Novel Peptide MPO Inhibitors for Treating Atherosclerosis

治疗动脉粥样硬化的新型肽 MPO 抑制剂

基本信息

批准号：
8208034
负责人：
Kirkwood Arthur Pritchard
金额：
$ 22.5万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8208034
关键词：
Alzheimer&apos 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

项目摘要

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.

项目概要该申请的长期目标是优化新型无毒髓过氧化物酶抑制剂的设计 (MPO) 抑制动脉粥样硬化 MPO 在炎症吞噬细胞中高表达，被认为具有抑制动脉粥样硬化的作用。在宿主防御机制中发挥重要作用，然而，吞噬细胞也会被氧化激活。血管壁中发现的生物分子在激活后会释放 MPO。并开始产生过氧化氢 (H2O2)，这是激活 MPO 所必需的。产生多种强效氧化剂和次级自由基，例如，活化的 MPO 与发生反应。氯化物 (Cl-) 生成次氯酸 (HOCl) 催化消耗一氧化氮 (¿NO) 和 MPO 又将其转化为亚硝酸盐 (NO2-)，氧化 NO2- 生成二氧化氮 (¿NO2)，这是一种自由基。 MPO 能够氧化脂质、蛋白质和 DNA，还可以氧化芳香族氨基酸，例如酪氨酸和 DNA。色氨酸产生细胞毒性的酪氨酰基和色氨酰基，正如 MPO 产生的那样，种类繁多。氧化剂和自由基会损害内皮依赖性血管舒张并加速动脉粥样硬化，开发有效、无毒的 MPO 抑制剂势在必行。在此应用中，我们建议设计。并开发新型 MPO 三肽竞争性抑制剂在目标 1 中，系统性氨基酸替代将实现。用于优化抑制剂设计。抑制剂对 MPO 活性的影响将在体外确定。系统使用纯化的 MPO 和从 MPO-/- 分离的巨噬细胞以及转基因人 MPO (Tg-h- MPO+/+)小鼠此外，将确定抑制剂对细胞毒性的影响。巨噬细胞的数量、细胞因子的产生和巨噬细胞泡沫细胞的形成有两个主要目的。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。目标 1 中确定。这些研究将在 C57BL/6J 小鼠中进行，将进行腹膜内注射。 (ip) 抑制剂的血浆水平将通过 HPLC 测定血浆 AST 和时间。 ALT 水平将用于监测肝功能，而组织学将用于监测药物的影响二、目的二：三肽对主要器官细胞结构的影响。 Aim 1 的抑制剂将改善嵌合 Ldlr-/-/MPO-/- 和 Ldlr-/- 的血管舒张并抑制动脉粥样硬化饲喂西方饮食的 Tg-h-MPO+/+ 小鼠将通过以下方式确定内皮依赖性血管舒张。将使用免疫荧光在组织学上确定动脉粥样硬化病变的形成。成功完成拟议的研究将导致开发出一种方法。新型无毒 MPO 抑制剂，可改善血管舒张并预防动脉粥样硬化。这里开发的 MPO 抑制剂也应该可用于治疗其他疾病的血管疾病和炎症疾病状态，如阿尔茨海默病、帕金森病、多发性硬化症、炎症性肠病 MPO 异常的疾病、肾脏疾病、类风湿性关节炎和慢性阻塞性肺疾病由于这些疾病状态代表了 NIH 的主要关注点，我们的研究结果表明。该提案将促进几个不同机构的研究和使命。