Screening for allosteric modulators of the protease activated receptor 4

描述（由申请人提供）：此资助是对PAR-12-058，高通量筛选以发现化学探针的试验征集的回应。血小板在血栓形成中起关键作用，血栓形成是死亡和发病的主要原因。凝血酶在神经创伤期间存在并激活蛋白酶激活受体，其引起局部氧化应激和炎症。PAR 4表达在这些条件下增加，这可能导致严重的神经退行性后遗症。急性冠状动脉综合征和脑血管损伤的药物治疗以抗血小板治疗为中心。目前可用的治疗，阿司匹林，氯吡格雷，和？联合调查局？3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效应，并与不良出血事件相关。凝血酶受体拮抗剂（TRA）在临床医学中备受期待，但靶向PAR 1的TRA Vorapaxar最近在III期临床试验中显示可增加临床显著出血，包括颅内出血。由于PAR 4对凝血酶的低亲和力，随着更多的凝血酶积聚，它在三维凝块的部位被局部激活。由于激活延迟，我们假设PAR 4拮抗作用可能不会有效影响止血，因此在血栓形成和脑血管损伤中可能是比PAR 1更好的治疗靶点。抑制PAR 4不会干扰通过PAR 1受体的信号传导，而PAR 1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PAR 4的抑制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PAR 4在生理环境中的作用的理解是有限的。为了解决这些问题，我们将1）筛选PAR 4的负变构调节剂/竞争性拮抗剂。这些化合物将避免来自试图置换以摩尔浓度存在的束缚配体的困难。我们将建立在范德比尔特在通过运行三重添加HTS筛选产生靶向GPCR的变构位点的新型配体方面的许多成功的基础上，2）我们将鉴定抑制凝血酶刺激PAR 4并且对内源性表达PAR 4的CHO细胞和人血小板中的PAR 4具有选择性的化合物，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，将具有时间和空间特异性的优势，成为有效的只有当凝血酶存在。

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.