Discovery of Selective Inhibitors for the EphA4 Kinase

阿尔茨海默氏症是一种毁灭性的疾病，涉及慢性、进行性的神经退行性过程和衰退 最终导致死亡的大脑认知功能。淀粉样β蛋白聚集物的积累和 大脑中过度磷酸化的tau蛋白会导致突触功能障碍和神经元丧失，尽管 这种疾病的确切发病机制尚不清楚。可用于治疗的药物很少 阿尔茨海默氏症，而且它们只能提供有限的好处。事实上，阿尔茨海默氏症造成的经济损失 仅在美国，疾病每年就高达数千亿美元。许多候选药物已经被 在过去十年的临床试验中进行了评估，但没有一种得到批准。因此，重要的是要确定新的 基于新靶点的药物。EphA4受体酪氨酸激酶最近被认为是一种新的有前途的 抗阿尔茨海默病和其他疾病的神经变性和认知缺陷的靶点 神经退行性疾病。EphA4受体信号在异常诱导时可促进神经毒性 淀粉样β-寡聚体和肾上腺素配体。EphA4的激活域，它负责 神经毒性作用，是一个可用药的靶点。然而，靶向EphA4的小分子激酶抑制剂与 选择性和高亲和力仍有待鉴定。在这里，我们建议执行两个高通量筛选 EphA4的运动。一个屏幕将使用蛋白质热漂移(PTS)作为分析格式，而另一个屏幕将使用蛋白质热漂移(PTS)作为分析格式 Screen将部署一种激酶活性分析格式。这些屏幕的目标是识别一组同样广泛的 EphA4的激酶依赖功能的调节器。使用两种格式应该会最大限度地提高我们的 能够识别具有不同作用模式的大量调节器，并最终开发出 对EphA4具有高选择性的抑制化合物。我们预计PTS屏幕将识别化合物 稳定EphA4胞内区的非活性构象，不仅通过与激酶结合 也适用于此域以外的监管区域。第二个屏幕将在体外部署一种新的 我们开发的激酶分析配置，其中不活跃的EphA4胞内区作为 具有催化活性的EphA4胞内区的生理底物。我们预计这个化验结果 将不仅鉴定以EphA4 ATP结合口袋为靶点的化合物，还将鉴定具有 新的作用机制。在两个屏幕及其衍生产品中确定的最高点击量将是 通过第二轮和第三轮生化和基于细胞的分析来表征和改进 已经在私人投资机构及其合作者的实验室中建立，并得到结构指导的补充 例如在硅胶对接和X射线结晶学方面的方法。这些化验将提供正式的命中确认， 神经元中作用和活动机制的选择性图谱和初步表征。我们期待着 该项目将导致发现几种新的EphA4选择性抑制剂，适用于 阿尔茨海默氏症和其他神经退行性疾病动物模型的未来研究进展。

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.