P450-Mediated Dehydrogenation Mechanisms

P450 介导的脱氢机制

• 批准号: 8399735
• 负责人: Garold S Yost
• 金额: $ 31.11万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8399735
• 关键词: Active Sites; Adverse effects; Alkylation; Behavior; Biochemical; CYP2D6 gene; CYP2E1 gene; CYP3A4 gene; Capsaicin; Characteristics; Charge; Chemicals; Chemistry; Computer Simulation; Cytochrome P450; Deuterium; Docking; Drug Industry; Drug Interactions; Electron Transport; Electronics; Environment; Enzyme Kinetics; Enzymes; Evaluation; Future; Glucocorticoids; Goals; Halogens; Health; Heme; Human; Injury; Isotopes; Knowledge; Liver; Measures; Mediating; Medicine; Metabolism; Methods; Modeling; Molecular; Molecular Conformation; Mutate; Mutation; Nature; Pathway interactions; Pharmaceutical Preparations; Poison; Predisposition; Process; Production; Proteins; Quantum Mechanics; Research; Site; Skatole; Structure; Tamoxifen; Toxic effect; Urine; Xenobiotics; adduct; base; dehydrogenation; drug development; drug metabolism; enzyme model; human SLC25A5 protein; improved; molecular dynamics; molecular mechanics; mutant; novel; prototype; public health relevance; stable isotope; toxicant

DESCRIPTION (provided by applicant): Bioactivation of xenobiotics to toxic intermediates through cytochrome P450 oxygenation mechanisms is a well recognized process. However, the production of electrophilic intermediates by several P450 enzymes (e.g. 1A2, 2B4, 2B6, 2C9, 2D6, 2E1, 2F1, 2F3, 2A13, and 3A4), through dehydrogenation pathways has only recently been investigated, and the mechanisms that govern selective dehydrogenation rather than oxygenation are not established. Several of the dehydrogenated intermediates are so reactive that they inactivate the P450 enzymes, generally through alkylation of active site nucleophilic residues. Recent convincing research on the P450 enzymes has documented the highly dynamic nature of these proteins that requires sophisticated computer-based simulations to model. Research concerning the catalytic behavior of these specific P450 enzymes and their propensity to dehydrogenate rather than oxygenate substrates is vitally needed. The hypothesis of this research is: the unique catalytic mechanism(s) of facilitated electron transport that determines dehydrogenation by certain P450 enzymes results in xenobiotic-mediated injury and altered drug metabolism in humans. The specific goals of this application are to determine the characteristics of the enzyme active-site and remote residue environments that direct dehydrogenation mechanisms of specific cytochrome P450 enzymes, and to define the substrate structural features that regulate selective dehydrogenation rather than oxygenation. These goals will be realized through the following aims: 1) to define the requisite chemical features of acceptable P450-mediated dehydrogenation substrates; 2) to characterize the chemical and biochemical mechanisms of dehydrogenation by evaluating the dehydrogenation of three prototype substrates, with the use of stable isotopes and identification of protein adducts; 3) to utilize integrated quantum mechanics-based models of substrates and intermediates with molecular mechanics and molecular dynamic simulations of P450 enzymes to predict critical dehydrogenation- specific residues and substrate reactivities; and 4) to validate specific P450 active site and remote residues by mutation of specific sites, followed by biochemical evaluations and x-ray structures of purified native and mutant enzymes. The long-term goals of this research are to elucidate the mechanisms of cytochrome P450- mediated dehydrogenation of xenobiotics in processes that generate toxic electrophilic intermediates, to assess the potential harm engendered by these toxic intermediates to human health, and to utilize mechanistic information to predict dehydrogenation, and concomitant toxicities and/or enzyme inactivation (altered drug metabolism), of new drugs and xenobiotics.

描述（由申请人提供）：通过细胞色素 P450 氧化机制将异生物质生物活化为有毒中间体是一个公认的过程。然而，最近才对几种 P450 酶（例如 1A2、2B4、2B6、2C9、2D6、2E1、2F1、2F3、2A13 和 3A4）通过脱氢途径产生亲电子中间体进行了研究，并且尚未建立控制选择性脱氢而不是氧化的机制。一些脱氢中间体的反应活性很高，通常会通过活性位点亲核残基的烷基化来使 P450 酶失活。最近对 P450 酶的令人信服的研究记录了这些蛋白质的高度动态性质，需要复杂的基于计算机的模拟来建模。迫切需要研究这些特定 P450 酶的催化行为及其脱氢而不是氧化底物的倾向。这项研究的假设是：促进电子传递的独特催化机制决定了某些 P450 酶的脱氢作用，导致外源性介导的损伤和人类药物代谢的改变。该应用的具体目标是确定指导特定细胞色素 P450 酶脱氢机制的酶活性位点和远程残基环境的特征，并定义调节选择性脱氢而不是氧合的底物结构特征。这些目标将通过以下目标实现：1）定义可接受的 P450 介导的脱氢底物的必要化学特征； 2) 通过使用稳定同位素和蛋白质加合物的鉴定，评估三种原型底物的脱氢，从而表征脱氢的化学和生物化学机制； 3）利用基于量子力学的底物和中间体的集成模型以及P450酶的分子力学和分子动力学模拟来预测关键的脱氢特异性残基和底物反应性； 4) 通过特定位点的突变来验证特定的 P450 活性位点和远程残基，然后对纯化的天然酶和突变酶进行生化评估和 X 射线结构。本研究的长期目标是阐明细胞色素 P450 介导的异生物质脱氢在产生有毒亲电子中间体的过程中的机制，评估这些有毒中间体对人类健康造成的潜在危害，并利用机制信息来预测脱氢以及伴随的毒性和/或酶失活（改变药物） 代谢），新药和外源性物质。