HTS for inhibitors of NADPH Oxidase 2 (NOX 2)

说明(申请人提供)：NOX2是NOX/DUOX NADPH氧化酶家族的七个成员之一，具有产生活性氧物种(ROS)的生物学功能。NOX2来源的ROS参与了多种疾病的发病，如急性和慢性炎症、心血管疾病和衰老。然而，细胞内的ROS也可以由NOx家族的其他成员和其他细胞酶产生，如黄嘌呤氧化酶、细胞色素P-450、线粒体加氧酶等。到目前为止，由于缺乏良好的特异性NOX2抑制剂，导致对NOX2衍生的ROS对其他来源产生的氧化剂的贡献的剖析变得复杂。这项拨款申请的目的是通过与休·罗森博士领导的TSRI分子图书馆筛选中心合作筛选化学文库，寻找新的NOX2小分子抑制剂。为此，我们开发了一种基于细胞的化学发光(CL)分析方法，采用96孔格式，用于检测佛波酯(PMA)刺激产生的NOX2。本实验将以COS-7Phox为细胞系。这些细胞经过基因改造，可以稳定表达NOX2酶活性所需的所有成分，它们代表了被广泛接受的能够高水平产生PMA诱导的ROS的整个细胞系统。我们计划在附近的MSLCN中心的384孔板上实施这种基于细胞的化学发光分析(CL)，用于高通量筛选(HTS)。此外，我们建议用我们在384孔板上成功开发的几种后续分析来验证初步的命中。这些二级筛选将：1)消除非特异性或毒性HITS；2)验证潜在HITS在独立细胞系(NOX2-HEK293)中阻断依赖于NOX2的ROS生成的能力；3)验证初级HITS的选择性，以阻断其他产生ROS的酶(即黄嘌呤氧化酶)的活性；4)评估NOX2活性与NOX家族其他成员活性的潜在HITS的特异性。作为有效的NOX2选择性HITS出现的化合物将成为研究NOX2衍生的ROS在炎症、心血管疾病和衰老中的参与的强大的研究工具。与此一致，我们计划将这些新产生的NOX2抑制剂与正在进行的生化和功能分析结合使用。此外，这些抑制剂有可能提供新的治疗方法来抑制高血压、主动脉瘤、心肌梗死、肺纤维化、关节炎、阿尔茨海默病、中风、癌症和炎症。 公共卫生相关性：已知NADPH氧化酶(NOX2)在健康和疾病中发挥重要作用，但关于NOX2在生物学中的作用仍有待进一步了解。我们将使用基于细胞的筛选来鉴定NOX2抑制剂，然后进行二次筛选来验证选择性、有效性和作用机制。这些抑制剂将有助于研究NOX2的生物学，并作为炎症性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.