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 Ks 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.
Footnotes
This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants 1R15-GM086767-01, 3R15-GM086767-01S1] (to L.L.F.); Kalamazoo College [Grant 52006304 from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program]; and the Hutchcroft Fund of Kalamazoo College.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.037630.
-
ABBREVIATIONS:
- P450
- cytochrome P450
- SCH 66712
- 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine
- HPLC
- high-performance liquid chromatography
- ACN
- acetonitrile
- MS
- mass spectrometry
- TFA
- trifluoroacetic acid
- LC
- liquid chromatography
- ESI
- electrospray ionization
- PAGE
- polyacrylamide gel electrophoresis
- PDB
- Protein Data Bank
- tBPA
- 4-tert-butylphenylacetylene
- amu
- atomic mass units
- NAC
- N-acetylcysteine
- EMTPP
- (1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)-methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine
- CID
- collision-induced dissociation.
- Received December 13, 2010.
- Accepted March 18, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|