Identification of small molecule inhibitors of protein disulfide isomerase

Beth Israel Deaconess Medical Center, Boston MA

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Abstract

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.