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

• 批准号: 8742012
• 负责人: HEIDI E HAMM
• 金额: $ 33.97万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 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/8742012
• 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

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(申请人提供)：这项资助是为了响应PAR-12-058，高通量筛选测试征集，以发现化学探针。血小板在血栓形成中起着关键作用，血栓是死亡和发病的主要原因。凝血酶存在于神经创伤中，并激活蛋白酶激活的受体，从而导致局部氧化应激和炎症。在这些情况下，PAR4的表达增加，这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处理以抗血小板治疗为中心。目前可用的治疗方法，阿司匹林、氯吡格雷和IIB？3抑制剂，不能完全减弱血小板的激活，可能具有延迟发病和长期效应，并与不良出血事件有关。凝血酶受体拮抗剂(Tras)在临床医学中被高度期待，但Vorapaxar，一种针对PAR1的TRA，最近在第三阶段临床试验中被证明可以增加临床上的重大出血，包括颅内出血。由于PAR4‘S对凝血酶的亲和力低，当更多的凝血酶积聚时，它会在三维血栓的部位被局部激活。由于激活的延迟，我们推测PAR4拮抗剂可能不会有效地影响止血，因此可能是比PAR1更好的治疗血栓和脑血管损伤的靶点。抑制PAR4不会干扰通过PAR1受体的信号，而PAR1受体是损伤过程中基本止血所必需的。我们的主要假设是，抑制PAR4是血栓形成和脑血管损伤中抑制血小板的一个潜在靶点。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。这些化合物将避免试图取代摩尔浓度的束缚配体所带来的困难。我们将在Vanderbilt的许多成功的基础上，通过运行三重ADD HTS筛选来生成针对GPCRs变构位点的新型配体；2)我们将在CHO细胞和内源性表达PAR4的人血小板中鉴定抑制凝血酶刺激PAR4并对PAR4具有选择性的化合物；3)我们将使用SChild分析来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，它们具有时间和空间特异性的优势，只有在凝血酶存在的情况下才有效。