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（申请人提供）：Nox2是NADPH氧化酶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参与了多种疾病的发病机制，例如急性和慢性炎症、心血管疾病和衰老。然而，细胞ROS也可以由Nox家族的其他成员和其他细胞酶产生，例如黄嘌呤氧化酶、细胞色素P-450、线粒体氧化酶等。迄今为止，由于缺乏良好的特异性Nox2抑制剂，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。 本次拨款申请的目的是与 Hugh Rosen 博士领导的 TSRI 分子库筛选中心合作，通过筛选化学库来识别 Nox2 的新型小分子抑制剂。为此，我们开发了一种强大的 96 孔形式的基于细胞的化学发光 (CL) 测定，用于检测佛波醇肉豆蔻酸酯乙酸酯 (PMA) 刺激后衍生的 Nox2。该测定将使用 COS-7phox 作为细胞系。此类细胞经过基因改造，可稳定表达 Nox2 酶活性所需的所有成分，它们代表了一种被广泛接受的全细胞系统，能够高水平 PMA 诱导的 ROS 生成。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，我们建议通过几种后续测定来验证主要命中，我们已在 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 (Nox2-HEK293) 中阻断 Nox2 依赖性 ROS 生成的能力 iii) 验证初级命中对阻断其他 ROS 产生酶（即黄嘌呤氧化酶）活性的选择性 iv) 评估潜在命中对 Nox2 活性与 Nox 其他成员活性的特异性 家庭。作为经过验证的 Nox2 选择性命中而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研究工具。与此一致，我们计划将这种新生成的 Nox2 抑制剂与正在进行的生化和功能分析相结合。 此外，这些抑制剂有可能提供新的治疗方法来抑制高血压、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。 公共健康相关性：众所周知，NADPH 氧化酶 (Nox2) 在健康和疾病中发挥着重要作用，但关于 Nox2 的生物学功能还有更多知识需要了解。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、功效和机制。此类抑制剂将有助于研究 Nox2 生物学，并作为炎症和神经退行性疾病的潜在治疗剂。