HTS for inhibitors of NADPH Oxidase 2 (NOX 2)

描述（申请人提供）：Nox 2是NADPH氧化酶NOX/DUOX家族的7个成员之一，具有产生活性氧（ROS）的生物学功能。Nox 2衍生的ROS参与了急性和慢性炎症、心血管疾病和衰老等多种疾病的发病机制。然而，细胞ROS也可以产生的其他成员的Nox家族和其他细胞的酶，如黄嘌呤氧化酶，细胞色素P-450，线粒体氧化酶等。到目前为止，解剖的贡献Nox 2衍生的ROS产生的其他来源的氧化剂已经复杂的缺乏良好的特异性Nox 2抑制剂。 该资助申请的目的是通过与由Hugh罗森博士领导的TSRI分子库筛选中心合作筛选化学库来鉴定新的Nox 2小分子抑制剂。为此，我们已经开发了一个强大的细胞为基础的化学发光（CL）测定96孔格式检测Nox 2-衍生佛波醇-肉豆蔻酸酯乙酸酯（PMA）刺激。本试验将使用COS-7 phox作为细胞系。此类细胞已被遗传修饰以稳定表达Nox 2酶活性所需的所有组分，并且它们代表了能够产生高水平PMA诱导的ROS的广泛接受的全细胞系统。我们计划在附近的MSLCN中心的384孔板中实施这种基于细胞的CL测定，用于高通量筛选（HTS）。此外，我们建议用几种后续测定来验证主要命中，我们已经在384孔板中成功开发了这些测定。这些二次筛选将i）消除非特异性或毒性命中，ii）验证潜在命中在独立细胞系中阻断Nox 2依赖性ROS产生的能力（Nox 2-HEK 293）iii）验证初级命中物的选择性以阻断其它ROS产生酶的活性iv）评估Nox 2活性相对于Nox家族其他成员活性的潜在命中的特异性。作为经验证的Nox 2选择性命中出现的化合物将是研究Nox 2衍生的ROS参与炎症，心血管疾病和衰老的强大研究工具。与此相一致，我们计划将这种新产生的Nox 2抑制剂与正在进行的生化和功能分析相结合。 此外，这些抑制剂具有提供抑制高血压、主动脉瘤、心肌梗塞、肺纤维化、关节炎、阿尔茨海默病、中风、癌症和炎症的新治疗方法的潜力。 公共卫生相关性：众所周知，NADPH氧化酶（Nox 2）在健康和疾病中起着重要作用，但关于Nox 2在生物学中的功能仍有很多需要了解。我们将使用基于细胞的筛选来鉴定Nox 2抑制剂，然后进行二次筛选以验证选择性、功效和机制。这些抑制剂将用于研究Nox 2生物学，并作为炎症和神经退行性疾病的潜在治疗剂。

DESCRIPTION (provided by applicant): Nox2 is one of the seven members of NOX/DUOX family of NADPH oxidases with the biological function of generating Reactive Oxygen Species (ROS). Nox2-derived ROS have been involved in the pathogenesis of several disease conditions such as acute and chronic inflammation, cardiovascular diseases and aging. However, cellular ROS can be also produced by other members of Nox family and by other cellular enzymes such as xanthine oxidase, cytochrome P-450, mitochondrial oxydases etc. To date, the dissection of the contribution of Nox2-derived ROS to oxidants generated by other sources has been complicated by the lack of good specific Nox2 inhibitors. The purpose of this grant application is to identify new small-molecule inhibitors for Nox2 by screening chemical libraries in collaboration with TSRI Molecular Library Screening Center headed by Dr. Hugh Rosen. To this aim, we have developed a robust cell-based chemoluminescence (CL) assay in 96-well format for the detection of Nox2-derived upon phorbol-myristate acetate (PMA) stimulation. This assay will use COS-7phox as a cell line. Such cells have been genetically modified to stably express all the components required for Nox2 enzyme activity, and they represent a widely- accepted whole cell system capable of high level of PMA-induced ROS generation. We plan to implement this cell-based CL assay for High-Throughput Screening (HTS) in 384-well plate at the nearby MSLCN center. Furthermore, we propose to validate the primary hits with several follow-up assays, which we have successfully developed in 384-well plate. These secondary screens will i) eliminate non-specific or toxic hits ii) validate potential hits for their ability to block Nox2-dependent ROS generation in an independent cell line (Nox2-HEK293) iii) verify selectivity of primary hits to block the activity of other ROS-producing enzymes (i.e. xanthine oxidase) iv) assess the specificity of potential hits for Nox2 activity vs. activities of other members of Nox family. The compounds that emerge as validated Nox2-selective hits will be powerful investigative tools to study the involvement of Nox2-derived ROS in inflammation, cardiovascular diseases and aging. Consistent with this, we plan to use such newly-generated Nox2 inhibitors in combination with ongoing biochemical and functional analyses. Additionally, these inhibitors have the potential to provide novel therapeutic approaches to inhibit hypertension, aortic aneurysm, myocardial infarction, pulmonary fibrosis, arthritis, Alzheimer's disease, stroke, cancer, and inflammation. PUBLIC HEALTH RELEVANCE: It is known that NADPH oxidases (Nox2) play important roles in health and disease, yet much more remains to be learned about Nox2 function in biology. We will use a cell-based screen to identify Nox2 inhibitors, followed by secondary screens to verify selectivity, efficacy, and mechanism. Such inhibitors will be useful to investigate Nox2 biology, and as potential therapeutic agents in inflammatory and neurodegenerative diseases.