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.