Elucidating the binding and metabolic interactions of sunitinib and sorafenib with Cytochrome P450s CYP2U1 and CYP2D6.

Cytochrome P450 2U1 (CYP2U1) is an extrahepatic monooxygenase that metabolizes both endogenous fatty acids and xenobiotic substrates. Because of its high expression in both healthy and cancerous thymus tissues, this study investigated CYP2U1-mediated biotransformation of 2 tumor-targeting tyrosine kinase inhibitors, sorafenib and sunitinib, both are commonly prescribed for thymus cancers. Recombinantly expressed CYP2U1 and CYP2D6 were incorporated into nanodiscs, and their metabolism of sorafenib and sunitinib was investigated. Liquid chromatography-tandem mass spectrometry metabolite profiling revealed that both CYP2U1 and CYP2D6 nanodiscs oxidized sorafenib to generate sorafenib N-oxide and both enzymes catalyzed the dealkylation of sunitinib to form N-desethyl sunitinib. Spectroscopic studies (UV-Visible and fluorescence) confirmed favorable binding interactions of CYP2U1 and CYP2D6 to both drugs tested. Molecular dynamics simulations demonstrated binding of sorafenib and sunitinib in the CYP2U1 active site and identified key interactions between the drug and key residues at the enzyme's active site. Rates of metabolite formation were quantified by targeted metabolomics, and inhibition of tyrosine kinase was assessed by ELISA assay. Both the parent compound sorafenib and metabolite sorafenib N-oxide showed similar inhibitory effects on cellular migration in HepG2 cells. The metabolite sorafenib N-oxide was approximately twice as potent as the parent compound in inhibiting cancer cell migration. In contrast, N-desethyl sunitinib failed to show similar extent of inhibition. Together, these findings highlight the potential role of extrahepatic CYP2U1 in the local metabolism of tyrosine kinase inhibitors and suggest that CYP2U1-mediated transformations directly influence antitumor efficacy at thymic tumor sites. SIGNIFICANCE STATEMENT: Understanding the interactions between cytochrome P450 2U1 and cytochrome P450 2D6 in nanodiscs and thymus tumor-targeting drugs, sorafenib and sunitinib, led to discovery of new bioactive metabolites that carry differential anticancer properties compared with their parent compounds.