P450-Mediated Dehydrogenation Mechanisms

描述（由申请方提供）：通过细胞色素P450氧化机制将外源性物质生物活化为毒性中间体是一种公认的过程。然而，通过几种P450酶（例如1A 2、2B 6、2 E1、2F 1、3A 4和4 B1）通过脱氢途径产生亲电中间体的研究最近才被研究，并且控制选择性脱氢而不是氧化的机制尚未建立。几种脱氢中间体的反应性很强，以至于它们通常通过活性位点亲核残基的烷基化作用而使P450酶失活。研究这些特定的P450酶的催化行为和他们的倾向，而不是催化底物是非常必要的。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，从而导致外源性介导的损伤和人类药物代谢的改变。本申请的具体目标是确定指导特定细胞色素P450酶的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来实现：1）确定由原型底物脱氢产生的活性中间体的结构，并表征酶对底物脱氢与氧化的偏好; 2）表征每种P450酶通过其特异性灭活剂的灭活机制;（3）确定P450酶对毒物的脱氢和生物活化机制的活性中心参数;和4）使用共价修饰P450脱辅基蛋白的脱氢底物来阐明P450的关键活性。位点残基指导脱氢机制，或控制抑制剂/底物进入通道、结合或产物释放。酶/底物对为CYP 2F 3/3-甲基吲哚、CYP 3A 4/扎鲁司特、CYP 2 E1/辣椒素和CYP 2B 6/他莫昔芬。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过程中产生有毒亲电中间体的机制，以评估这些有毒中间体对人类健康的潜在危害，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），新药和异生物质。

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.