Mechanistic Study of Icaritin-Induced Inactivation of Cytochrome P450 2C9

Icaritin (ICT) is a prenylflavonoid derivative that has been approved by National Medical Products Administration for the treatment of hepatocellular carcinoma. This study aims to evaluate the potential inhibitory effect of ICT against cytochrome P450 (CYP) enzymes and to elucidate the inactivation mechanisms. Results showed that ICT inactivated CYP2C9 in a time-, concentration-, and NADPH-dependent manner with K-i = 1.896 lM, K-inact = 0.02298 minutes(-1), and K-inact/K-i = 12 minutes(-1) mM(-1), whereas the activities of other CYP isozymes was minimally affected. Additionally, the presence of CYP2C9 competitive inhibitor, sulfaphenazole, superoxide dis-mutase/catalase system, and GSH all protected CYP2C9 from ICT-induced activity loss. Moreover, the activity loss was neither recov-ered by washing the ICT-CYP2C9 preincubation mixture nor the addition of potassium ferricyanide. These results, collectively, im-plied the underlying inactivation mechanism involved the covalent binding of ICT to the apoprotein and/or the prosthetic heme of CYP2C9. Furthermore, an ICT-quinone methide (QM)-derived GSH adduct was identified, and human glutathione S-transferases (GST) isozymes GSTA1-1, GSTM1-1, and GSTP1-1 were shown to be sub-stantially involved in the detoxification of ICT-QM. Interestingly, our systematic molecular modeling work predicted that ICT-QM was covalently bound to C216, a cysteine residue located in the F-G loop downstream of substrate recognition site (SRS) 2 in CYP2C9. The sequential molecular dynamics simulation con-firmed the binding to C216 induced a conformational change in the active catalytic center of CYP2C9. Lastly, the potential risks of clinical drug-drug interactions triggered by ICT as a perpetrator were extrapolated. In summary, this work confirmed that ICT was an inactivator of CYP2C9.SIGNIFICANCE STATEMENT This study is the first to report the time-dependent inhibition of CYP2C9 by icaritin (ICT) and the intrinsic molecular mechanism be-hind it. Experimental data indicated that the inactivation was via ir-reversible covalent binding of ICT-quinone methide to CYP2C9, while molecular modeling analysis provided additional evidence by predicting C216 as the key binding site which influenced the structural confirmation of CYP2C9's catalytic center. These find-ings suggest the potential of drug-drug interactions when ICT is co-administered with CYP2C9 substrates clinically.