The microglial potassium channels Kv1.3 and KCa3.1 as therapeutic targets for neu

小胶质细胞钾通道 Kv1.3 和 KCa3.1 作为 neu 的治疗靶点

• 批准号: 8286872
• 负责人: HEIKE WULFF
• 金额: $ 31.13万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8286872
• 关键词: Affect; Alzheimer' s Disease; Anti-Inflammatory Agents; Anti-inflammatory; Area; Atherosclerosis; Autoimmune Diseases; Bacterial Infections; Binding Sites; Biological Availability; Brain; Calcium; Calcium Signaling; Cells; Contact Dermatitis; Data; Delayed Hypersensitivity; Drug Delivery Systems; Drug Kinetics; Encephalitis; Evaluation; Experimental Autoimmune Encephalomyelitis; Fever; Genetic; Glutamates; Goals; Grant; Hour; Human; Hypoxia; Immune system; Immunohistochemistry; Immunosuppressive Agents; In Situ Nick-End Labeling; In Vitro; Infarction; Infiltration; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Inhibitory Concentration 50; Insulin-Dependent Diabetes Mellitus; Ischemic Stroke; Knockout Mice; Kv1.3 potassium channel; Laboratories; Lead; Learning; Lymphocyte; Maps; Measures; Mediating; Membrane Potentials; Microglia; Middle Cerebral Artery Occlusion; Minocycline; Motor; Multiple Sclerosis; Neurologic; Neurons; Optic Nerve Transections; Oral; Pathogenesis; Pathology; Patients; Performance; Phagocytosis; Pharmaceutical Chemistry; Pharmaceutical Preparations; Play; Positioning Attribute; Potassium Channel; Primates; Production; Rattus; Reaction; Reactive Oxygen Species; Recovery of Function; Reperfusion Therapy; Reporting; Research; Respiratory Burst; Retina; Retinal Ganglion Cells; Rodent; Role; Secondary to; Staining method; Stains; Stroke; T-Cell Activation; Testing; Therapeutic; Time; Toxic effect; Translating; Transplantation; Vaccines; Vascular Diseases; Virus Diseases; Wild Type Mouse; Work; base; cyclooxygenase 2; cytokine; design; expectation; improved; in vivo; inhibitor/antagonist; killings; lipophilicity; macrophage; migration; neurogenesis; neuronal survival; neurotoxic; neurotoxicity; neurotrophic factor; novel; prevent; programs; research study; response; small molecule; therapeutic evaluation; therapeutic target; translational study; voltage; week trial

DESCRIPTION (provided by applicant): In addition to directly causing neuronal damage ischemic stroke elicits a delayed neuroinflammatory response that is characterized by lymphocyte infiltration, hyperthermia and robust microglia activation. "Reactive" microglia in particular contribute to this "secondary damage" by producing inflammatory cytokines, reactive oxygen species, NO, and cyclooxygenase-2 reaction products. However, activated microglia might also be neuroprotective by releasing neurotrophic factors and phagocytosing cellular debris. The goal of microgliatargeted therapies therefore should be to reduce the neurotoxic effects of activated microglia while at the same time maintaining their beneficial functions. We here hypothesize, that blockers of the microglial K+ channels Kv1.3 and KCa3.1 might be able to do exactly this based on the preliminary data presented in this application. We previously designed potent and selective small molecule inhibitors for both channels, PAP-1 for Kv1.3 and TRAM-34 for KCa3.1, and demonstrated that these compounds can prevent or treat various autoimmune diseases and inflammatory conditions in rodents such as contact dermatitis, type-1 diabetes, inflammatory bowel disease, atherosclerosis and EAE. More recently we made the exciting observation that our KCa3.1 blocker TRAM-34 reduces infarct area and neurological deficit scores following ischemic stroke in rats even if treatment is commenced 12 hours after reperfusion. Another strong rationale for our study is a report that TRAM-34 does not prevent microglia from phagocytosing damaged neurons but increases the number of surviving retinal ganglion cells following optic nerve transection in rats by reducing the production and/or secretion of neurotoxic molecules in the retina. Taken together with previous work from our laboratory and other groups implicating both Kv1.3 and KCa3.1 in microglia mediated neuronal killing, these results suggest Kv1.3 and KCa3.1 as novel targets for CNS pathologies involving inflammation. With the help of this grant we therefore intend to test the hypothesis that both channels constitute novel targets for the treatment of stroke. Under Aim-1 we will more rigorously evaluate Kv1.3 and KCa3.1 as targets for stroke by testing the effect of both pharmacological blockade and genetic deletion in reperfusion MCAO and by performing parallel in vitro studies to investigate the role Kv1.3 and KCa3.1 in microglia functions. Under Aim-2 we will use our expertise in medicinal chemistry to design a less lipophilic and more brain penetrant small molecule Kv1.3 inhibitor than our existing lead compound PAP-1 (IC50 2 nM). We further will resynthesize a brain-penetrant KCa3.1 inhibitor, which was abandoned by Bayer, when the company pulled out of stroke research. Under Aim-3 we will directly compare the new Kv1.3 and KCa3.1 blockers to minocycline in a 4-week trial by assessing in vivo cytokine production, neurogenesis and functional recovery. As a first step towards translating our findings to humans, we will further obtain brain sections from stroke patients and controls and perform immunohistochemistry for KCa3.1, Kv1.3, and microglia activation markers.

