Molecular analysis and modeling of inactivation of human CYP2D6 by four mechanism based inactivators.

Human cytochrome P450 2D6 (CYP2D6) is involved in metabolism of approximately 25% of pharmaceutical drugs. Inactivation of CYP2D6 can lead to adverse drug interactions. Four inactivators of CYP2D6 have previously been identified: 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine(SCH66712), (1-[(2-ethyl- 4-methyl-1H-imidazol-5-yl)-methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine(EMTPP), paroxetine, and 3,4- methylenedioxymethamphetamine (MDMA). All four contain planar, aromatic groups as well as basic nitrogens common to CYP2D6 substrates. SCH66712 and EMTPP also contain piperazine groups and substituted imidazole rings that are common in pharmaceutical agents, though neither of these compounds is clinically relevant. Paroxetine and MDMA contain methylenedioxyphenyls. SCH66712 and EMTPP are both known protein adductors while paroxetine and MDMA are probable heme modifiers. The current study shows that each inactivator displays Type I binding with Ks values that vary by 2-orders of magnitude with lower Ks values associated with greater inactivation. Comparison of KI, kinact, and partition ratio values shows SCH66712 is the most potent inactivator. Molecular modeling experiments using AutoDock identify Phe120 as a key interaction for all four inactivators with face-to-face and edge-to-face pi interactions apparent. Distance between the ligand and heme iron correlates with potency of inhibition. Ligand conformations were scored according to their binding energies as calculated by AutoDock and correlation was observed between molecular models and Ks values.

Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 µM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.