Differences in reactive metabolite formation and cytochrome P450 binding between acetaminophen and its...

Bicyclo[1.1.1]pentane (BCP) is widely used as a bioisostere of 1,4-disubstituted benzene to prevent oxidative metabolism of the benzene ring by cytochrome P450s (P450s). In this study, we investigated its effectiveness in reducing metabolism-dependent toxicity using acetaminophen (APAP) as a model compound, which causes hepatotoxicity by forming the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). In cultured rat hepatocytes, APAP markedly reduced intracellular glutathione levels, whereas N-{3-hydroxybicyclo[1.1.1]pentan-1-yl}acetamide (BCP-APAP), in which the benzene ring of APAP is replaced with BCP, had only a minor effect, suggesting that BCP-APAP does not form NAPQI-like reactive metabolites. We then investigated the interactions of these compounds with human CYP1A2, CYP2E1, and CYP3A4, the major P450s involved in NAPQI formation, using inhibition assays. While both compounds similarly inhibited CYP1A2 and CYP3A4, only APAP inhibited CYP2E1, suggesting that BCP-APAP does not bind to CYP2E1. Docking simulations with 3D crystal structures of these P450s revealed that both compounds can bind to CYP1A2 and CYP3A4 in a similar orientation for heme-mediated metabolism, whereas BCP-APAP did not adopt an APAP-like docking pose in CYP2E1. These findings indicate that replacing the benzene ring in APAP with BCP prevents the formation of reactive metabolites and may subtly alter P450 binding properties in an isoform-dependent manner.