描述（由申请人提供）：除了直接引起神经元损伤外，缺血性卒中还引起迟发性神经炎症反应，其特征在于淋巴细胞浸润、高热和强烈的小胶质细胞活化。“反应性”小胶质细胞通过产生炎性细胞因子、活性氧、NO和环氧合酶-2反应产物而特别促成这种“二次损伤”。然而，活化的小胶质细胞也可能通过释放神经营养因子和吞噬细胞碎片而具有神经保护作用。因此，小胶质细胞靶向治疗的目标应该是减少激活的小胶质细胞的神经毒性作用，同时保持其有益功能。我们在这里假设，小胶质细胞K+通道Kv1.3和KCa3.1的阻断剂可能能够根据本申请中提供的初步数据准确地做到这一点。 我们之前设计了两种通道的有效和选择性小分子抑制剂，PAP-1用于Kv1.3和TRAM-34用于KCa3.1，并证明这些化合物可以预防或治疗啮齿动物中的各种自身免疫性疾病和炎症性疾病，如接触性皮炎，1型糖尿病，炎症性肠病，动脉粥样硬化和EAE。最近，我们进行了令人兴奋的观察，我们的KCa3.1阻断剂TRAM-34减少了大鼠缺血性中风后的梗死面积和神经功能缺损评分，即使在再灌注后12小时开始治疗。我们研究的另一个强有力的理由是，有报道称，TRAM-34不能阻止小胶质细胞吞噬受损的神经元，但通过减少视网膜中神经毒性分子的产生和/或分泌，增加了大鼠视神经横断后存活的视网膜神经节细胞的数量。结合我们实验室和其他小组先前的工作，这些结果表明Kv1.3和KCa3.1都参与了小胶质细胞介导的神经元杀伤，这些结果表明Kv1.3和KCa3.1是涉及炎症的CNS病理学的新靶点。因此，在这笔赠款的帮助下，我们打算测试这两个通道构成中风治疗新靶点的假设。在Aim-1中，我们将通过测试药物阻断和基因缺失在再灌注MCAO中的作用，并通过进行平行的体外研究来研究Kv1.3和KCa3.1在小胶质细胞功能中的作用，更严格地评估Kv1.3和KCa3.1作为中风的靶点。根据Aim-2，我们将利用我们在药物化学方面的专业知识，设计一种比我们现有的先导化合物PAP-1（IC 50 2 nM）亲脂性更低、脑渗透性更强的小分子Kv1.3抑制剂。我们还将重新合成一种脑渗透剂KCa3.1抑制剂，这是拜耳公司在退出中风研究时放弃的。在Aim-3中，我们将通过评估体内细胞因子产生、神经发生和功能恢复，在为期4周的试验中直接比较新的Kv1.3和KCa3.1阻滞剂与米诺环素。作为将我们的发现转化为人类的第一步，我们将进一步从中风患者和对照组中获得脑切片，并对KCa3.1，Kv1.3和小胶质细胞活化标记物进行免疫组织化学。