Amgen Inc., Department of Phamacokinetics and Drug Metabolism, 1201 Amgen Court West, Seattle, WA 98119, USA.
Arch Biochem Biophys. 2011 Sep 15;513(2):110-8. doi: 10.1016/j.abb.2011.06.016. Epub 2011 Jul 13.
Raloxifene is a polyaromatic compound which has been reported to form radicals when incubated with horseradish peroxidase resulting in formation of a homo-dimer product. Polyaromatic phenols have also been reported to undergo oxidation by P450 enzymes to form reactive intermediates, presumably through the formation of phenoxy radical species. Recently, we observed that a raloxifene homo-dimer was formed in vitro when incubated with CYP3A4. In response to this finding, a series of experiments were designed to determine whether the observed raloxifene homo-dimer was formed via solution phase chemistry similar to that previously documented with horseradish peroxidase or if generation of the homo-dimer occurred within the P450 active site. To this end, a series of experiments were carried out to determine the structure of the CYP3A4 generated raloxifene homo-dimer using analytical techniques including: high resolution MS, NMR and H/D exchange. In addition, a variety of in vitro techniques were applied to characterize the mechanism responsible for formation of the raloxifene homo-dimer. Collectively, the results of these experiments suggest that unlike the homo-dimer formed by peroxidase enzymes, raloxifene homo-dimer formation mediated by CYP3A4 is a consequence of two raloxifene molecules binding simultaneously within the active site of a catalytically competent P450 enzyme.
雷洛昔芬是一种多环芳香族化合物，据报道，当与辣根过氧化物酶孵育时，它会形成自由基，从而形成同二聚体产物。多环酚类也被报道通过 P450 酶氧化形成反应性中间体，推测是通过形成苯氧自由基物种。最近，我们观察到当与 CYP3A4 孵育时，雷洛昔芬同二聚体在体外形成。针对这一发现，设计了一系列实验来确定观察到的雷洛昔芬同二聚体是否是通过类似于先前用辣根过氧化物酶记录的溶液相化学形成的，或者同二聚体的生成是否发生在 P450 活性部位内。为此，进行了一系列实验，使用包括高分辨率 MS、NMR 和 H/D 交换在内的分析技术来确定 CYP3A4 生成的雷洛昔芬同二聚体的结构。此外，还应用了各种体外技术来表征形成雷洛昔芬同二聚体的机制。总的来说，这些实验的结果表明，与过氧化物酶酶形成的同二聚体不同，CYP3A4 介导的雷洛昔芬同二聚体形成是两个雷洛昔芬分子同时结合在催化有效 P450 酶的活性部位内的结果。
