title: Mechanism-based inactivation of human cytochrome P450 2B6 by clopidogrel: involvement of both covalent modification of cysteinyl residue 475 and loss of heme.
abstract: We have investigated the mechanisms by which clopidogrel inactivates human cytochrome P450 2B6 (CYP2B6) in a reconstituted system. It was found that clopidogrel and its thiolactone metabolite, 2-oxo-clopidogrel, both inactivate CYP2B6 in a time- and concentration-dependent manner. On the basis of k(inact)/K(I) ratios, clopidogrel is approximately 5 times more efficient than 2-oxo-clopidogrel in inactivating CYP2B6. Analysis of the molecular mass of the CYP2B6 wild-type (WT) protein that had been inactivated by either clopidogrel or 2-oxo-clopidogrel showed an increase in the mass of the protein by ∼350 Da. This increase in the protein mass corresponds to the addition of the active metabolite of clopidogrel to CYP2B6. It is noteworthy that this adduct can be cleaved from the protein matrix by incubation with dithiothreitol, confirming that the active metabolite is linked to a cysteinyl residue of CYP2B6 via a disulfide bond. Peptide mapping of tryptic digests of the inactivated CYP2B6 using electrospray ionization liquid chromatography-tandem mass spectrometry identified Cys475 as the site of covalent modification by the active metabolite. This was further confirmed by the observation that mutation of Cys475 to a serine residue eliminates the formation of the protein adduct and prevents the C475S variant from mechanism-based inactivation by 2-oxo-clopidogrel. However, this mutation did not prevent the C475S variant from being inactivated by clopidogrel. Furthermore, inactivation of both CYP2B6 WT and C475S by clopidogrel, but not by 2-oxo-clopidogrel, led to the loss of the heme, which accounts for most of the loss of the catalytic activity. Collectively, these results suggest that clopidogrel inactivates CYP2B6 primarily through destruction of the heme, whereas 2-oxo-clopidogrel inactivates CYP2B6 through covalent modification of Cys475.
authors.affiliation: Department of Pharmacology, The University of Michigan, Ann Arbor, MI 48109-5632, USA.
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.forename: Hemali
authors.initials: H
authors.lastname: Amunugama
authors.forename: Sarah
authors.initials: S
authors.lastname: Ney
authors.forename: Nyemade
authors.initials: N
authors.lastname: Cooper
authors.forename: Paul F
authors.initials: PF
authors.lastname: Hollenberg
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D000077144:Clopidogrel; D003545:Cysteine / Q000737:chemistry*; D065702:Cytochrome P-450 CYP2B6; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D004791:Enzyme Inhibitors; D006418:Heme; D000077144:Clopidogrel; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D003545:Cysteine; D013988:Ticlopidine