P450-Mediated Dehydrogenation Mechanisms

P450 介导的脱氢机制

• 批准号: 7048271
• 负责人: Garold S Yost
• 金额: $ 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 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048271
• 关键词: active sites; capsaicin; catalyst; chemical kinetics; chemical structure; cytochrome P450; drug /agent; drug metabolism; electron transport; electrospray ionization mass spectrometry; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate; indoles; mass spectrometry; matrix assisted laser desorption ionization; pharmacology; protein purification; protein structure function; protonation; site directed mutagenesis; tamoxifen; toxin

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, 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) 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 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.

描述（由申请人提供）：通过细胞色素P450氧化机制将外源药物生物活化为有毒中间体是一个公认的过程。然而，几种P450酶（如1A2, 2B6, 2E1, 2F1, 3A4和4B1）通过脱氢途径产生亲电中间体的研究直到最近才开始，并且控制选择性脱氢而不是氧化的机制尚未确定。一些脱氢中间体的反应性很强，通常通过活性位点亲核残基的烷基化使P450酶失活。研究这些特定的P450酶的催化行为以及它们对底物脱氢而不是氧化的倾向是非常必要的。本研究的假设是：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，导致了外源介导的人体损伤和药物代谢的改变。本应用程序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环境的特征，并定义调节选择性脱氢而不是氧化的底物结构特征。这些目标将通过以下目标实现：1)确定由原型底物脱氢产生的反应中间体的结构，并表征底物脱氢与氧化的酶偏好；2)表征P450酶被其特异性失活剂失活的机制；3)确定控制P450酶对有毒物质脱氢和生物活化机制的活性位点参数；4)利用共价修饰P450载脂蛋白的脱氢底物来阐明指导脱氢机制或控制抑制剂/底物进入通道、结合或产物释放的关键活性位点残基。酶/底物对是cyp2f3 /3-甲基吲哚、CYP3A4/zafirlukast、CYP2El/辣椒素和CYP2B6/他莫昔芬。本研究的长期目标是阐明细胞色素p450介导的异种生物在产生有毒亲电中间体的过程中脱氢的机制，评估这些有毒中间体对人体健康的潜在危害，并利用机制信息预测新药和异种生物的脱氢以及伴随的毒性和/或酶失活（改变药物代谢）。