Discovery of Selective Inhibitors for the EphA4 Kinase

阿尔茨海默氏症是一种破坏性的疾病，涉及慢性，进行性神经退行性过程和下降 最终导致死亡淀粉样蛋白-β聚集体的积聚， 脑中过度磷酸化的tau蛋白引起突触功能障碍和神经元损失，尽管 这种疾病的确切机制仍然未知。几乎没有药物可以治疗 老年痴呆症，它们只能提供有限的好处。事实上，老年痴呆症造成的经济损失 每年仅在美国，疾病就达到数千亿美元。许多候选药物已经 在过去的十年里，在临床试验中进行了评估，但没有一个被批准。因此，重要的是确定新的 新靶点药物。EphA 4受体酪氨酸激酶最近已成为一种新的有前途的 用于对抗阿尔茨海默病和其它疾病中神经变性和认知缺陷的靶点 神经退行性疾病EphA 4受体信号传导可以在由EphA 4受体异常诱导时促进神经毒性。 β淀粉样蛋白低聚物和肝配蛋白配体。EphA 4的激酶结构域，其负责EphA 4的蛋白质合成。 神经毒性作用，是一个药物的目标。然而，靶向EphA 4的小分子激酶抑制剂， 选择性和高亲和力仍有待鉴定。在这里，我们建议进行两个高通量筛选 EphA 4的活动。一个屏幕将部署蛋白质热位移（PTS）作为测定格式，另一个屏幕将部署蛋白质热位移（PTS）作为测定格式。 屏幕将部署激酶活性测定格式。这些屏幕的目标是确定广泛的面板， EphA 4的激酶依赖性功能的调节剂。使用两种格式应最大限度地提高我们的 能够识别具有不同作用模式的调节剂的大量库，并最终开发出一种 对EphA 4具有高选择性抑制化合物。我们预计PTS屏幕将识别化合物 其不仅通过结合激酶， 域，但也适用于该域以外的调控区域。第二个屏幕将部署一个新的体外 我们已经开发的激酶测定配置，其中无活性的EphA 4细胞内区域用作 EphA 4细胞内区域的催化活性生理底物。我们预计， 将不仅鉴定靶向EphA 4 ATP结合口袋的化合物，而且鉴定具有以下特征的变构抑制剂： 新的作用机制。在两个屏幕中确定的热门歌曲及其衍生物将是 通过一轮又一轮的二级和三级生化和基于细胞的检测， 已经在PI及其合作者的实验室中建立，并辅以结构指导 方法，如计算机对接和X射线晶体学。这些分析将提供正式的命中验证， 神经元中作用和活性机制的选择性分析和初步表征。我们预计 该项目将导致几种新的EphA 4选择性抑制剂的鉴定， 在阿尔茨海默氏症和其他神经退行性疾病的动物模型的未来研究的进展。

Alzheimer’s is a devastating disease involving chronic, progressive neurodegenerative processes and decline in brain cognitive function that ultimately lead to death. Accumulation of aggregates of amyloid-beta and hyperphosphorylated tau protein in the brain causes synaptic dysfunction and loss of neurons, although the precise mechanisms underlying the disease remain unknown. There are few drugs available to treat Alzheimer’s disease, and they provide only limited benefits. In fact, the economic damages from Alzheimer’s disease reach hundreds of billion dollars every year in the US alone. Many candidate drugs have been evaluated in clinical trials in the last decade, but none has been approved. Thus, it is important to identify new drugs based on novel targets. The EphA4 receptor tyrosine kinase has recently emerged as a novel promising target for counteracting neurodegeneration and cognitive deficits in Alzheimer’s disease and other neurodegenerative diseases. EphA4 receptor signaling can promote neurotoxicity when aberrantly induced by amyloid-beta oligomers and ephrin ligands. The kinase domain of EphA4, which is responsible for the neurotoxic effects, is a druggable target. However, small molecule kinase inhibitors targeting EphA4 with selectivity and high affinity remain to be identified. Here we propose to perform two high-throughput screening campaigns of EphA4. One screen will deploy Protein Thermal Shift (PTS) as the assay format and the other screen will deploy a kinase activity assay format. The goal of these screens is to identify as broad a panel of modulators of the kinase-dependent functions of EphA4 as possible. Use of two formats should maximize our ability to identify a large repertoire of modulators with diverse modes of action and ultimately develop an inhibitory compound with high selectivity for EphA4. We anticipate that the PTS screen will identify compounds that stabilize the inactive conformation of the EphA4 intracellular region by binding not only to the kinase domain but also to regulatory regions outside this domain. The second screen will deploy a novel in vitro kinase assay configuration we have developed, where the inactive EphA4 intracellular region serves as the physiological substrate for the catalytically competent EphA4 intracellular region. We anticipate that this assay will identify not only compounds that target the EphA4 ATP binding pocket, but also allosteric inhibitors with novel mechanisms of action. The top hits identified in the two screens and their derivatives will be characterized and improved through rounds of secondary and tertiary biochemical and cell-based assays already established in the laboratories of the PIs and their collaborators, complemented by structure-guided approaches such as in silico docking and X-ray crystallography. These assays will provide formal hit validation, selectivity profiling and initial characterization of mechanisms of action and activities in neurons. We anticipate that this project will result in the identification of several novel EphA4 selective inhibitors that are suitable for advancement to future studies in animal models of Alzheimer’s and other neurodegenerative diseases.