Novel pathways in eicosanoid biosynthesis and metabolism

摘要 花生四烯酸通过环氧合酶、脂氧合酶和细胞色素P450的氧化转化， 产生调节体内平衡和疾病中细胞过程的内源性脂质介质。这些脂质 介体形成称为类二十烷酸的进化和扩展的家族。该应用程序包括两个 这两个项目都涉及类花生酸生物化学的新转变， 用于抑制其生物合成的非甾体抗炎药（NSAID）的衍生物。的 第一个项目是围绕5-LOX/考克斯-2交叉生物合成途径，而第二个项目是 关注类二十烷酸的新代谢转化，以及这些对血浆的使用意味着什么。 和尿前列腺素类代谢物作为药物反应的标志物。今天，类花生酸的新成员 在脂质组学方法中，经常通过它们与已知的类二十烷酸的相似性来发现家族。我们有 采用了一种基于对生物合成途径，酶， 和基质，使我们能够预测新的转变。这导致了 5-LOX/考克斯-2交叉生物合成途径形成半缩酮类二十烷酸（HKE 2和HKD 2）和5- 羟基-甘草素，由阿司匹林-乙酰化考克斯-2形成的15 R-甘草素的鉴定， 鉴定PGD2代谢为11-β-D-葡萄糖的Baeyer-Villiger氧化途径。 血栓素类强调我们方法的相关性是，我们拥有的新类花生酸 在脂质组学分析中尚未鉴定出在体外和体内形成，可能是由于它们的 不寻常的属性，使他们难以检测在标准分析。我们设计了分析 允许在体外和体内检测和定量新的类二十烷酸的方法。我们计划继续 鉴定新类二十烷酸并建立它们的细胞靶点和在体内平衡中的生物学作用， 在炎症性疾病的模型中。我们正在进行的交叉感染的生物学效应的调查 类花生酸已经将受体酪氨酸激酶（RTK）VEGFR2鉴定为促血管生成的靶点。 HKE2的活性，以及其他RTK和介导血小板活化抑制的未知靶点， 有待进一步分析。对于5-羟基-甘草素，我们计划采用筛选方法，以及 作为前列腺素类受体的靶向测试，以鉴定其细胞靶点并确定生物学活性。 方面的影响.我们的目标是鉴定可用于操纵5-LOX/考克斯-2交叉酶的生物合成的化合物， 相对于类花生酸，独立于类花生酸和白三烯的形成。我们将继续 表征前列腺素类化合物的新代谢途径，并建立这些途径在体内的相关性。 新型类二十烷酸的鉴定和表征、其生物学效应以及新型前列腺素代谢 途径将导致对NSAID可能适用的病理生理条件的精确理解。 阿司匹林和其他非甾体类抗炎药在体内对前列腺素类生物合成的作用。

Abstract Oxidative transformation of arachidonic acid by cyclooxygenases, lipoxygenases, and cytochromes P450 gives rise to endogenous lipid mediators that regulate cellular processes in homeostasis and disease. These lipid mediators form an evolving and expanding family referred to as eicosanoids. This application comprises two projects that both are concerned with novel transformations in eicosanoid biochemistry and present important ramifications for the use of non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit their biosynthesis. The first project is centered around the 5-LOX/COX-2 cross-over biosynthetic pathway while the second project is concerned with novel metabolic transformations of eicosanoids, and what these mean for the use of plasma and urinary prostanoid metabolites as markers of drug response. Today, novel members of the eicosanoid family are often discovered in lipidomics approaches by their similarity with known eicosanoids. We have employed an approach based on understanding the structure-function of the biosynthetic pathways, enzymes, and substrates that allowed us to make predictions of novel transformations. This has led to the discovery of the 5-LOX/COX-2 cross-over biosynthetic pathway forming hemiketal eicosanoids (HKE2 and HKD2) and 5- hydroxy-prostaglandins, the identification of 15R-prostaglandins formed by aspirin-acetylated COX-2, and the identification of the Baeyer-Villiger oxidative pathway underlying the metabolism of PGD2 to 11-dehydro- thromboxanes. Underscoring the relevance of our approach is the fact that the novel eicosanoids we have shown to be formed in vitro and in vivo have not been identified in lipidomics analyses, likely due to their unusual properties that make them difficult to detect in standard analyses. We have designed analytical procedures that allow to detect and quantify the novel eicosanoids in vitro and in vivo. We plan to continue the identification of novel eicosanoids and to establish their cellular targets and biological role in homeostasis and in models of inflammatory disease. Our ongoing investigation into the biological effects of the cross-over eicosanoids has identified the receptor tyrosine kinase (RTK) VEGFR2 as a target for the pro-angiogenic activity of HKE2, as well as other RTK and an unknown target that mediates inhibition of platelet activation that are to be further analyzed. For the 5-hydroxy-prostaglandins we plan to employ screening approaches as well as targeted testing of prostanoid receptors in order to identify their cellular targets and determine biological effects. We aim to identify compounds that can be used to manipulate biosynthesis of 5-LOX/COX-2 cross- over eicosanoids independent from the formation of prostaglandins and leukotrienes. We will continue to characterize novel metabolic pathways of prostanoids and establish the relevance of these pathways in vivo. Identification and characterization of novel eicosanoids, their biological effects, and novel prostanoid metabolic pathways will result in a refined understanding of the pathophysiologic conditions for which NSAIDs may be used and effect of aspirin and other NSAIDs on prostanoid biosynthesis in vivo.