Identification of small molecule inhibitors of protein disulfide isomerase

蛋白质二硫键异构酶小分子抑制剂的鉴定

• 批准号: 8135133
• 负责人: Robert C Flaumenhaft
• 金额: $ 4.35万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8135133
• 关键词: Agonist; Animal Model; Antibodies; Biological; Biological Assay; Blood Platelets; Blood Vessels; Cells; Cellular biology; Cerebrovascular Disorders; Chemicals; Chemistry; Collection; Coronary Arteriosclerosis; Development; Disulfides; ERp57; Endothelial Cells; Family; Fibrin; Flavonols; Fluorescence; Generations; Goals; Human; Infarction; Inhibitory Concentration 50; Injury; Institutes; Insulin; Isomerase; Laboratories; Lasers; Lead; Libraries; Mediating; Morbidity - disease rate; Myocardial Infarction; Oxidases; Oxidoreductase; Peripheral Vascular Diseases; Platelet Activation; Protein Disulfide Isomerase; Quercetin; Recurrence; Regulation; Role; Rutin; Screening Result; Screening procedure; Specificity; Stroke; Sulfhydryl Compounds; Surface; System; Testing; Thrombosis; Thrombus; Tissues; United States; Work; atherothrombosis; base; counterscreen; endoplasmic reticulum glycoprotein p72; extracellular; glutathione peroxidase; high throughput screening; improved; in vivo; inhibitor/antagonist; intravital microscopy; member; mortality; mouse model; prevent; programs; small molecule

DESCRIPTION (provided by applicant): Arterial thrombosis mediates tissue infarction in coronary artery disease, cerebrovascular disease, and peripheral vascular disease, and thus is the single most common cause of morbidity and mortality in the United States. The high rate of recurrence following heart attacks and strokes despite current therapies indicates a need to identify new targets with improved efficacy for inhibiting arterial thrombosis. We and others have determined that protein disulfide isomerase (PDI) is released onto the extracellular surface of endothelial cells and platelets following vascular injury and serves a critical role in thrombus formation. PDI is the founding member of a large family of oxidoreductases that catalyze posttranslational disulfide exchange. Antibodies directed at PDI inhibit arterial thrombosis in animal models. However, there are presently no known potent and selective small molecule inhibitors of PDI to test in these systems. In an effort to identify PDI inhibitors, we have developed an insulin-based turbidometric assay for high throughput screening. We have performed a pilot screen of 5000 compounds from the Known Bioactives Collection at the Institute of Chemistry and Cell Biology. This assay demonstrated a Z' factor of 0.89 and a coefficient of variance of 4.9%. Twenty new PDI inhibitors were identified during this pilot screen. Among them was the quercetin flavonol, rutin. Rutin inhibited PDI with an IC50 of approximately 1 <M and displayed specificity in inhibition of oxidoreductases, demonstrating inhibition of PDI but not ERp57. Studies using intravital microscopy showed that rutin inhibited thrombus formation following laser-induced vascular injury with an IC50 of <3.5 mg/kg. We propose to collaborate with the MLPCN to conduct a large-scale, high throughput screen for more potent and selective PDI inhibitors. Aim 1 of this project is to transfer the insulin-based turbidometric assay to the MLPCN collaborator, who will conduct a screen of 300,000-500,000 compounds. Aim 2 is to validate active compounds using a fluorescence-based assay of insulin aggregation, remove non-specific oxidase inhibitors using a glutathione peroxidase assay, and determine the specificity of inhibitors using alternative oxidoreductases, including ERp5, ERp46, ERp57, and ERp72, in the insulin-based turbidometric assay. Aim 3 is to use a platelet-based assay to identify biologically active compounds and determine reversibility of compounds that inhibit platelet activation. Active compounds will be tested in vivo in a mouse model of thrombus formation. These studies will establish PDI as a valid target for antithrombotic therapy and provide lead compounds for development of PDI-targeted antithrombotics. PUBLIC HEALTH RELEVANCE: Protein disulfide isomerase is absolutely required for thrombus formation in animal models of arterial thrombosis. We will develop inhibitors to protein disulfide isomerase to study the role of thiol isomerases in the regulation of thrombus formation and to interfere with atherothrombosis, which causes heart attacks and stroke and thus is the most prevalent cause of morbidity and mortality in the United States.

描述（由申请人提供）：动脉血栓形成介导冠状动脉疾病、脑血管疾病和外周血管疾病中的组织梗死，因此是美国发病率和死亡率的单一最常见原因。尽管有目前的治疗，但心脏病发作和中风后的高复发率表明需要鉴定具有改善的抑制动脉血栓形成功效的新靶标。我们和其他人已经确定，蛋白质二硫键异构酶（PDI）被释放到血管损伤后内皮细胞和血小板的细胞外表面，并在血栓形成中发挥关键作用。PDI是催化翻译后二硫键交换的氧化还原酶大家族的创始成员。针对PDI的抗体在动物模型中抑制动脉血栓形成。然而，目前还没有已知的有效的和选择性的小分子PDI抑制剂在这些系统中进行测试。为了鉴定PDI抑制剂，我们开发了一种用于高通量筛选的基于胰岛素的浊度测定法。我们已经从化学和细胞生物学研究所的已知生物活性物质库中对5000种化合物进行了初步筛选。该测定证明Z'因子为0.89，变异系数为4.9%。在该中试筛选期间鉴定了20种新的PDI抑制剂。其中有槲皮素黄酮醇，芦丁。罗格列酮抑制PDI的IC 50约为1 μ M，并在抑制氧化还原酶方面表现出特异性，表明抑制PDI但不抑制ERp 57。使用活体显微镜的研究表明，芦丁抑制激光诱导的血管损伤后的血栓形成，IC 50 <3.5 mg/kg。我们建议与MLPCN合作进行大规模，高通量筛选更有效和选择性的PDI抑制剂。该项目的目标1是将基于胰岛素的浊度测定法转移给MLPCN合作者，后者将对300，000 - 500，000种化合物进行筛选。目的2是使用基于荧光的胰岛素聚集测定法验证活性化合物，使用谷胱甘肽过氧化物酶测定法去除非特异性氧化酶抑制剂，并在基于胰岛素的浊度测定法中使用替代氧化还原酶（包括ERp 5、ERp 46、ERp 57和ERp 72）测定抑制剂的特异性。目的3是使用基于血小板的测定来鉴定生物活性化合物并确定抑制血小板活化的化合物的可逆性。将在血栓形成的小鼠模型中体内测试活性化合物。这些研究将确立PDI作为抗血栓治疗的有效靶点，并为开发PDI靶向抗血栓药物提供先导化合物。 公共卫生相关性：在动脉血栓形成的动物模型中，蛋白质二硫键异构酶是血栓形成所必需的。我们将开发蛋白质二硫键异构酶的抑制剂，以研究硫醇异构酶在血栓形成调节中的作用，并干扰动脉粥样硬化血栓形成，动脉粥样硬化血栓形成导致心脏病发作和中风，因此是美国发病率和死亡率最普遍的原因。