Molecular docking and in vitro evaluations reveal the role of human cytochrome P450 3A4 in the cross-coupling metabolism of phenolic xenobiotics

Metabolism generally transforms xenobiotics into more polar and hydrophilic products, facilitating their elim-ination from the body. Recently, a new metabolic pathway that transforms phenolic xenobiotics into more lipophilic and bioactive dimer products was discovered. To elucidate the role of cytochrome P450 (CYP) enzymes in mediating this cross-coupling metabolism, we used high-throughput screening to identify the metabolites generated from the coupling of 20 xenobiotics with four endogenous metabolites in liver microsomes. Endog-enous vitamin E (VE) was the most reactive metabolite, as VE reacted with seven phenolic xenobiotics containing various structures (e.g., an imidazoline ring or a diphenol group) to generate novel lipophilic ethers such as bakuchiol-O-VE, phentolamine-O-VE, phenylethyl resorcinol-O-VE, 2-propanol-O-VE, and resveratrol-O-VE. Seven recombinant CYP enzymes were successfully expressed and purified in Escherichia coli. Integration of the results of recombinant human CYP incubation and molecular docking identified the central role of CYP3A4 in the cross-coupling metabolic pathway. Structural analysis revealed the 7C-7C interactions, hydrogen bonds, and hy-drophobic interactions between reactive xenobiotics and VE in the malleable active sites of CYP3A4. The con-sistency between the molecular docking results and the in vitro human cytochrome P450 evaluation shows that docking calculations can be used to screen molecules participating in cross-coupling metabolism. The results of this study provide supporting evidence for the overlooked toxicological effects induced by direct reactions be-tween xenobiotics and endogenous metabolites during metabolic processes.

Automated summary

A new metabolic pathway has been discovered that transforms phenolic xenobiotics into more lipophilic and bioactive dimer products. Cytochrome P450 (CYP) enzymes play a central role in mediating this cross-coupling metabolism. This study provides supporting evidence for overlooked toxicological effects induced by direct reactions between xenobiotics and endogenous metabolites during metabolic processes.