Shahrokh Kiumars, Cheatham Thomas E, Yost Garold S
Department of Pharmacology and Toxicology, College of Pharmacy, Skaggs Hall 201, University of Utah, Salt Lake City, UT 84112, USA.
Biochim Biophys Acta. 2012 Oct;1820(10):1605-17. doi: 10.1016/j.bbagen.2012.05.011. Epub 2012 Jun 4.
Structure-based methods for P450 substrates are commonly used during drug development to identify sites of metabolism. However, docking studies using available X-ray structures for the major drug-metabolizing P450, CYP3A4, do not always identify binding modes supportive of the production of high-energy toxic metabolites. Minor pathways such as P450-catalyzed dehydrogenation have been experimentally shown to produce reactive products capable of forming biomolecular adducts which can lead to increased risk toxicities. 4-Hydroxy-tamoxifen (4OHT) is metabolized by CYP3A4 via competing hydroxylation and dehydrogenation reactions.
Ab initio gas-phase electronic structural characterization of 4OHT was used to develop a docking scoring scheme. Conformational sampling of CYP3A4 with molecular dynamics simulations along multiple trajectories were used to generate representative structures for docking studies using recently published heme parameters. A key predicted binding mode was tested experimentally using site-directed mutagenesis of CYP3A4 and liquid chromatography-mass spectroscopy analysis.
Docking with MD-refined CYP3A4 structures incorporating hexa-coordinate heme parameters identifies a unique binding mode involving ARG212 and channel 4, unobserved in the starting PDB ID: 1TQN X-ray structure. The models supporting dehydrogenation are consistent with results from in vitro incubations.
Our models indicate that coupled structural contributions of the ingress, egress and solvent channels to the CYP3A4 active site geometries play key roles in the observed 4OHT binding modes. Thus adequate sampling of the conformational space of these drug-metabolizing promiscuous enzymes is important for substrates that may bind in malleable regions of the enzyme active-site.
在药物研发过程中，基于结构的P450底物方法常用于识别代谢位点。然而，使用主要药物代谢P450（CYP3A4）的现有X射线结构进行对接研究，并不总能识别出支持高能毒性代谢物产生的结合模式。实验表明，诸如P450催化脱氢等次要途径会产生能够形成生物分子加合物的反应性产物，这可能导致毒性风险增加。4-羟基他莫昔芬（4OHT）通过竞争羟基化和脱氢反应被CYP3A4代谢。
利用4OHT的从头算气相电子结构表征来开发对接评分方案。通过沿多条轨迹的分子动力学模拟对CYP3A4进行构象采样，以生成用于对接研究的代表性结构，该研究使用了最近公布的血红素参数。使用CYP3A4的定点诱变和液相色谱-质谱分析对一种关键的预测结合模式进行了实验测试。
与纳入六配位血红素参数的MD优化CYP3A4结构进行对接，确定了一种独特的结合模式，涉及ARG212和通道4，这在起始的PDB ID：1TQN X射线结构中未观察到。支持脱氢的模型与体外孵育结果一致。
我们的模型表明，入口、出口和溶剂通道对CYP3A4活性位点几何结构的耦合结构贡献在观察到的4OHT结合模式中起关键作用。因此，对这些药物代谢多底物酶的构象空间进行充分采样，对于可能结合在酶活性位点可塑性区域的底物来说很重要。
