Targeting CaM Kinase II for Neuroprotection in Ischemic Stroke

描述(申请人提供)：中风是一个主要的公共卫生问题，美国的死亡率排名第三，仅次于其他涉及心血管系统的疾病和癌症。我们建议通过靶向钙稳态的关键调节因子--钙/钙调素依赖的蛋白激酶II(CaMKII)来寻求缺血性卒中的神经保护。抑制CaMKII代表了一种新的缺血性卒中神经保护范式，其基础是阻断CaMKII对钙超载的影响及其对神经毒性介质的更直接作用。两项进展为我们提供了测试有效的小分子CaMKII抑制剂治疗缺血性中风的理论基础和机会。首先，在谷氨酸兴奋性毒性中，CaMKII的高活性被证明促进了细胞的死亡，而在原位和大脑中动脉阻塞(MCAO)模型中，CaMKII的抑制剂具有神经保护作用。其次，我们已经借此机会重新启动了一项CaMKII抑制剂计划，该计划最初是在PARM推进的，现在我们正在根据对人类酶的第一个晶体结构的洞察来加速该计划。我们的建议相对简单，即增加我们开发的有效抑制剂的中枢神经系统渗透率，并在永久性闭塞MCAO模型中测试优化的先导化合物。我们已经设计了一些修饰来增加我们化合物的亲脂性，目的是增加它们的中枢神经系统渗透性。这些化合物将在体外分析激酶抑制和选择性，以及在神经元培养中抑制和保护谷氨酸兴奋毒性。将根据对MDR-MDCK单层的双向渗透性来选择具有最大CNS渗透潜力的抑制剂，并将量化其在体内的脑/血浆分布。铅中枢神经系统穿透抑制剂将在大鼠永久性闭塞MCAO模型中进行测试。我们将使用激酶活性的生物标志物来监测体内实现的CaMKII抑制程度，并量化该抑制剂对梗塞面积的影响。在缺血性中风中成功地证明-概念将使我们能够进入第二阶段SBIR提案，以优化药物-特性并对先导化合物进行IND使能研究。 与公共健康相关：中风是一个主要的公共健康问题，美国的死亡率位居第三，仅次于其他涉及心血管系统和癌症的疾病。最近的数据表明，作为钙信号的主要中介，钙/钙调素依赖的蛋白激酶II(CaMKII)在缺血性损伤中具有高活性。我们的目标是增加我们强大的CaMKII抑制剂的大脑渗透率，并在人类缺血性中风的动物模型中测试优化的铅化合物的有效性。

DESCRIPTION (provided by applicant): Stroke is a major public health problem, with a US mortality rate that ranks third behind other diseases involving the cardiovascular system and cancer. We propose to pursue neuroprotection in ischemic stroke by targeting a key regulator of Ca2+ homeostasis, Ca2+/CaM-dependent protein kinase II (CaMKII). Inhibition of CaMKII represents a novel paradigm in ischemic stroke-neuroprotection based on blocking the effects of CaMKII on Ca2+ overload as well as its more direct effect on mediators of neurotoxicity. Two developments provide us with the rationale and opportunity to test potent small molecule CaMKII inhibitors in ischemic stroke. First, hyperactivity of CaMKII has been shown to promote cell death in glutamate excitotoxicity while inhibitors of the kinase are neuroprotective in situ and in the middle cerebral artery occlusion (MCAO) model. Second we have taken the opportunity to restart a CaMKII inhibitor program initially advanced at a pharm and which we are now accelerating based on insights from the first crystal structures of the human enzyme. Our proposal is relatively straightforward, to increase CNS penetration of the potent inhibitors we developed and test the optimized lead compound in a permanent occlusion MCAO model. We have designed a number of modifications that increase lipophilicity of our compounds with the aim of increasing their CNS penetration. The compounds will be analyzed for kinase inhibition and selectivity in vitro, and for inhibition and protection from glutamate excitotoxicity in neuronal cultures. The inhibitor with the greatest potential for CNS penetration will be selected based on bidirectional permeability through MDR----MDCK monolayers and its brain/plasma distribution in vivo will be quantified. The lead CNS penetrating inhibitor will be tested in the rat permanent occlusion MCAO model. We will monitor the degree of CaMKII inhibition achieved in vivo using biomarkers of kinase activity and quantify the effect of the inhibitor on infarct size. A successfully proof----of----concept in ischemic stroke will position us for a Phase II SBIR proposal to optimize the drug----like properties and conduct IND enabling studies of the lead compound. PUBLIC HEALTH RELEVANCE: Stroke is a major public health problem with a US mortality rate that ranks third behind other diseases involving the cardiovascular system and cancer. Recent data implicate hyperactivity of Ca2+/CaM- dependent protein kinase II (CaMKII), a major mediator of Ca2+ signaling, in ischemic damage. We aim to increase the brain penetration of our potent CaMKII inhibitors and test the optimized lead compound for efficacy in an animal model of human ischemic stroke.ischemic stroke.