Identification of small molecule inhibitors of protein disulfide isomerase

描述（由申请人提供）：动脉血栓形成介导冠状动脉疾病、脑血管疾病和周围血管疾病的组织梗死，因此是美国发病率和死亡率的最常见原因。尽管目前的治疗方法，心脏病发作和中风后的高复发率表明需要确定新的靶点，以提高抑制动脉血栓形成的疗效。我们和其他人已经确定，在血管损伤后，蛋白二硫异构酶（PDI）被释放到内皮细胞和血小板的细胞外表面，并在血栓形成中起关键作用。PDI是催化翻译后二硫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和选择性的PDI小分子抑制剂在这些系统中进行测试。为了鉴定PDI抑制剂，我们开发了一种基于胰岛素的浊度测定法，用于高通量筛选。我们已经从化学和细胞生物学研究所的已知生物活性收集中对5000种化合物进行了试点筛选。Z′因子为0.89，方差系数为4.9%。在这个中试筛选过程中，发现了20种新的PDI抑制剂。其中有槲皮素、黄酮醇、芦丁。芦丁抑制PDI的IC50值约为1 <M，对氧化还原酶有特异性抑制，对PDI有抑制作用，但对ERp57无抑制作用。活体显微镜研究显示，芦丁抑制激光血管损伤后血栓形成的IC50 <3.5 mg/kg。我们建议与MLPCN合作，进行大规模、高通量筛选，以寻找更有效、更有选择性的PDI抑制剂。该项目的目标1是将基于胰岛素的浊度测定转移给MLPCN的合作者，后者将进行30万至50万种化合物的筛选。目的2是使用基于胰岛素聚集的荧光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ERp5， ERp46， ERp57和ERp72）确定抑制剂的特异性。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。活性化合物将在血栓形成的小鼠模型中进行体内测试。这些研究将确立PDI作为抗血栓治疗的有效靶点，并为PDI靶向抗血栓药物的开发提供先导化合物。

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.