Novel Peptide MPO Inhibitors for Treating Atherosclerosis

治疗动脉粥样硬化的新型肽 MPO 抑制剂

• 批准号: 8046699
• 负责人: Kirkwood Arthur Pritchard
• 金额: $ 18.75万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8046699
• 关键词: Alzheimer' s Disease; Amides; Amino Acid Substitution; Amino Acids; Aromatic Amino Acids; Arterial Fatty Streak; Asthma; Atherosclerosis; Binding; Biochemistry; Blood Circulation; Blood Vessels; Bone Marrow Transplantation; Cardiovascular Diseases; Cell Count; Cell-Mediated Cytolysis; Cellular Structures; Chloride Ion; Chlorides; Chronic Obstructive Airway Disease; Complex; Confocal Microscopy; Cysteine; DNA; Development; Diet; Disease; Disulfides; Dose; Drug Kinetics; Electron Transport; Electrons; Endothelium; Foam Cells; Free Radicals; Generations; Glutathione; Goals; Heart Diseases; Heme; High Pressure Liquid Chromatography; Histologic; Histology; Host Defense Mechanism; Human; Hydrogen Peroxide; Hypochlorous Acid; Immunofluorescence Microscopy; In Vitro; Inflammatory; Inflammatory Bowel Diseases; Institutes; Kidney Diseases; Knowledge; Lesion; Low Density Lipoprotein oxidation; Lupus; Lysine; Mediating; Mediator of activation protein; Medical; Melatonin; Mission; Mitochondria; Modeling; Monitor; Multiple Sclerosis; Mus; Myocardial Infarction; Natural regeneration; Nitric Oxide; Nitrites; Nitrogen Dioxide; Organ; Oxidants; Parkinson Disease; Peptides; Peroxidases; Phagocytes; Plasma; Play; Positioning Attribute; Production; Proteins; Reporting; Research; Respiration; Rheumatoid Arthritis; Role; Sickle Cell Anemia; Structure; Sulfhydryl Compounds; System; Testing; Time; Toxic effect; Transgenic Organisms; Tryptophan; Tube; Tyrosine; United States National Institutes of Health; Vascular Diseases; Vasodilation; Ventricular Remodeling; Video Microscopy; base; cytokine; cytotoxic; cytotoxicity; design; feeding; improved; in vitro Assay; in vitro activity; in vivo; inhibitor/antagonist; insight; liver function; macrophage; monocyte; monomer; neutrophil; novel; oxidation; oxidative damage; oxidized lipid; oxidized low density lipoprotein; prevent; suicide inhibitor; vascular inflammation

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.

描述（由申请人提供）：本申请的长期目标是优化新型无毒髓过氧化物酶（MPO）抑制剂的设计，以抑制动脉粥样硬化。MPO在炎症吞噬细胞中高度表达，被认为在宿主防御机制中起重要作用。然而，吞噬细胞也被血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（H2O2），这是激活MPO所需的。一旦激活，MPO产生各种各样的强效氧化剂和次级自由基。例如，活化的MPO与氯化物（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-）。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还氧化芳香族氨基酸如酪氨酸和色氨酸以产生细胞毒性酪氨酰基和色氨酸基。由于MPO会产生多种氧化剂和自由基，从而损害内皮依赖性血管舒张并加速动脉粥样硬化，因此开发有效、无毒的MPO抑制剂势在必行。在本申请中，我们提出设计和开发新的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。将使用纯化的MPO和从MPO-/-和转基因人MPO（Tg-h-MPO +/+）小鼠分离的巨噬细胞在体外系统中测定抑制剂对MPO活性的影响。此外，将根据巨噬细胞数量、细胞因子产生和巨噬细胞泡沫细胞形成来确定抑制剂对细胞毒性的影响。目标2有两个主要目标。首先，目标2将确定目标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C57 BL/6J小鼠中进行。将对小鼠进行腹膜内（ip）注射，并通过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将确定来自目标1的三肽抑制剂是否将改善喂食西方饮食的嵌合Ldlr-/-/MPO-/-和Ldlr-/-Tg-h-MPO +/+小鼠中的血管舒张并抑制动脉粥样硬化。将通过视频显微镜确定内皮依赖性血管舒张。将使用免疫荧光和共聚焦显微镜在组织学上确定动脉粥样硬化病变形成。成功完成拟议的研究将导致一类新的无毒MPO抑制剂的开发，改善血管舒张和预防动脉粥样硬化。在此开发的优化的MPO抑制剂还应该可用于治疗血管疾病和其他疾病状态中的炎症，例如阿尔茨海默病、帕金森病、多发性硬化症、炎性肠病、肾病、类风湿性关节炎和慢性阻塞性肺病，其中异常的MPO活性已经被牵连。由于这些疾病状态代表了NIH的一个主要焦点，我们提案的发现将推动几个不同研究所的研究和使命。 公共卫生相关性：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中起重要作用，从而增加心脏病。本申请的目的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，从而预防心脏病。