Baer Brian R, Wienkers Larry C, Rock Dan A
Amgen, Department of Pharmacokinetics and Drug Metabolism, 1201 Amgen Court West, Seattle, Washington 98119, USA.
Chem Res Toxicol. 2007 Jun;20(6):954-64. doi: 10.1021/tx700037e. Epub 2007 May 12.
Time-dependent inactivation of cytochrome P450s is typically a result of substrate bioactivation to form reactive species that subsequently alkylate the heme group, apoprotein, or both. The chemical identity of many reactive intermediates is generally proposed based on the products of trapping reactions with nucleophilic agents as only a few P450-drug adducts have been directly characterized. We describe the use of mass spectrometry to show that a single equivalent of raloxifene is bound to the intact P450 apoprotein. Furthermore, mass analysis of peptides following digestion with proteinase K revealed that the covalently bound drug is localized to residue Cys239. A mass shift of 471 Da to the intact protein and peptide, relative to control samples, indicated that time-dependent inactivation of P450 3A4 occurred through the raloxifene diquinone methide intermediately prior to nucleophilic attack of the sulfur of Cys239. Association between raloxifene adduction to P450 3A4 apoprotein and the observed time-dependent inactivation was further investigated with the use of cysteine-specific modifying reagents. When P450 3A4 was treated with iodoacetamide or N-(1-pyrene)iodoacetamide, which alkylated residue Cys239 exclusively, time-dependent inactivation of P450 3A4 by raloxifene was prevented. The change in protein mass of 471 Da combined with the protection from inactivation that occurred through pre-alkylation of Cys239 provided conclusive evidence that raloxifene-mediated P450 3A4 inactivation occurred through the bioactivation of raloxifene to the diquinone methide and subsequent alkylation of Cys239.
细胞色素P450的时间依赖性失活通常是底物生物活化形成反应性物种的结果，这些反应性物种随后会烷基化血红素基团、脱辅基蛋白或两者。许多反应性中间体的化学特性通常是根据与亲核试剂捕获反应的产物提出的，因为只有少数P450-药物加合物得到了直接表征。我们描述了使用质谱法来表明一当量的雷洛昔芬与完整的P450脱辅基蛋白结合。此外，用蛋白酶K消化后的肽段的质谱分析表明，共价结合的药物定位于残基Cys239。相对于对照样品，完整蛋白质和肽段的质量位移为471 Da，这表明P450 3A4的时间依赖性失活是通过雷洛昔芬二醌甲基化物中间体在Cys239的硫亲核攻击之前发生的。使用半胱氨酸特异性修饰试剂进一步研究了雷洛昔芬与P450 3A4脱辅基蛋白加合与观察到的时间依赖性失活之间的关联。当用碘乙酰胺或N-(1-芘基)碘乙酰胺处理P450 3A4时，它们仅烷基化残基Cys239，从而防止了雷洛昔芬对P450 3A4的时间依赖性失活。471 Da的蛋白质质量变化以及通过Cys239的预烷基化防止失活，提供了确凿的证据，表明雷洛昔芬介导的P450 3A4失活是通过雷洛昔芬生物活化成二醌甲基化物并随后烷基化Cys239而发生的。
