P450 Allosterism and Drug Interactions

P450 变构和药物相互作用

基本信息

批准号：
7559323
负责人：
WILLIAM M ATKINS
金额：
$ 23.71万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7559323
关键词：
Active Sites Allosteric Regulation Allosteric Site Antiepileptic Agents Behavior Binding Binding Sites Biological Models CYP17A1 gene CYP2C9 gene CYP2E1 gene CYP3A4 gene Carbamazepine Chemicals Clinical Research Collaborations Complex Computer Simulation Consumption Coupling Cytochrome P450 Cytochromes Data Dose Drug Interactions Electron Transport Exhibits Experimental Designs Fluorescence Fluorescence Spectroscopy Fluorescent Probes Goals Grant Heme Human Hydration status In Vitro Lead Ligands Link Maps Mass Spectrum Analysis Measures Metabolic Metabolic Activation Metabolism Methods Midazolam Modeling Molecular Molecular Conformation NADP Object Attachment Oral Oral Administration Oxidoreductase Oxygen P-Glycoprotein P-Glycoproteins Peripheral Personal Satisfaction Pharmaceutical Preparations Property Protein Isoforms Proteins Protons Radioisotope Dilution Technique Rate Reaction Regulation Relative (related person)Relaxation Reporter Reporting Site Source Structure Sulfonic Acids Surface Testosterone Time base clinically relevant comparative cost crosslink drug metabolism improved in vivo inhibitor/antagonist molecular dynamics nile red novel oxidation predictive modeling prevent programs protein protein interaction small molecule tertiary amine

项目摘要

Cytochrome P450's (CYPs) are a major source of metabolic drug interactions. The allosteric behavior of CYPs, including the major drug metabolizing isoform CYP3A4, continues to confound in vitro - In vivo correlations and the prediction of drug interactions based on in vitro data. The molecular mechanisms that confer allosteric behavior are incompletely understood, and improved mechanistic models are likely to improve predictive drug metabolism. Moreover, although allosteric behavior in vitro is well known and widely observed, examples of in vivo allosterism are limited. Among the multiple mechanisms that contribute, occupancy by small molecule drugs at a peripheral effector site of CYP3A4, and conformational changes induced by the electron transfer partner Cytochrome bs (Cyt bs), are likely to converge on a set of common effects, wherein the active site is more efficiently desolvated or 'well-packed' through protein-protein interactions or multiple drug binding. In turn this, hypothetically, leads to more efficient coupling of NADPH consumption and O2 reduction, concomitant with drug oxidation, with decreased 'uncoupling' to form reduced oxygen species. This proposal aims to: 1) increase our understanding of allosteric CYP mechanisms with model probe drugs, and 2) to complete a comparative, and mechanistic, analysis of CYP-Cyt b5 interactions. The fluorescent probes Nile Red and TNS provide a comparison of the active site hydration and steric constraints of CYP3A4 when the peripheral allosteric site is occupied or empty. Mass spectrometry and computational models provide methods to map binding interactions in Cyt b5-CYP complexes and the resulting conformational changes linked to effector functions of Cyt bs. Each of these approaches will be combined with functional studies to characterize reaction intermediates that determine the efficiency of the flux through the catalytic cycle. A major goal of these studies is to develop their translational utility in conceptually analogous studies with P-glycoprotein in Project 4 of this Program. In addition, the importance of in vivo allosteric effects remains speculative, and the final aim, aim 3, of this proposal will utilize a novel in vivo, human, clinical study to explore heterotropic effects between the antiepileptic drug carbamazapine and the antianxiety drug midazolam. This study not only seeks to establish definitive proof-of-principle for in vivo allosteric effects in human CYPs, but also to provide a platform experimental design to be exploited in other in vivo studies.

细胞色素 P450 (CYP) 是代谢药物相互作用的主要来源。的变构行为 CYP，包括主要药物代谢亚型 CYP3A4，在体外和体内继续混淆基于体外数据的相关性和药物相互作用的预测。分子机制赋予变构行为尚未完全理解，改进的机械模型可能会改善预测药物代谢。此外，尽管体外变构行为是众所周知且广泛的据观察，体内变构的例子是有限的。在多种做出贡献的机制中，小分子药物在 CYP3A4 外周效应位点的占据以及构象变化由电子转移伙伴细胞色素 bs (Cyt bs) 诱导，可能会收敛于一组常见的效应，其中活性位点通过蛋白质-蛋白质更有效地去溶剂化或“充分堆积” 相互作用或多种药物结合。反过来，假设这会导致 NADPH 更有效的偶联消耗和 O2 减少，伴随药物氧化，“解偶联”减少形成还原态氧物种。该提案旨在：1）增加我们对变构 CYP 机制的理解模型探针药物，2) 完成 CYP-Cyt b5 相互作用的比较和机制分析。荧光探针尼罗红和 TNS 提供了活性位点水合和空间位阻的比较当外周变构位点被占据或空时，CYP3A4 的限制。质谱分析和计算模型提供了绘制 Cyt b5-CYP 复合物中结合相互作用的方法以及由此产生的构象变化与 Cyt bs 的效应器功能相关。这些方法中的每一种都将与功能研究相结合，表征决定反应效率的反应中间体通过催化循环的通量。这些研究的一个主要目标是开发它们的转化效用本计划项目 4 中 P-糖蛋白概念上的类似研究。此外，重要性体内变构效应的影响仍然是推测性的，该提案的最终目标，即目标 3，将利用一种新颖的体内、人体、临床研究，探讨抗癫痫药物卡马扎平和抗焦虑药咪达唑仑。这项研究不仅旨在为体内实验建立明确的原理证明人类 CYP 中的变构效应，同时也提供了一个可在其他领域利用的平台实验设计体内研究。