Screening for allosteric modulators of the protease activated receptor 4

筛选蛋白酶激活受体 4 的变构调节剂

基本信息

批准号：
8629339
负责人：
HEIDI E HAMM
金额：
$ 34.11万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8629339
关键词：
3-Dimensional Address Affect Affinity Allosteric Site Aspirin Attenuated Binding Sites Biological Assay Blood Platelets Cell Line Chemicals Chinese Hamster Ovary Cell Clinical Medicine Coagulation Process Coupled Developed Countries Drug or chemical Tissue Distribution Environment Event F2R gene G Protein-Coupled Receptor Genes GTP-Binding Proteins Generations Grant Hemorrhage Hemostatic function Human Inflammation Injury Integrins Intracranial Hemorrhages Lead Life Ligand Binding Ligands Link Measures Mediating Medical Morbidity - disease rate Nerve Degeneration Oxidative Stress P-Selectin PAR-1 Receptor PAWR gene Parents Pathologic Peptide Hydrolases Peptides Phase III Clinical Trials Physiological Platelet Activation Platelet aggregation Play Proteinase-Activated Receptors Purinoceptor Reading Recruitment Activity Role Running Shapes Signal Transduction Site Specificity Stroke Testing Thrombin Thrombin Receptor Thrombosis Thrombus Time United States Work acute coronary syndrome autocrine cerebrovascular clinically significant clopidogrel counterscreen design high throughput screening inhibitor/antagonist mortality novel protease-activated receptor 4 public health relevance receptor receptor coupling response screening success therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): This grant is in response to PAR-12-058, Solicitation of Assays for High Throughput Screening to Discover Chemical Probes. Platelets play a critical role in thrombosis, the major cause of mortality and morbidity. Thrombin is present during neurotrauma and activates protease activated receptors, which causes oxidative stress and inflammation locally. PAR4 expression is increased under these conditions, which could lead to serious neurodegenerative sequelae. Medical management of acute coronary syndrome and cerebrovascular injury is centered on anti-platelet therapies. Currently available therapies, aspirin, clopidogrel, and ?IIb?3 inhibitors, do not fully attenuate platelet activation, can have delayed onset and long lived effects, and are linked to adverse bleeding events. Thrombin receptor antagonists (TRAs) are highly awaited in clinical medicine but Vorapaxar, a TRA that targets PAR1, was recently shown in Phase III clinical trials to increase clinically significant bleeding including intracranial hemorrhage. Because of PAR4's low affinity for thrombin, it is activated locally at the site of the 3-dimensional clot as more thrombin builds up. Because of the delay in activation we hypothesize that PAR4 antagonism might not affect hemostasis as potently and thus may be a better therapeutic target than PAR1 in thrombosis and cerebrovascular injury. Inhibition of PAR4 would not perturb signaling through the PAR1 receptor, which is essential for basic hemostasis during injury. Our major hypothesis is that inhibition of PAR4 is a potential target for platelet inhibition in thrombosis and cerebrovascular injury. Due to the lack of tool compounds, the field's understanding of the role of PAR4 in physiological environments is limited. To address these questions, we will 1) screen for negative allosteric modulators/competitive antagonists of PAR4. These compounds will avoid the difficulty that comes from trying to displace the tethered ligand which is present at molar concentrations. We will build on Vanderbilt's many successes in generating novel ligands that target the allosteric sites of GPCRs by running a triple add HTS screen, 2) we will identify compounds which inhibit thrombin stimulation of PAR4 and are selective for PAR4 in CHO cells and human platelets which express PAR4 endogenously, 3) we will determine whether the mechanism of inhibition is competitive or non-competitive using Schild analysis. These studies should provide valuable tool compounds which would have advantages of temporal and spatial specificity, becoming efficacious only when thrombin is present.

描述（由申请人提供）：此项资助是为了响应 PAR-12-058，“高通量筛选发现化学探针的测定征集”。血小板在血栓形成中发挥着关键作用，血栓形成是死亡和发病的主要原因。凝血酶存在于神经创伤期间，并激活蛋白酶激活的受体，从而导致局部氧化应激和炎症。在这些条件下 PAR4 表达增加，可能导致严重的神经退行性后遗症。急性冠脉综合征和脑血管损伤的医疗治疗以抗血小板治疗为中心。目前可用的疗法，阿司匹林、氯吡格雷和IIb3抑制剂，不能完全减弱血小板活化，可能具有延迟起效和长期作用，并且与不良出血事件有关。凝血酶受体拮抗剂 (TRA) 在临床医学中备受期待，但 Vorapaxar（一种针对 PAR1 的 TRA）最近在 III 期临床试验中显示，可增加临床上显着的出血，包括颅内出血。由于 PAR4 对凝血酶的亲和力较低，因此随着更多凝血酶的积聚，它会在 3 维凝块部位被局部激活。由于激活延迟，我们推测 PAR4 拮抗作用可能不会对止血产生有效影响，因此在血栓形成和脑血管损伤中可能是比 PAR1 更好的治疗靶点。抑制 PAR4 不会干扰通过 PAR1 受体的信号传导，而 PAR1 受体对于损伤期间的基本止血至关重要。我们的主要假设是，抑制 PAR4 是血栓形成和脑血管损伤中血小板抑制的潜在靶点。由于缺乏工具化合物，该领域对 PAR4 在生理环境中的作用的了解有限。为了解决这些问题，我们将 1) 筛选 PAR4 的负变构调节剂/竞争性拮抗剂。这些化合物将避免因试图取代以摩尔浓度存在的束缚配体而产生的困难。我们将在范德比尔特公司通过运行三重添加 HTS 筛选生成靶向 GPCR 变构位点的新型配体方面取得的许多成功的基础上，2) 我们将鉴定出抑制 PAR4 凝血酶刺激并对 CHO 细胞和人血小板中的 PAR4 具有选择性的化合物，内源性表达 PAR4，3) 我们将使用 Schild 分析确定抑制机制是竞争性的还是非竞争性的。这些研究应该提供有价值的工具化合物，其具有时间和空间特异性的优势，只有在凝血酶存在时才有效。