Novel Peptide MPO Inhibitors for Treating Atherosclerosis

描述(申请人提供)：本申请的长期目标是优化设计新型的、无毒的髓过氧化物酶(MPO)抑制剂来抑制动脉粥样硬化。MPO在炎性吞噬细胞中高度表达，被认为在宿主防御机制中发挥重要作用。然而，吞噬细胞也被管壁中发现的氧化生物分子激活。一旦激活，这些炎性吞噬细胞释放MPO并开始产生过氧化氢(H_2O_2)，这是激活MPO所必需的。一旦被激活，MPO就会产生各种各样的强氧化剂和次级自由基。例如，活化的MPO与氯(Cl-)反应生成次氯酸(HOCl)。MPO催化消耗一氧化氮(7NO)，并将其转化为亚硝酸盐(NO2-)。反过来，MPO氧化NO2-生成二氧化氮(7NO2)，这是一种能够氧化脂质、蛋白质和DNA的自由基。MPO还氧化酪氨酸和色氨酸等芳香族氨基酸，生成细胞毒性的酪氨酰和色氨酸自由基。由于MPO产生如此广泛的氧化剂和自由基，损害内皮依赖的血管扩张并加速动脉粥样硬化，开发有效的、无毒的MPO抑制剂势在必行。在这一应用中，我们建议设计和开发新型的MPO三肽竞争性抑制剂。在目标1中，将使用系统的氨基酸取代来优化抑制剂设计。这些抑制剂对MPO活性的影响将在体外系统中用纯化的MPO和从MPO-/-和转基因的人MPO(TG-h-MPO+/+)小鼠中分离的巨噬细胞来确定。此外，将根据巨噬细胞数量、细胞因子的产生和巨噬细胞泡沫细胞的形成来确定这些抑制剂对细胞毒性的影响。AIM 2有两个主要目标。首先，AIM 2将确定AIM 1中确定的最有效的MPO抑制剂的药代动力学和细胞毒性。这些研究将在C57BL/6J小鼠身上进行。小鼠将被腹腔注射(IP)，血浆中抑制物的水平将通过高效液相色谱(HPLC)与剂量和时间进行测定。血浆AST和ALT水平将被用来监测肝功能，而组织学将被用来监测三肽对主要器官细胞结构的影响。其次，AIM 2将确定来自AIM 1的三肽抑制剂是否会改善Ldlr-/-/MPO-/-和Ldlr-/-TG-h-MPO+/+嵌合小鼠的血管扩张和抑制动脉粥样硬化。内皮依赖性血管扩张将通过视频显微镜进行测定。动脉粥样硬化病变的形成将使用免疫荧光和共聚焦显微镜进行组织学测定。拟议研究的成功完成将导致开发一类新的无毒MPO抑制剂，以改善血管扩张和预防动脉粥样硬化。这里开发的优化的MPO抑制剂也应该有助于治疗其他疾病状态的血管疾病和炎症，如阿尔茨海默病、帕金森氏病、多发性硬化症、炎症性肠道疾病、肾脏疾病、类风湿性关节炎和慢性阻塞性肺疾病，这些疾病都与MPO活性异常有关。由于这些疾病状态代表了NIH的主要关注点，我们提案中的发现将推进几个不同研究所的研究和任务。 与公众健康相关：髓过氧化物酶产生氧化剂和自由基，在氧化血管中的脂质、蛋白质和DNA以增加心脏病的过程中发挥重要作用。这项应用的目标是开发无毒的髓过氧化物酶抑制剂，以抑制对血管的氧化损伤，以预防心脏病。

DESCRIPTION (provided by applicant): 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 (7NO) and converts it to nitrite (NO2-). In turn, MPO oxidizes NO2- to generate nitrogen dioxide (7NO2), 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. PUBLIC HEALTH RELEVANCE: Myeloperoxidase generates oxidants and free radicals that play important roles in oxidizing lipids, protein and DNA in blood vessels to increase heart disease. The goal of this application is to develop non-toxic inhibitors of myeloperoxidase to inhibit oxidative damage to blood vessels to prevent heart disease.