title: Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.
abstract: 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.
authors.affiliation: Department of Chemistry, Kalamazoo College, 1200 Academy Street, Kalamazoo, MI 49006. laura.furge@kzoo.edu.
authors.forename: Leslie D
authors.initials: LD
authors.lastname: Nagy
authors.forename: Catherine S
authors.initials: CS
authors.lastname: Mocny
authors.forename: Laura E
authors.initials: LE
authors.lastname: Diffenderfer
authors.forename: David J
authors.initials: DJ
authors.lastname: Hsi
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Butler
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Arthur
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Fletke
authors.forename: Jairam R
authors.initials: JR
authors.lastname: Palamanda
authors.forename: Amin A
authors.initials: AA
authors.lastname: Nomeir
authors.forename: Laura Lowe
authors.initials: LL
authors.lastname: Furge
mesh_terms: D002851:Chromatography, High Pressure Liquid; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D008958:Models, Molecular; D011485:Protein Binding; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics
chemical_list: D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011743:Pyrimidines; D011994:Recombinant Proteins; C403229:SCH 66712; D006418:Heme; D019389:Cytochrome P-450 CYP2D6