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，几种P450酶(如1A2、2B4、2B6、2C9、2D6、2E1、2F1、2F3、2A13和3A4)通过脱氢途径产生亲电中间体的研究直到最近才被研究，而控制选择性脱氢而不是加氧的机制尚未建立。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。最近对P450酶的令人信服的研究证明了这些蛋白质的高度动态性质，需要复杂的基于计算机的模拟来建模。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。本申请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和远程残基环境的特征，并确定调节选择性脱氢而不是加氧的底物结构特征。这些目标将通过下列目标实现：1)确定可接受的P450介导的脱氢底物的必要化学特征；2)通过使用稳定同位素和鉴定蛋白质加合物来评估三种原型底物的脱氢作用，从而表征脱氢的化学和生化机制；3)利用基于底物和中间体的综合量子力学模型以及对P450酶的分子力学和分子动力学模拟来预测关键的脱氢特定残基和底物的活性；以及4)通过特定位点的突变来验证特定的P450活性位点和远程残基，然后对纯化的天然和突变酶进行生化评估和X射线结构分析。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)。 与公共卫生相关：药物是理想的具有有益效果且副作用很少的化学物质，并且不存在药物/药物相互作用，即当它们共同作用导致药物失去效力时。在服用药物并发挥其有益作用后，它通常被肝脏中的细胞色素P450酶代谢，即化学改变，以帮助其消除。尽管P450酶通常会将药物转化为无害的代谢物，这些代谢物会在尿液中排出，但这些酶往往会通过一种名为脱氢的化学机制，将药物的结构改变为高度活性的有毒产物。脱氢产物经常是有毒的，通过使代谢药物的P450酶失活而导致药物/药物相互作用。然而，人们对脱氢过程知之甚少。因此，本研究的目标是准确地描述P450介导的脱氢的机制，以及已知通过这一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，当推出新药时应该避免。这一知识将显著改善制药行业未来的药物开发流程。

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.