating a strong kinetic preference. Additionally, the γ-ketoenal is thermodynamically more stable than the epoxide. Thus, even if the epoxide forms initially, it is likely to rearrange into the γ-ketoenal, either within the enzyme or in aqueous solution. Collectively, these findings suggest that the γ-ketoenal is the sole ultimate product of DHB oxidation by CYP3A4. This study provides valuable insights into CYP3A4 inactivation by grapefruit constituents and advances our understanding of food-drug interactions.