Assay Development for Substrate and Phosphorylation State Specific JNK Inhibitors

描述(申请人提供)：阿尔茨海默氏症、心脏病、中风、帕金森氏病和糖尿病是美国的主要死亡原因，因此对人类健康和生活质量有很大影响。大量的体外和体内研究表明，c-jun-N末端激酶(JNK)对神经元、心肌细胞和胰岛等疾病的关键细胞类型的细胞死亡和生存具有极其重要的作用。这种细胞死亡很大程度上是通过氧化应激机制和线粒体功能障碍发生的。已经发现了许多ATP竞争性激酶抑制剂，但几乎没有竞争底物被开发出来。因此，这项工作的目标是发展高通量的生化分析方法，能够发现选择性底物竞争性(非ATP竞争性)c-Jun-N末端激酶(JNK)抑制剂和磷酸化状态选择性抑制剂。这将把激酶分析的开发和药物发现带向一个大胆的新方向。此外，还将建立高通量的基于细胞的分析和功能分析，以了解这些抑制物在预防线粒体功能障碍和细胞死亡方面的功能。开发发现JNK底物竞争性抑制剂的分析方法为设计高度选择性的JNK抑制剂提供了一种新的机制，这些抑制剂可能没有与严格的ATP竞争性激酶抑制剂相关的潜在副作用。为了实现这一目标，我们将：1)建立用于发现底物和双齿竞争性选择性抑制剂的荧光偏振(FP)分析，以及用于激活(磷酸化)状态选择抑制的荧光共振能量转移(FRET)分析。2)建立检测SHSY5Y细胞c-jun磷酸化抑制的免疫细胞化学方法(In-Cell Western)；3)建立SHSY5Y细胞的功能性细胞检测方法，以监测活性氧(ROS)的产生、线粒体功能障碍和细胞死亡。总的来说，这三个目标将为一个新的药物筛选平台提供骨干，该平台将允许独特的化合物发现和基于神经细胞的功能分析。

DESCRIPTION (provided by applicant): Alzheimer's disease, heart disease, stroke, Parkinson's disease, and diabetes are leadings causes of death in the United States and therefore have a large impact on human health and quality of life. Numerous studies, both in vitro and in vivo have implicated c-jun-N-terminal kinase (JNK) as being extremely important for cell death and survival in key cell types affecting these diseases such as neurons, cardiomyocytes, and ß-islets. Much of this cell death occurs through oxidative stress mechanisms and mitochondrial dysfunction. Many ATP-competitive kinase inhibitors have been discovered, but few, if any substrate competitive have been advanced. The goal of this work therefore is to develop high-throughput biochemical assays that can discover selective substrate competitive (non-ATP competitive) inhibitors of c-jun-N-terminal Kinase (JNK) and phosphorylation state selective inhibitors. This will take kinase assay development and drug discovery in a bold new direction. In addition, high-throughput cell-based assays and functional assays will be established to understand the function of these inhibitors in preventing mitochondrial dysfunction and cell death. Developing assays to discover compounds that are substrate competitive inhibitors of JNK provides a novel mechanism to design highly selective JNK inhibitors that likely may not have potential side effects associated with strict ATP competitive kinase inhibitors. To accomplish this we will: 1) Develop a fluorescence polarization (FP) assay for discovering substrate and bidentate competitive selective inhibitors, and fluorescence resonance energy transfer (FRET) assays for activation (phosphorylation) state selective inhibitors. 2) Develop an immunocytochemical cell-based (In-Cell Western) to detect inhibition of c-Jun phosphorylation in SHSY5Y cells, and 3) Establish functional cell-based assays in SHSY5Y cells to monitor reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cell death. Collectively, these three aims will provide the backbone for a novel drug screening platform that will allow for unique compound discovery and neuronal cell-based functional assays.