Identification and characterization of novel antithrombotic PDI inhibitors

新型抗血栓 PDI 抑制剂的鉴定和表征

• 批准号: 8656769
• 负责人: Bruce Furie
• 金额: $ 45.2万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8656769
• 关键词: Antibodies; Binding; Binding Proteins; Biological Assay; Biological Availability; Blood Platelets; Blood coagulation; Cell physiology; Cells; Characteristics; Cytoplasmic Granules; Deep Vein Thrombosis; Development; ERp57; Endothelial Cells; Evaluation; Fibrin; Flavonoids; Goals; Hemostatic function; Injury; Instruction; Integrins; Isomerase; Knowledge; Laser injury; Lasers; Lead; Mediating; Modeling; Modification; Molecular Chaperones; Mus; Myocardial Infarction; NMR Spectroscopy; Oxidoreductase; Permeability; Plasma; Platelet Activation; Platelet aggregation; Protein Disulfide Isomerase; Pulmonary Embolism; Role; Rutin; Site; Solubility; Stroke; Structure-Activity Relationship; Testing; Thioredoxin; Thromboembolism; Thromboplastin; Thrombosis; Thrombus; United States; Venous; base; cytotoxicity; dietary supplements; endoplasmic reticulum glycoprotein p72; high throughput screening; improved; in vivo; inhibitor/antagonist; intravital microscopy; mortality; mouse model; novel; prevent; small molecule; therapy development

PROJECT SUMMARY (See instructions): Inhibition of protein disulfide isomerase (PDI) using antibodies prevents both platelet accumulation and fibrin formation in murine models of thrombus formation. This observation indicates that inhibition of PDI could represent a viable strategy for control of pathological thrombus formation. However, potent, selective small molecule inhibitors to test this hypothesis are not presently available. We have begun high throughput screening to identify compounds that inhibit PDI. A preliminary screen of ~5000 compounds identified PDI inhibitors with a hit rate of 0.3%. Among the active compounds were several flavonoids, including the widely used nutritional supplement quercetin-3-rutinoside. Quercetin-3-rutinoside was markedly antithrombotic in murine models. The fact that this PDI inhibitor is well-tolerated and potently antithrombotic in vivo supports the feasibility of inhibition of PDI for antithrombotic therapy. However, more selective, potent compounds with improved bioavailability are required. We will perform a large scale high throughput screen to identify novel PDI inhibitors. The objective of this project is to characterize a set of potent and selective PDI inhibitors as probes to study the role of PDI in thrombus formation and identify lead compounds that could be developed as antithrombotics. PDI demonstrates multiple functions in the vasculature including oxidoreductase/isomerase, chaperone, and denitrosation activities. In Aim 1, we will characterize PDI inhibitors on the basis of their ability to block these different activities. Studies performed in Aim 2 will use NMR spectroscopy to determine the structural basis of PDI inhibitor activity. The effect of PDI inhibitors on platelet activation and endothelial cell function will be detennined in Aim 3. Select compounds will then be tested for their inhibitory activity in a mouse model of thrombus formation using intravital microscopy (Aim 4). Evaluation of PDI inhibitors in enzymatic and cell-based assays will enable the identification of characteristics that are essential for the antithrombotic activity of PDI inhibitors. Such information will be critical for further development of PDI inhibitors as a novel class of antithrombotics.

项目摘要（参见说明）： 在血栓形成的小鼠模型中，使用抗体抑制蛋白质二硫键异构酶 (PDI) 可防止血小板积聚和纤维蛋白形成。这一观察结果表明，抑制 PDI 可能是控制病理性血栓形成的可行策略。然而，目前还没有有效的、选择性的小分子抑制剂来检验这一假设。我们已经开始高吞吐量 筛选以确定抑制 PDI 的化合物。对约 5000 种化合物进行初步筛选，鉴定出 PDI 抑制剂，命中率为 0.3%。活性化合物中有多种黄酮类化合物，包括广泛使用的营养补充剂槲皮素-3-芸香苷。 Quercetin-3-rutinoside 在小鼠模型中具有显着的抗血栓作用。这种 PDI 抑制剂具有良好的耐受性和体内有效的抗血栓作用，这一事实支持了抑制 PDI 用于抗血栓治疗的可行性。然而，需要更具选择性、更有效且具有改善的生物利用度的化合物。我们将进行大规模高通量筛选来鉴定新型 PDI 抑制剂。该项目的目标是表征一组有效且选择性的 PDI 抑制剂： 研究 PDI 在血栓形成中的作用并鉴定可开发为抗血栓药物的先导化合物的探针。 PDI 在脉管系统中表现出多种功能，包括氧化还原酶/异构酶、分子伴侣和脱亚硝化活性。在目标 1 中，我们将根据 PDI 抑制剂阻断这些不同活性的能力来表征它们。目标 2 中进行的研究将使用 NMR 光谱来确定 PDI 抑制剂活性的结构基础。 PDI抑制剂的作用 血小板活化和内皮细胞功能将在目标 3 中确定。然后将使用活体显微镜（目标 4）在血栓形成小鼠模型中测试选定化合物的抑制活性。 在酶法和细胞分析中对 PDI 抑制剂进行评估将能够识别 PDI 抑制剂抗血栓活性所必需的特征。这些信息对于 PDI 抑制剂作为一类新型抗血栓药物的进一步开发至关重要。