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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.

SUBMITTER: Zhang H

PROVIDER: S-EPMC3198913 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Mechanism-based inactivation of human cytochrome P450 2B6 by clopidogrel: involvement of both covalent modification of cysteinyl residue 475 and loss of heme.

Zhang Haoming H Amunugama Hemali H Ney Sarah S Cooper Nyemade N Hollenberg Paul F PF

Molecular pharmacology 20110823 5

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 ...[more]

PMID: 21862689

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Project description:Cytochrome P450 2B6 (CYP2B6) belongs to the minor drug metabolizing P450s in human liver. Expression is highly variable both between individuals and within individuals, owing to non-genetic factors, genetic polymorphisms, inducibility, and irreversible inhibition by many compounds. Drugs metabolized mainly by CYP2B6 include artemisinin, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone. CYP2B6 is one of the most polymorphic CYP genes in humans and variants have been shown to affect transcriptional regulation, splicing, mRNA and protein expression, and catalytic activity. Some variants appear to affect several functional levels simultaneously, thus, combined in haplotypes, leading to complex interactions between substrate-dependent and -independent mechanisms. The most common functionally deficient allele is CYP2B6*6 [Q172H, K262R], which occurs at frequencies of 15 to over 60% in different populations. The allele leads to lower expression in liver due to erroneous splicing. Recent investigations suggest that the amino acid changes contribute complex substrate-dependent effects at the activity level, although data from recombinant systems used by different researchers are not well in agreement with each other. Another important variant, CYP2B6*18 [I328T], occurs predominantly in Africans (4-12%) and does not express functional protein. A large number of uncharacterized variants are currently emerging from different ethnicities in the course of the 1000 Genomes Project. The CYP2B6 polymorphism is clinically relevant for HIV-infected patients treated with the reverse transcriptase inhibitor efavirenz, but it is increasingly being recognized for other drug substrates. This review summarizes recent advances on the functional and clinical significance of CYP2B6 and its genetic polymorphism, with particular emphasis on the comparison of kinetic data obtained with different substrates for variants expressed in different recombinant expression systems.

Dataset Information

Mechanism-based inactivation of human cytochrome P450 2B6 by clopidogrel: involvement of both covalent modification of cysteinyl residue 475 and loss of heme.

Publications

Mechanism-based inactivation of human cytochrome P450 2B6 by clopidogrel: involvement of both covalent modification of cysteinyl residue 475 and loss of heme.

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