Novel Peptide MPO Inhibitors for Treating Atherosclerosis

项目摘要 这项申请的长期目标是优化设计新型的、无毒的髓过氧化物酶抑制剂 (MPO)抑制动脉粥样硬化。MPO在炎性吞噬细胞中高度表达， 在宿主防御机制中发挥重要作用。然而，吞噬细胞也被氧化 血管壁中的生物分子。一旦激活，这些炎性吞噬细胞释放MPO 并且开始产生活化MPO所需的过氧化氢（H2 O2）。一旦启动，MPO 产生各种各样的强氧化剂和次级自由基。例如，激活的MPO与 氯化物（Cl-）生成次氯酸（HOCl）。MPO催化消耗一氧化氮（NO）， 将其转化为亚硝酸盐（NO2-）。反过来，MPO氧化NO2-产生二氧化氮（NO2），这是一种自由基， 能够氧化脂质蛋白质和DNA MPO还氧化芳香族氨基酸，如酪氨酸， 色氨酸以产生细胞毒性酪氨酰和色氨酸自由基。由于MPO产生了如此广泛的各种 氧化剂和自由基，损害内皮依赖性血管舒张和加速动脉粥样硬化， 迫切需要开发有效、无毒的MPO抑制剂。在本申请中，我们建议设计 开发新型MPO三肽竞争性抑制剂。在目标1中，系统性氨基酸取代将 用于优化抑制剂设计。将在体外测定抑制剂对MPO活性的影响 使用纯化的MPO和从MPO-/-和转基因人MPO（Tg-h-）分离的巨噬细胞的系统 MPO+/+）小鼠。此外，抑制剂对细胞毒性的影响将根据以下方面来确定： 巨噬细胞数量、细胞因子产生和巨噬细胞泡沫细胞形成。目标2有两个主要目标 目标.首先，目标2将确定最有效的MPO抑制剂的药代动力学和细胞毒性 在目标1中确定。这些研究将在C57 BL/6 J小鼠中进行。小鼠将腹腔注射 (ip)通过HPLC测定抑制剂的血浆水平与剂量和时间的关系。血浆AST和 ALT水平将用于监测肝功能，而组织学将用于监测 三肽对主要器官细胞结构的影响。第二，目标2将确定三肽是否 来自Aim 1的抑制剂将改善嵌合Ldlr-/-/MPO-/-和Ldlr-/-中的血管舒张并抑制动脉粥样硬化。 喂食西方饮食的Tg-h-MPO+/+小鼠。内皮依赖性血管舒张将通过 视频显微镜将使用免疫荧光法在组织学上确定动脉粥样硬化病变形成 和共聚焦显微镜。成功完成拟议的研究将导致制定一个 新型无毒MPO抑制剂，可改善血管舒张并预防动脉粥样硬化。优化 在此开发的MPO抑制剂也应该可用于治疗血管疾病和其他炎症。 疾病状态如阿尔茨海默病、帕金森病、多发性硬化症、炎症性肠 疾病，肾脏疾病，类风湿性关节炎和慢性阻塞性肺疾病，其中异常MPO 活动受到牵连。由于这些疾病状态代表了NIH的主要关注点，我们的研究结果 该提案将推动几个不同研究所的研究和使命。

Project Summary The long-term goal of this application is to optimize the design of novel, non-toxic inhibitors of myeloperoxidase (MPO) to inhibit atherosclerosis. MPO is highly expressed in inflammatory phagocytes and is considered to play an important role in host defense mechanisms. However, phagocytes are also activated by oxidized biomolecules that are found in the vessel wall. Upon activation these inflammatory phagocytes release MPO and begin to generate hydrogen peroxide (H2O2), which is required to activate MPO. Once activated, MPO generates a wide variety of potent oxidants and secondary radicals. For example, activated MPO reacts with chloride (Cl-) to generate hypochlorous acid (HOCl). MPO catalytically consumes nitric oxide (¿NO) and converts it to nitrite (NO2-). In turn, MPO oxidizes NO2- to generate nitrogen dioxide (¿NO2), a radical that is capable of oxidizing lipids, proteins and DNA. MPO also oxidizes aromatic amino acids such as tyrosine and tryptophan to generate cytotoxic tyrosyl and tryptophanyl radicals. As MPO generates such a wide variety of oxidants and radicals that impair endothelial-dependent vasodilatation and accelerate atherosclerosis, it is imperative that effective, non-toxic inhibitors of MPO be developed. In this application, we propose to design and develop novel tripeptide competitive inhibitors of MPO. In Aim 1, systematic amino acid substitutions will be used to optimize inhibitor design. Effects of the inhibitors on MPO activity will be determined in in vitro systems using both purified MPO and macrophages isolated from MPO-/- and transgenic human MPO (Tg-h- MPO+/+) mice. Further, effects of the inhibitors on cellular cytotoxicity will be determined with respect to macrophage cell number, cytokine production and macrophage foam cell formation. Aim 2 has two major goals. First, Aim 2 will determine the pharmacokinetics and cytotoxicity of the most effective MPO inhibitors identified in Aim 1. These studies will be performed in C57BL/6J mice. Mice will be injected intraperitoneally (ip) and plasma levels of inhibitor will be determined by HPLC with respect to dose and time. Plasma AST and ALT levels will be used to monitor liver function while histology will be used to monitor the effects of the tripeptides on cell structure of the major organs. Second, Aim 2 will determine whether the tripeptide inhibitors from Aim 1 will improve vasodilatation and inhibit atherosclerosis in chimeric Ldlr-/-/MPO-/- and Ldlr-/- Tg-h-MPO+/+ mice fed western diet. Endothelium-dependent vasodilatation will be determined by videomicroscopy. Atherosclerotic lesion formation will be determined histologically using immunofluorescence and confocal microscopy. Successful completion of the proposed studies will result in the development of a new class of non-toxic MPO inhibitors that improve vasodilatation and prevent atherosclerosis. The optimized MPO inhibitors developed here should also be useful for treating vascular disease and inflammation in other disease states such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, inflammatory bowel disease, kidney disease, rheumatoid arthritis and chronic obstructive pulmonary disease, where aberrant MPO activity has been implicated. As these disease states represent a major focus of the NIH, findings from our proposal will advance the research and mission of several different institutes.