Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Clopidogrel: Involvement of Both Covalent Modification of Cysteinyl Residue 475 and Loss of Heme
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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DOI：
10.1124/mol.111.073783
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发表时间：
2011-11
影响因子：
3.6
通讯作者：
Haoming Zhang;Hemali Amunugama;S. Ney;Nyemade Cooper;P. Hollenberg
中科院分区：
文献类型：
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作者：
Haoming Zhang;Hemali Amunugama;S. Ney;Nyemade Cooper;P. Hollenberg
我们研究了氯吡格雷在重组系统中灭活人细胞色素P450 2B 6（CYP 2B 6）的机制。结果发现，氯吡格雷及其硫内酯代谢产物2-氧代氯吡格雷均以时间和浓度依赖性方式抑制CYP 2B 6。根据kinact/KI比值，氯吡格雷灭活CYP 2B 6的效率约为2-氧代-氯吡格雷的5倍。对已被氯吡格雷或2-氧代-氯吡格雷灭活的CYP 2B 6野生型（WT）蛋白的分子量进行分析，结果显示蛋白质质量增加了约350 Da。蛋白质质量的增加对应于向CYP 2B 6中添加了氯吡格雷的活性代谢产物。值得注意的是，该加合物可通过与二硫苏糖醇孵育从蛋白质基质中裂解，证实活性代谢产物通过二硫键与CYP 2B 6的半胱氨酰残基连接。使用电喷雾离子化液相色谱-串联质谱法对灭活CYP 2B 6的胰蛋白酶解酶进行肽图谱分析，确定Cys 475为活性代谢产物共价修饰的位点。这一点进一步得到了Cys 475突变为丝氨酸残基消除了蛋白加合物的形成，并防止C475 S变体被2-氧代-氯吡格雷基于机制的失活的观察结果的证实。然而，这种突变并不能阻止C475 S变异体被氯吡格雷灭活。此外，氯吡格雷可灭活CYP 2B 6 WT和C475 S，但2-氧代-氯吡格雷不能，导致血红素损失，这是催化活性损失的主要原因。总的来说，这些结果表明氯吡格雷主要通过破坏血红素使CYP 2B 6失活，而2-氧代-氯吡格雷通过共价修饰Cys 475使CYP 2B 6失活。
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 kinact/KI 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.