P450-Mediated Dehydrogenation Mechanisms

P450 介导的脱氢机制

• 批准号: 8210904
• 负责人: Garold S Yost
• 金额: $ 32.29万
• 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/8210904
• 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. PUBLIC HEALTH RELEVANCE: Medicines are chemicals that ideally have beneficial effects with few side effects, and that don't have drug/drug interactions, which is when they act together to cause the medicines to lose their efficacy. After a medicine is taken and has its beneficial action, it is usually metabolized, i.e. chemically altered, by cytochrome P450 enzymes in the liver to aid in its elimination. Even though the P450 enzymes generally convert the medicines to harmless metabolites that are excreted in the urine, frequently these enzymes change the structures of the medicines to highly reactive, toxic products, through a chemical mechanism called dehydrogenation. Dehydrogenation products are frequently toxic and cause drug/drug interactions by inactivating the P450 enzymes that metabolized the medicines. However, very little is known about the dehydrogenation process. Thus, the goal of this research is to precisely delineate the mechanisms of P450-mediated dehydrogenation, with medicines and toxic chemicals that are known to be metabolized by this process. Our long term objective is to predict which chemical motifs are likely to be dehydrogenation substrates, and should be avoided when new drugs are introduced. This knowledge will significantly improve the drug development process by the pharmaceutical industry in the future.

描述（由申请人提供）：通过细胞色素P450氧合机制将异种生物学生物活化至有毒中间体是一个众所周知的过程。但是，通过几种P450酶（例如1A2、2B4、2B6、2C9、2D6、2D6、2E1、2F1、2F3、2A13和3A4）生产亲电的中间体，通过脱水途径仅通过脱水途径进行了研究，并且没有被脱氢途径进行了调查，而不是在选择性脱水的机制。几种脱氢中间体具有反应性，以至于它们通常通过活性位点亲核残基的烷基化使P450酶失活。对P450酶的最新研究记录了这些蛋白质的高度动态性质，这些蛋白需要基于计算机的基于计算机的模拟才能进行建模。关于这些特定P450酶的催化行为及其对脱氢而不是氧化底物的倾向的研究非常需要。这项研究的假设是：促进电子转运的独特催化机制，该促进的电子转运决定了某些P450酶的脱氢作用会导致异种生物介导的损伤和人类药物代谢改变。本应用的具体目标是确定酶的活性位点和远程残基环境的特征，这些环境直接指导特定细胞色素P450酶的脱氢机制，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的实现：1）定义可接受的P450介导的脱氢底物的必要化学特征； 2）通过使用稳定的同位素和蛋白质加合物的鉴定来评估三种原型底物的脱氢作用来表征脱氢作用的化学和生化机制； 3）利用基于量子力学的综合力学模型和中间体与P450酶的分子力学和分子动力学模拟，以预测关键的脱氢特异性残基和底物激活性； 4）通过特定部位的突变来验证特定的P450活性位点和远程残基，然后进行纯化的天然和突变酶的生化评估和X射线结构。这项研究的长期目标是阐明细胞色素p450-在产生有毒的亲电中间体的过程中介导的异种生物学介导的脱氢，以评估这些有毒中间体对人类健康产生的潜在伤害，并利用机械性信息来预测机械性（Orniz and）的作用（Ornz anz），以促进脱水的毒性（ORTREDITIS），或者，或者是抗浓度的有毒率和综合性毒性毒性和综合性毒性毒性毒性毒性和综合性毒性毒性。代谢），新药和异种生物。 公共卫生相关性：药物是理想情况下具有有益作用而具有很少副作用的化学物质，并且没有药物/药物相互作用，这是当它们共同起作用以使药物失去疗效时。服用药物并采取有益作用后，通常通过细胞色素P450酶在肝脏中代谢，即化学改变，以帮助消除其。即使P450酶通常将药物转化为尿液中排出的无害代谢产物，但这些酶经常通过称为脱氢作用的化学机制将药物的结构转化为高度反应性的有毒产物。脱氢产物经常有毒，并通过灭活对药物的P450酶来引起药物/药物相互作用。但是，关于脱氢过程知之甚少。因此，这项研究的目的是精确地描述P450介导的脱氢作用的机制，该过程已知被该过程代谢的药物和有毒化学物质。我们的长期目标是预测哪些化学基序可能是脱氢底物，应在引入新药时避免。这些知识将在未来大大改善制药行业的药物开发过程。