Optimization of PAR4 antagonists for thrombotic disorders

描述（由申请人提供）：这项拨款是为了响应PAR-12-060，征求体内化学探针发现的验证命中值。血小板在血栓形成中起关键作用，血栓形成是死亡率和发病率的主要原因。急性冠状动脉综合征和脑血管损伤的医疗管理以抗血小板治疗为中心。凝血酶受体拮抗剂（TRAs）在临床医学中备受期待，但最近在III期临床试验中显示，针对PAR1的TRA Vorapaxar可增加包括颅内出血在内的临床显著性出血。PAR4是抗血小板治疗血栓和脑血管损伤的一个有吸引力的靶点。由于PAR4对凝血酶的亲和力较低，当更多的凝血酶积聚时，它在3D凝块的局部位置被激活。由于PAR4激活的延迟，我们假设PAR4拮抗剂可能不会像PAR1那样有效地影响止血，因此可能是比PAR1更好的治疗靶点。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。然而，目前还没有有效的体内PAR4拮抗剂来研究PAR4在血栓形成、缺血性卒中和神经退行性疾病中的作用。由于缺乏工具化合物，该领域对PAR4在生理环境中的作用的理解有限。PAR4的抑制不会干扰通过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。PAR4拮抗剂YD-3具有较差的物理化学性质，在可接受的载体中几乎没有溶解度；因此，它不适合体内研究。我们建议优化YD- 3及其相关分子的溶解度、效价和选择性，以便更好地了解PAR4在血栓形成和止血中的作用。我们建议1)使用文库方法优化PAR4的小分子拮抗剂，2)确定优化后的化合物对PAR4的选择性，3)使用体外和体内DMPK技术确定新型PAR4拮抗剂的代谢和处置，4)在血栓形成模型中测试优化后的化合物。优化后的化合物将使我们能够确定PAR4的基本药理学信息，包括受体密度、PAR4拮抗剂和激动剂的作用机制以及对体内血栓模型的影响，这些数据目前还无法收集到。这些研究应该提供一个有价值的工具化合物，这将使研究可能揭示PAR4作为预防和治疗抑制病理性血栓形成的替代靶点。

DESCRIPTION (provided by applicant): This grant is in response to PAR-12-060, Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes. Platelets play a critical role in thrombosis, the major cause of mortality and morbidity. Medical management of acute coronary syndrome and cerebrovascular injury is centered on anti-platelet therapies. 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. PAR4 is an attractive target for anti-platelet therapy in thrombosis and cerebrovascular injury. Because of PAR4's low affinity for thrombin, it is activated locally at the site of the 3D clot as more thrombin builds up. Because of this delay in activation of PAR4, we hypothesize that PAR4 antagonism might not affect hemostasis as potently and thus may be a better therapeutic target than PAR1. Our major hypothesis is that inhibition of PAR4 is a potential target for platelet inhibition in thrombosis and cerebrovascular injury. However, there are no PAR4 antagonists that are efficacious in vivo to study the role of PAR4 in thrombosis, ischemic stroke and neurodegeneration. Due to the lack of tool compounds, the field's understanding of the role of PAR4 in physiological environments is limited. Inhibition of PAR4 would not perturb signaling through the PAR1 receptor, which is essential for basic hemostasis during injury. The PAR4 antagonist YD-3 has poor physiochemical properties and virtually no solubility in acceptable vehicles; therefore, it is not suitable for in vivo studies. We propose to optimize solubility, potency, and selectivity of YD- 3 and related molecules in order to better understand the role of PAR4 in thrombosis and hemostasis. We propose to 1) optimize a small molecule antagonist of PAR4 using a library approach, 2) determine selectivity of optimized compounds for PAR4, 3) determine the metabolism and disposition of novel PAR4 antagonists using in vitro and in vivo DMPK techniques, and 4) test the optimized compound in models of thrombosis. An optimized compound will allow us to determine basic pharmacological information about PAR4 including receptor density, mechanism of action for PAR4 antagonists and agonists, and effects on in vivo models of thrombosis, data which currently cannot be collected. These studies should provide a valuable tool compound which will enable studies that may reveal PAR4 as an alternative target for preventive and therapeutic suppression of pathologic thrombus formation.