P450-Mediated Dehydrogenation Mechanisms

描述(由申请人提供)：通过细胞色素P450氧化机制将外源物质生物激活为有毒中间体是一个公认的过程。然而，最近才对几种P450酶(如1A2、2B6、2E1、2F1、3A4和4B1)通过脱氢途径产生亲电中间体进行了研究，并且没有建立起控制选择性脱氢而不是氧合的机制。一些脱氢中间体是如此活跃，以至于它们通常通过活性部位亲核残基的烷基化来使P450酶失活。关于这些特定的P450酶的催化行为及其脱氢而不是氧合底物的倾向的研究是至关重要的。本研究的假设是：决定某些P450酶脱氢的独特的促进电子传递的催化机制(S)会导致异物介导的损伤和人类药物代谢的改变。这项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部位环境的特征，并确定调节选择性脱氢而不是加氧的底物结构特征。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活性中间体的结构，并表征底物脱氢与氧化的酶偏好；2)表征每一种P450酶被其特定的失活剂失活的机制；3)确定控制P450酶脱氢和生物激活毒物的机制的活性部位参数；以及4)使用共价修饰P450载脂蛋白的脱氢底物来阐明指导脱氢机制的关键活性部位残基，或控制抑制剂/底物访问通道、结合或产物释放的关键活性部位残基。酶/底物对分别为：CYP2F3/3-甲基吲哚、CYP3A4/扎鲁司特、CYP2EL/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介导的外源物质在产生有毒亲电中间体的过程中的脱氢机制，评估这些有毒中间体对人类健康的潜在危害，并利用机制信息预测新药和外源物质的脱氢以及伴随的毒性和/或酶失活(改变的药物代谢)。

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.