Discovery of Potent 12-Lipoxygenase Inhibitors of Platelet Activation

描述（由申请人提供）：本研究的主要目的是开发选择性/有效的人12-LOX抑制剂，并探索血小板活化中炎症的生物化学，从而获得治疗心血管疾病的候选药物。最近的失败，在测试中的抗凝剂强调了需要新的方法来治疗急性凝血。在这些新的靶点中，12-脂氧合酶（12-LOX）由于许多原因而突出：1）相对于循环中的其他细胞，它在血小板中高度表达，2）我们先前已经表明12-LOX对于止血和血栓形成都是重要的，以及3）12-LOX在人和小鼠的血小板中表达。目前的建议的广泛目标是推进一个或多个候选分子对血小板活化/聚集的治疗发展，利用我们的测定，以确定新的选择性抑制剂的人12-LOX。我们已经与NIH合作成功进行了12-LOX高通量（HTP）筛选，发现了超过1000个潜在的命中。在这约1000种化合物中，发现7种是有效的12-LOX抑制剂。这七种抑制剂中的两种被重新合成，并显示出在体外对人12-LOX是有效的和选择性的，并且在许多人血小板细胞测定中是有效的，证实它们是“经验证的命中”。我们的研究计划以以下四个具体目标为指导。首先，我们建议优化我们的两个“验证命中”，并发现额外的化学型，这对人类12-LOX的效力增加。其次，我们将优化我们的“命中”的ADME/PK特性，保持LOX同工酶的选择性，并确定其抑制机制。第三，一旦我们优化了我们的各种抑制剂化学型，我们将评估它们在各种血小板活化筛选中的保护效力。我们将在我们的血小板模型中利用这些体外抑制剂，并确定单个抑制剂是否减少聚集，以及确定我们的抑制剂是否减少12- HETE的生化产生，作为12-LOX抑制的直接标志物。第四，我们将评估这些12-LOX抑制剂对抑制小鼠血栓形成同时保持正常止血水平的体内功效。我们将通过评估用12-LOX抑制剂处理的小鼠的12-HETE产生和离体血小板功能来进一步确认12-LOX抑制剂的体内活性。这些抑制剂还将检测其预防肝素诱导的血小板减少症（HIT）的能力，因为最近的研究表明HIT以12-LOX依赖性方式调节。我们相信这些研究将发现一种12-LOX抑制剂，它可能是一种有效的治疗血小板聚集的药物，在许多病理环境中，包括动脉粥样硬化血栓形成、静脉血栓形成和HIT，并帮助我们更好地了解心血管疾病的生物学，心血管疾病是一种随年龄增长而改变的病理生理状况。

DESCRIPTION (provided by applicant): The principle aim of this research is to develop selective/potent human 12-LOX inhibitors and probe the biochemistry of inflammation in platelet activation, leading to drug candidates for cardiovascular disease. Recent failures in testing of anti-coagulant agents have highlighted the need for new approaches to acute blood coagulation treatment. Among the novel targets, 12-lipoxygenase (12-LOX) stands out for numerous reasons: 1) It is highly expressed in platelets relative to other cells in circulation, 2) we have previously shown 12-LOX to be important for both hemostasis and thrombosis, and 3) 12-LOX is expressed in the platelets of both human and mice. The broad objective of the current proposal is to advance one or more candidate molecules toward therapeutic development against platelet activation/aggregation, utilizing our assays to identify novel selective inhibitor for the human 12-LOX. We have already performed a successful 12-LOX high-throughput (HTP) screen in collaboration with the NIH, which found over ~1000 potential hits. Of these ~1000 compounds, seven were found to be potent 12-LOX inhibitors. Two of these seven inhibitors were re- synthesized and shown to be potent and selective against human 12-LOX in vitro and effective in numerous human platelet cell assays, confirming them as "validated hits". Our research plan is guided by the following four specific aims. First, we propose to optimize our two "validated hits" and discover additional chemotypes, which have increased potency against human 12-LOX. Second, we shall optimize the ADME/PK properties of our "hits", maintain LOX isozyme selectivity and determine their mechanism of inhibition. Third, once we have optimized our various inhibitor chemotypes, we shall evaluate their protective potency in a variety of platelet activation screens. We will utilize these in vitro inhibitors in our platelet models and determine if individual inhibitors reduce aggregation, as well as ascertain if our inhibitors reduc the biochemical production of 12- HETE, as a direct marker of 12-LOX inhibition. Fourth, we will evaluate the in vivo efficacy of these 12-LOX inhibitors toward inhibition of thrombosis in mice while retaining normal levels of hemostasis. We will further confirm the activity of the 12-LOX inhibitors in vivo through assessment of 12-HETE production and ex vivo platelet function from mice treated with 12-LOX inhibitors. These inhibitors will additionally be tested for their abilityto prevent heparin-induced thrombocytopenia (HIT), as recent work suggests HIT is regulated in a 12- LOX-dependent manner. We are confident these studies will discover a 12-LOX inhibitor that is potentially an effective therapeutic against platelet aggregation in a number of pathological settings including athero- thrombosis, venous thrombosis, and HIT, and help us to better understand the biology of cardiovascular disease, a pathophysiological condition which worsens with age.