P450-Mediated Dehydrogenation Mechanisms

P450 介导的脱氢机制

基本信息

批准号：
7544947
负责人：
Garold S Yost
金额：
$ 28.16万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7544947
关键词：
Acetylcysteine Active Sites Alkylation Apoproteins Behavior Binding Biochemical CYP2B6 gene CYP2E1 gene CYP2F3 gene CYP3A4 gene Capsaicin Characteristics Chemicals Cleaved cell Complementary DNA Cyanogen Bromide Cytochrome P450 Deuterium Drug Interactions Electron Transport Environment Enzymes Escherichia coli Exhibits Glutathione Goals Health Human Injury Kinetics Label MALDI-TOF Mass Spectrometry Mediating Methods Molecular Molecular Models Pathway interactions Peptide Fragments Pharmaceutical Preparations Predisposition Process Production Property Recombinants Research Research Personnel Site Skatole Specificity Structure Tamoxifen Time Toxic effect Xenobiotics analog base dehydrogenation drug metabolism enzyme substrate human SLC25A5 protein inhibitor/antagonist insight liquid chromatography mass spectrometry molecular modeling mutant preference programs toxicant zafirlukast

项目摘要

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, 2B6, 2E1, 2F1, 3A4, and 4B1), 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. 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) offacilitated 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 environment 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 determine the structures of the reactive intermediates that are produced by dehydrogenation of prototypical substrates, and characterize enzyme preferences for dehydrogenation vs. oxygenation of the substrates; 2) To characterize the mechanisms of inactivation of each P450 enzyme by its specific inactivator; 3) To define the active-site parameters that control the mechanisms of dehydrogenation and bioactivation of toxicants by P450 enzymes; and 4) To use the dehydrogenation substrates that covalently modify the P450 apoproteins to elucidate critical active-site residues that direct the dehydrogenation mechanism, or that control inhibitor/substrate access channels, binding, or product release. The enzyme/substrate pairs are CYP2F3/3-methylindole, CYP3A4/zafirlukast, CYP2El/capsaicin, and CYP2B6/tamoxifen. 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.

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种很好的方法认可的过程。然而，几种 P450 酶（例如 1A2、 2B6、2E1、2F1、3A4 和 4B1)，通过脱氢途径最近才被研究，并且尚未建立控制选择性脱氢而不是氧化的机制。其中几个脱氢中间体的反应活性很高，通常会通过烷基化使 P450 酶失活活性位点亲核残基。有关这些特定 P450 酶催化行为的研究并且它们对底物脱氢而不是氧化的倾向是非常需要的。对此的假设研究是：决定脱氢的促进电子传输的独特催化机制某些 P450 酶导致异生素介导的损伤并改变人类的药物代谢。这该应用的具体目标是确定酶活性位点环境的特征特定细胞色素 P450 酶的直接脱氢机制，并确定底物结构调节选择性脱氢而不是氧化的功能。这些目标将通过以下目标： 1) 确定脱氢产生的反应中间体的结构原型底物，并表征酶对脱氢与氧化的偏好基材； 2) 表征每种P450酶被其特定灭活剂灭活的机制； 3）定义控制毒物脱氢和生物活化机制的活性位点参数通过 P450 酶； 4) 使用共价修饰 P450 脱辅基蛋白的脱氢底物阐明指导脱氢机制或控制抑制剂/底物的关键活性位点残基访问渠道、绑定或产品发布。酶/底物对是 CYP2F3/3-甲基吲哚， CYP3A4/扎鲁司特、CYP2E1/辣椒素和CYP2B6/他莫昔芬。这项研究的长期目标是阐明细胞色素 P450 介导的异生素脱氢过程中的机制产生有毒的亲电子中间体，以评估这些有毒中间体对人类健康，并利用机械信息来预测脱氢和伴随的毒性和/或新药和异生素的酶失活（改变药物代谢）。