Functional Dynamics of Cytochrome P4503A4

项目摘要 拟议的工作旨在澄清长期存在的机制不确定性药物代谢细胞色素 P450（CYP）。这些CYP是非常底物混杂的，它们是细胞增殖的主要决定因素。 异生物质解毒、药物代谢和药物相互作用。由于它们在药物代谢中的作用， 也是药物靶点，其中它们的药理学操作可以提供对其他药物治疗的控制。 这里的一个焦点是关于配体依赖的血红素自旋状态之间的关系的知识缺口 和催化性能。在为底物特异性而开发的单正则催化反应循环中， 异构体，底物置换血红素结合水，并导致从低自旋到高自旋血红素的转变， 伴随的血红素性质的转变，促进还原和通过催化循环的进展。在 相反，对于药物代谢CYP，许多药物不引起高自旋血红素的转变，但它们有效地 代谢或导致过氧化氢的形成，这两者都需要通过催化剂的进展， 周期最近的结果表明，许多这些基板氢键的轴向水，而不是 这些水桥络合物的催化性能尚未确定。因此，我们认为， 所提出的工作将确定CYP 3A 4的血红素氧化还原特性和催化能力， 水桥复合物，使用计算方法和先进的生物化学方法，包括 光谱电位法和停流反应动力学。还将采用计算方法， 了解水桥络合物对血红素还原过程的影响。 拟议工作的第二个重点是澄清复杂的构象动力学的作用 CYP的变构行为及其显著的底物混杂性。这两个特征都与蛋白质有关 动力学，这仍然是不好的特点，并通过可用的晶体结构不明确。变构 性质混淆了药物清除和药物相互作用的预测，因此对翻译的兴趣很大。 基于精细变构模型更好地预测药物相互作用。在这里，实验 氢-氘交换质谱（H/DX）和预稳态配体结合的方法 脂质双层纳米盘中的CYP 3A 4方法与计算方法相结合， 加速分子动力学模拟（aMD）和操纵分子动力学。建议的研究填补 在理解以前实验上无法实现的脂质中的脂质动力学方面存在重大差距， 膜和构象动力学在实现底物混杂和变构中的作用。

Project Summary The proposed work aims to clarify longstanding mechanistic uncertainties about drug metabolizing cytochrome P450s (CYPs). These CYPs are extraordinarily substrate promiscuous and they are major determinants of xenobiotic detoxication, drug metabolism and drug interactions. Because of their role in drug metabolism, they are also drug targets, wherein their pharmacological manipulation can afford control of therapy with other drugs. One focus here is the knowledge gap concerning the relationship between ligand-dependent heme spin state and catalytic properties. In the CYP canonical catalytic reaction cycle developed for substrate specific isoforms, the substrate displaces heme bound water and causes a shift from low spin to high spin heme with concomitant shift in the heme properties that facilitate reduction and progress through the catalytic cycle. In contrast, with drug metabolizing CYPs, many drugs cause no shift to high spin heme, but they are efficiently metabolized or cause hydrogen peroxide formation, both of which require progression through the catalytic cycle. Recent results demonstrate that many of these substrates hydrogen bond to the axial water, rather than displace it. The catalytic properties of these water-bridged complexes have not been determined. Therefore, the proposed work will determine for CYP3A4 the heme redox properties and the catalytic competence of water-bridged complexes, using computational approaches and advanced biochemical methods including spectropotentiometry and stopped-flow reaction kinetics. Computational approaches will also be employed to understand the effect of water-bridged complexes on heme reduction processes. A second focus of the proposed work aims to clarify the role of conformational dynamics in the complex allosteric behavior of CYPs and their remarkable substrate promiscuity. Both traits are linked to the protein dynamics, which remain poorly characterized, and unclarified by the available crystal structures. The allosteric properties confound prediction of drug clearance and drug interactions, so there is great interest in translational models that better predict drug interactions based on refined allosteric models. Here, the experimental methods of hydrogen-deuterium exchange mass spectrometry (H/DX) and pre-steady state ligand binding methods with CYP3A4 in lipid bilayer nanodiscs are combined with computational approaches such as accelerated Molecular Dynamics simulations (aMD) and steered molecular dynamics. The proposed studies fill a significant gap in understanding the, previously experimentally inaccessible, CYP dynamics in a lipid membrane and the role of conformational dynamics in achieving substrate promiscuity and allostery.