HTS for inhibitors of NADPH Oxidase 2 (NOX 2)

NADPH 氧化酶 2 (NOX 2) 抑制剂的 HTS

• 批准号: 7991279
• 负责人: HUGH ROSEN
• 金额: $ 18.99万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7991279
• 关键词: Acute; Age; Aging; Alzheimer' s Disease; Aortic Aneurysm; Applications Grants; Arthritis; Biochemical; Biological; Biological Assay; Biological Process; Biology; COS-7 Cell; Cardiovascular Diseases; Cardiovascular system; Cell Death; Cell Line; Cells; Chronic; Collaborations; Cytochrome P450; Data; Detection; Disease; Dissection; Dose; Enzymes; Family; Family member; Future; Generations; HL-60 Cells; Head; Health; Housing; Hypertension; Inflammation; Inflammatory; Investigation; Learning; Libraries; Lipoxygenase; Luminol; Malignant Neoplasms; Mitochondria; Molecular Bank; Myocardial Infarction; NADPH Oxidase; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Oxidants; Oxidases; Pathogenesis; Play; Pulmonary Fibrosis; Reactive Oxygen Species; Reperfusion Injury; Role; Screening procedure; Series; Signal Transduction; Solid; Source; Specificity; Stroke; System; Testing; Tetradecanoylphorbol Acetate; Therapeutic Agents; Toxic effect; Xanthine Oxidase; base; diphenyleneiodonium; enzyme activity; follow-up; high throughput screening; inhibitor/antagonist; insight; member; miniaturize; novel therapeutic intervention; public health relevance; reconstitution; response; small molecule; small molecule libraries; tool

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.

描述（由申请人提供）：NOX2是NADPH氧化酶的NOX/DUOX家族的七个成员之一，具有产生活性氧（ROS）的生物学功能。 NOX2衍生的ROS参与了几种疾病的发病机理，例如急性和慢性炎症，心血管疾病和衰老。然而，NOX家族的其他成员以及其他细胞酶（例如黄氨酸氧化酶，细胞色素p-450，线粒体氧酶等）也可以生产细胞ROS。迄今为止，由于缺乏良好的nox2，由于其他来源所产生的NOX2衍生的ROS对其他来源产生的氧化剂对其他来源产生的氧化剂的贡献是复杂的。 该赠款申请的目的是通过与Hugh Rosen博士领导的TSRI分子图书馆筛选中心合作筛选化学库来鉴定NOX2的新小分子抑制剂。为此，我们以96孔格式开发了一种稳健的基于细胞的化学发光（CL）测定，以检测NOX2衍生的乙酸凤梨酸 - 厌食症（PMA）刺激。该测定法将使用cos-7phox作为细胞系。此类细胞已经过遗传修饰，以稳定表达NOX2酶活性所需的所有成分，并且它们代表了能够高水平PMA诱导的ROS生成的广泛接受的全细胞系统。我们计划在附近的MSLCN中心的384孔板中实施此基于细胞的CL分析，以进行高通量筛选（HTS）。此外，我们建议通过几个后续测定来验证主要命中率，这已经在384孔板中成功开发。这些次要筛选将i）消除非特异性或有毒的命中ii）i ii）验证潜在的命中，以阻止独立的细胞系列中Nox2依赖性ROS产生的能力（NOX2-HEK293）iii）验证主要命中的选择性以阻止其他ROS产生酶的活性（即其他Xanthine氧化酶）的活性（Xanthine oxidase）IV的特定性，并评估HIN的其他活性。 家庭。验证的NOX2选择性命中的化合物将是研究NOX2衍生的ROS参与炎症，心血管疾病和衰老的强大研究工具。与此一致，我们计划将这种新生成的NOX2抑制剂与持续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型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，阿尔茨海默氏病，中风，癌症和炎症。 公共卫生相关性：众所周知，NADPH氧化酶（NOX2）在健康和疾病中起着重要作用，但有关生物学中NOX2功能的更多尚待了解。我们将使用基于细胞的屏幕来识别NOX2抑制剂，然后使用辅助屏幕来验证选择性，功效和机制。这种抑制剂对于研究NOX2生物学以及作为炎症和神经退行性疾病的潜在治疗剂将是有用的。