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，并探索血小板活化炎症的生物化学，从而开发心血管疾病的候选药物。最近抗凝剂试验的失败突出了对急性凝血治疗新方法的需求。在这些新的靶标中，12-脂氧合酶（12-LOX）脱颖而出的原因有很多：1)相对于循环中的其他细胞，它在血小板中的表达量很高；2)我们之前已经证明12-LOX对止血和血栓形成都很重要；3)12-LOX在人和小鼠的血小板中都有表达。目前的主要目标是推进一种或多种候选分子用于抗血小板活化/聚集的治疗开发，利用我们的检测方法鉴定新的人类12-LOX选择性抑制剂。我们已经与美国国立卫生研究院合作，成功地进行了12-LOX高通量（HTP）筛选，发现了超过1000个潜在的hit。在这1000种化合物中，有7种被发现是有效的12-LOX抑制剂。这七种抑制剂中的两种被重新合成，并在体外显示出对人12-LOX的强效和选择性，并在许多人血小板细胞试验中有效，证实它们是“有效的命中”。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，这些化学型对人类12-LOX具有更高的效力。其次，优化“命中”的ADME/PK特性，保持LOX同工酶的选择性，确定其抑制机制。第三，一旦我们优化了各种抑制剂的化学型，我们将在各种血小板激活筛选中评估它们的保护效力。我们将在我们的血小板模型中使用这些体外抑制剂，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的生化生产，作为12- lox抑制的直接标志。第四，我们将评估这些12-LOX抑制剂在保持正常止血水平的同时抑制小鼠血栓形成的体内功效。我们将通过评估12-LOX抑制剂治疗小鼠的12-HETE生成和体外血小板功能来进一步证实12-LOX抑制剂在体内的活性。这些抑制剂还将被测试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是以12- lox依赖的方式调节的。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病理环境，包括动脉粥样硬化血栓形成、静脉血栓形成和HIT，并帮助我们更好地理解心血管疾病的生物学，这是一种随着年龄的增长而恶化的病理生理状况。

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.