Functional Dynamics of Cytochrome P4503A4

细胞色素 P4503A4 的功能动力学

基本信息

批准号：
9638812
负责人：
WILLIAM M ATKINS
金额：
$ 49.63万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-18 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9638812
关键词：
Active Sites Adverse effects Affinity Anaerobic Bacteria Behavior Binding Binding Sites Biochemical Biochemistry Biophysics Bromocriptine CYP3A4 gene Catalysis Cessation of life Characteristics Clinical Research Competence Complex Crystallization Cytochromes Data Deuterium Drug Design Drug Interactions Drug Targeting Equilibrium Exhibits Face Fluconazole Frequencies Heme Hepatic Human Hydrogen Hydrogen Bonding Hydrogen Peroxide In Vitro Kinetics Knowledge Ligand Binding Ligands Link Lipid Bilayers Lipids Maps Mass Spectrum Analysis Measures Mechanics Membrane Membrane Lipids Methods Midazolam Modeling Molecular Molecular Conformation Monitor Motion Optics Oxidation-Reduction Pathway interactions Pharmaceutical Preparations Pharmacology Play Process Proline Property Protein Dynamics Protein Isoforms Reaction Recording of previous events Regulation Resolution Role Source Specificity Structure Toxic effect Uncertainty Water Work Xenobiotics base cofactor detoxication drug clearance drug metabolism experimental study improved inhibitor/antagonist interest molecular dynamics molecular mechanics nanodisk novel predictive modeling prevent quantum response trait translational model

项目摘要

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.

项目概要拟议的工作旨在澄清药物代谢细胞色素长期存在的机制不确定性 P450（CYP）。这些 CYP 的底物非常混杂，它们是外源物解毒、药物代谢和药物相互作用。由于它们在药物代谢中的作用，也是药物靶标，其中它们的药理学操作可以控制其他药物的治疗。这里的一个焦点是有关配体依赖性血红素自旋状态之间关系的知识差距和催化性能。在针对特定底物开发的 CYP 标准催化反应循环中同种型，底物取代血红素结合水并导致从低自旋血红素转变为高自旋血红素血红素特性的伴随变化有利于催化循环的还原和进展。在相比之下，对于药物代谢 CYP，许多药物不会导致向高自旋血红素的转变，但它们可以有效地代谢或导致过氧化氢形成，两者都需要通过催化过程进行循环。最近的结果表明，许多这些底物与轴向水形成氢键，而不是取代它。这些水桥配合物的催化性能尚未确定。所以，拟议的工作将确定 CYP3A4 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化方法，包括分光电位法和停流反应动力学。计算方法也将用于了解水桥复合物对血红素还原过程的影响。拟议工作的第二个重点旨在阐明构象动力学在复杂的过程中的作用 CYP 的变构行为及其显着的底物混杂性。这两个特征都与蛋白质有关动力学，其特征仍然很差，并且可用晶体结构尚不清楚。变构性质混淆了药物清除率和药物相互作用的预测，因此人们对转化有很大兴趣基于精细的变构模型更好地预测药物相互作用的模型。这里，实验氢-氘交换质谱 (H/DX) 和预稳态配体结合方法脂质双层纳米盘中 CYP3A4 的方法与计算方法相结合，例如加速分子动力学模拟 (AMD) 和引导分子动力学。拟议的研究填补了在理解以前无法通过实验实现的脂质中 CYP 动力学方面存在显着差距膜以及构象动力学在实现底物混杂和变构中的作用。