Assay Development for Substrate and Phosphorylation State Specific JNK Inhibitors

描述（由申请人提供）：阿尔茨海默病、心脏病、中风、帕金森病和糖尿病是美国的主要死因，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）对影响这些疾病的关键细胞类型（如神经元、心肌细胞和心肌细胞）的细胞死亡和存活极其重要。小岛这种细胞死亡大部分通过氧化应激机制和线粒体功能障碍发生。许多ATP竞争性激酶抑制剂已被发现，但很少，如果任何底物竞争性已被推进。因此，这项工作的目标是开发高通量的生化分析，可以发现c-jun-N-末端激酶（JNK）的选择性底物竞争性（非ATP竞争性）抑制剂和磷酸化状态选择性抑制剂。这将使激酶检测开发和药物发现朝着一个大胆的新方向发展。此外，将建立高通量的基于细胞的测定和功能测定，以了解这些抑制剂在预防线粒体功能障碍和细胞死亡中的功能。开发用于发现作为JNK的底物竞争性抑制剂的化合物的测定提供了设计高度选择性JNK抑制剂的新机制，所述高度选择性JNK抑制剂可能不具有与严格ATP竞争性激酶抑制剂相关的潜在副作用。为此，我们将：1）开发用于发现底物和双齿竞争性选择性抑制剂的荧光偏振（FP）测定，以及用于活化（磷酸化）状态选择性抑制剂的荧光共振能量转移（FRET）测定。2)开发基于细胞的免疫细胞化学（In-Cell Western）以检测SHSY 5 Y细胞中c-Jun磷酸化的抑制，以及3）在SHSY 5 Y细胞中建立基于功能细胞的测定以监测活性氧（ROS）产生、线粒体功能障碍和细胞死亡。总的来说，这三个目标将提供一个新的药物筛选平台，将允许独特的化合物发现和神经元细胞为基础的功能测定的骨干。

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 ?-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.