Development of Potent/Selective Lipoxygenase Therapeutics Against Stroke Injury

针对中风损伤的有效/选择性脂氧合酶疗法的开发

• 批准号: 8632065
• 负责人: Theodore R Holman
• 金额: $ 56.08万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8632065
• 关键词: 12-HETE; ALOX15 gene; Adjuvant; Affect; Affinity; Animal Model; Animals; Arachidonate 15-Lipoxygenase; Biochemical; Biological Assay; Blood - brain barrier anatomy; Blood Clot; Blood Vessels; Blood coagulation; Brain Injuries; Cell Line; Cells; Characteristics; Chemicals; Coagulation Process; Collaborations; Data; Development; Drug Interactions; Drug Kinetics; Edema; Endothelial Cells; Enzymes; Evaluation; Excretory function; Extravasation; Failure; Goals; Human; In Vitro; Individual; Infarction; Inflammation; Injury; Iron; Ischemia; Ischemic Brain Injury; Ischemic Penumbra; Ischemic Stroke; Isoenzymes; Knockout Mice; LOX gene; Lead; Ligands; Lipoxygenase; Lipoxygenase Inhibitors; Liquid substance; Metabolic; Metabolic Clearance Rate; Metabolism; Middle Cerebral Artery Occlusion; Modeling; Molecular Bank; Monitor; Mus; Neurons; Neuroprotective Agents; Oxidative Stress; Patients; Pattern; Permeability; Pharmaceutical Preparations; Physiological; Plasma; Process; Production; Property; Prostaglandin-Endoperoxide Synthase; Protein Binding; Reperfusion Therapy; Research; Rodent Model; Role; Series; Simulate; Solubility; Solutions; Stroke; Stroke prevention; Subarachnoid Hemorrhage; Testing; Therapeutic; United States National Institutes of Health; absorption; acute stroke; aqueous; base; chemical property; design; drug candidate; drug development; high throughput screening; human disease; in vivo; inhibitor/antagonist; mouse model; neuron loss; novel; novel strategies; pharmacophore; public health relevance; repository; scaffold; small molecule; therapeutic development

DESCRIPTION (provided by applicant): The principle aim of this research is to develop selective and potent human 12/15-LOX inhibitors, which will probe the role of inflammation in stroke, leading to drug candidates for this devastating human disease. Recent failures in testing of neuroprotective agents have highlighted the need for new approaches to acute stroke treatment. Among the novel targets, 12/15-lipoxygenase (12/15-LOX or 15-LOX-1) stands out for numerous reasons. 12/15-LOX expression is increased in the peri-infarct region following middle cerebral artery occlusion (MCAO) in mice, both in neurons and in endothelial cells, which contributes to ischemic brain injury by increasing neuronal cell death, vascular leakage, and edema formation. ALOX15 gene (i.e. 12/15-LOX) knock out mice are protected against these detrimental effects and human 12/15-LOX expression is increased in the ischemic penumbra of ischemic stroke patients, with a cellular pattern very similar to that found in mice. Furthermore, increased levels of the 12/15-LOX metabolite 12-HETE have been found in patients with stroke caused by subarachnoid hemorrhage, suggesting 12/15-LOX may be a target for subarachnoid hemorrhage as well. Targeting this one enzyme may thus provide pleiotropic benefits. The broad objective of the current proposal is to advance one or more candidate molecules toward therapeutic development against stroke, utilizing our assays to identify novel selective inhibitors for the human 12/15-LOX. We have already performed a successful 12/15-LOX high-throughput (HTP) screen in collaboration with the NIH which found over ~1000 potent inhibitors. Of these ~1000 compounds, 50 were re-screened and six potent 12/15-LOX inhibitors were discovered. One of these six inhibitors was re-synthesized and shown to be potent and selective against human 12/15-LOX in vitro, effective in mouse HT-22 neuronal cells and in a mouse stroke animal model, confirming it as a "validated hit". Our research plan is guided by the following three specific aims. First, we propose to design new molecules based on our "validated hit", which will have increased potency against human 12/15-LOX. Second, we shall confirm the optimized "validated hit's" potency in a neuronal cell line, optimize ADME properties, maintain LOX isozyme selectivity and determine its mechanism of inhibition. Third, once we have optimized our inhibitor scaffold, we will evaluate its protective potency in rodent models of stroke. We will utilize these in vitro inhibitors in our transient focal ischemia mouse model and determine if individual inhibitors reduce infarct size, as well as ascertain if our inhibitors redue the biochemical production of 12-HETE, as a direct marker of 12/15-LOX inhibition. Once this is achieved, we will test these compounds in a model of permanent focal ischemia through iron-induced blood clotting, and a clotting ischemia model with tPA-induced reperfusion, where we test the compounds as adjuvants to tPA treatment. Considering that our "validated hit" manifests a 30% reduction in infarct size, we are confident these studies will discover a 12/15- LOX inhibitor as an effective therapeutic against stroke damage.

