P450-Mediated Dehydrogenation Mechanisms

描述（由申请人提供）：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个公认的过程。然而，最近才对几种 P450 酶（例如 1A2、2B4、2B6、2C9、2D6、2E1、2F1、2F3、2A13 和 3A4）通过脱氢途径产生亲电子中间体进行了研究，并且尚未建立控制选择性脱氢而不是氧化的机制。一些脱氢中间体的反应活性很高，通常会通过活性位点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记录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。迫切需要研究这些特定 P450 酶的催化行为及其脱氢而不是氧化底物的倾向。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，导致外源性介导的损伤和人类药物代谢的改变。该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位点和远程残基环境的特征，并定义调节选择性脱氢而不是氧合的底物结构特征。这些目标将通过以下目标实现：1）定义可接受的 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蛋白质加合物的鉴定评估三种原型底物的脱氢，来表征脱氢的化学和生物化学机制； 3) 利用基于量子力学的底物和中间体的集成模型以及 P450 酶的分子力学和分子动力学模拟来预测关键的脱氢特异性残基和底物反应性； 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，然后对纯化的天然酶和突变酶进行生化评估和 X 射线结构。本研究的长期目标是阐明细胞色素 P450 介导的异生物质在产生有毒亲电子中间体的过程中脱氢的机制，评估这些有毒中间体对人类健康造成的潜在危害，并利用机制信息来预测新药和异生物质的脱氢以及伴随的毒性和/或酶失活（改变药物代谢）。 公共健康相关性：药物是理想情况下具有有益作用且副作用很少的化学物质，并且不存在药物/药物相互作用，即它们共同作用导致药物失去功效。服用药物并产生有益作用后，通常会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并通过尿液排出，但这些酶经常通过一种称为脱氢的化学机制将药物的结构改变为高反应性的有毒产物。脱氢产物通常有毒，并通过使代谢药物的 P450 酶失活而引起药物/药物相互作用。然而，人们对脱氢过程知之甚少。因此，这项研究的目标是精确描述 P450 介导的脱氢机制，以及已知通过该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，并且在引入新药时应避免。这些知识将在未来显着改善制药行业的药物开发流程。

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.