描述（由申请人提供）：本研究的主要目的是开发选择性和有效的人类12/15-LOX抑制剂，这将探索炎症在中风中的作用，从而为这种毁灭性的人类疾病提供候选药物。最近在神经保护剂的测试失败，突出了需要新的方法来治疗急性中风。在新的靶点中，12/15-脂氧合酶（12/15-LOX或15-LOX-1）由于许多原因而突出。在小鼠大脑中动脉闭塞（MCAO）后，12/15-LOX表达在梗死周围区域中增加，在神经元和内皮细胞中均增加，这通过增加神经元细胞死亡、血管渗漏和水肿形成而促成缺血性脑损伤。ALOX 15基因（即12/15-LOX）敲除小鼠被保护免受这些有害作用，并且人12/15-LOX表达在缺血性中风患者的缺血半暗带中增加，其细胞模式与小鼠中发现的非常相似。此外，在患有由蛛网膜下腔出血引起的中风的患者中发现12/15-LOX代谢物12-HETE的水平增加，这表明12/15-LOX也可能是蛛网膜下腔出血的靶点。因此，靶向这一种酶可以提供多效性益处。目前建议的广泛目标是利用我们的检测方法来鉴定新的选择性抑制剂，以促进一种或多种候选分子的抗中风治疗开发 人类12/15-LOX的基因我们已经与NIH合作进行了成功的12/15-LOX高通量（HTP）筛选，发现了超过1000种有效的抑制剂。在这约1000种化合物中，重新筛选了50种，发现了6种有效的12/15-LOX抑制剂。这六种抑制剂中的一种被重新合成，并显示出在体外对人12/15-LOX是有效的和选择性的，在小鼠HT-22神经元细胞和小鼠中风动物模型中是有效的，确认其为“经验证的命中”。我们的研究计划以以下三个具体目标为指导。首先，我们建议基于我们的“验证命中”设计新的分子，其将具有针对人12/15-LOX的增加的效力。其次，我们将确认优化的“验证命中”的效力在神经元细胞系，优化ADME性能，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抑制剂支架，我们将评估其在啮齿动物中风模型中的保护效力。我们将在我们的短暂局灶性缺血小鼠模型中利用这些体外抑制剂，并确定单独的抑制剂是否减少梗死面积，以及确定我们的抑制剂是否减少12-HETE的生物化学产生，作为12/15-LOX抑制的直接标志物。一旦实现这一点，我们将通过铁诱导的血液凝固在永久性局灶性缺血模型中测试这些化合物，并在tPA诱导的再灌注的凝血缺血模型中测试这些化合物作为tPA治疗的佐剂。考虑到我们的“有效命中”表明梗死面积减少30%，我们相信这些研究将发现12/15- LOX抑制剂作为对抗中风损伤的有效治疗剂。