Small Molecule targeting of NADPH oxidase in neutrophils

中性粒细胞中 NADPH 氧化酶的小分子靶向

• 批准号: 8003097
• 负责人: Marie-Dominique Filippi
• 金额: $ 31.87万
• 依托单位: P2D, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003097
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Acute; Acute Disease; Acute Lung Injury; Adult Respiratory Distress Syndrome; Adverse event; Affinity; Americas; Anti-Inflammatory Agents; Anti-inflammatory; Binding; Biological Assay; Blood Cells; Catalysis; Cell Lineage; Cell Survival; Cell membrane; Cells; Cessation of life; Chronic; Chronic Obstructive Airway Disease; Clinical; Complex; Cytoskeleton; Cytosol; Data; Development; Disease; Dose; Drug Delivery Systems; Drug Kinetics; Edema; Electron Transport; Event; Fluorescence; Free Radicals; Functional disorder; Gene Targeting; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Human; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Inhibitory Concentration 50; Isoenzymes; Lead; Lung; Lung Inflammation; Lung diseases; Membrane; Modality; Molecular; Morbidity - disease rate; Multiple Trauma; Mus; NADP; NADPH Oxidase; Neutrophil Activation; Neutrophil Infiltration; Operative Surgical Procedures; Oxidases; Oxygen; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phenotype; Process; Production; Reactive Oxygen Species; Regulation; Research; Respiratory Failure; Safety; Screening procedure; Sepsis; Severities; Signal Transduction; Small Business Technology Transfer Research; Specificity; Stimulus; Superoxides; Toxic effect; Toxicity Tests; Transfusion; Tumor Necrosis Factor-alpha; United States; Validation; chemokine; cytochrome b558; cytokine; design; efficacy testing; human TNF protein; in vivo; inhibitor/antagonist; lung injury; migration; mortality; mouse model; neutrophil; neutrophil cytosol factor 40K; neutrophil cytosol factor 67K; novel; novel therapeutics; prognostic; protein protein interaction; public health relevance; response; small molecule; superoxide-generating NADPH oxidase; treatment site

DESCRIPTION (provided by applicant): Acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are characterized by a rapid and severe respiratory failure, arising after clinical events including major surgery, trauma, multiple transfusions and sepsis. ALI/ARDS have an associated mortality of 40-80% and current pharmacological modalities have been unsuccessful in decreasing mortality. ARDS, ALI (and other airway diseases including the Chronic Obstructive Pulmonary Disease (COPD)) are associated with neutrophil infiltration of the airway wall. Considerable observational and experimental data support a tight correlation between neutrophils and the severity and progression of the above airways diseases. The goal of the proposed research is to develop a novel class of anti-inflammatory drugs specifically targeting neutrophil functions for amelioration of lung diseases including acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and chronic obstructive pulmonary disease (COPD). We propose to develop small molecule inhibitors that target Rac GTPase and Rac-p67phox interaction inhibitors to downmodulate neutrophil functions in lung diseases. The principal clinical manifestation of ARDS and ALI is edema, which results from endothelial dysfunction and microvasculature permeability. This is caused by release of free radical and reactive oxygen species (ROS) and inflammatory cytokines (e.g., TNF-1) from the activated neutrophils infiltrating the lungs. ROS production results from the activation of the neutrophil NADPH superoxide complex. The NADPH superoxide complex consists of a plasma membrane-bound flavocytochrome b558 (cyt b) and a regulatory complex, termed p47phox-p67phox-p40phox, taht is located in the cytosol of neutrophils. The p47phox-p67phox- p40phox complex translocates to the membrane in response to inflammatory signal-initiated Rac GTPase activation to produce superoxide anion (O2-) and other deleterious oxygen radicals. The direct interaction between activated Rac GTPase and p67phox is an essential step in this neutrophil inflammatory process and are the targets for our small molecule lead compounds: 1) NSC23766, a selective Rac GTPase inhibitor, and 2) phox-I, a selective p67phox inhibitor phox-I. In preliminary studies, we have established that (1) the Rac GTPases, Rac1 and Rac2, are essential regulators of neutrophil migration and superoxide production in gene targeting studies, (2) our rationally designed Rac-specific small molecule inhibitor, NSC23766, functionally suppressed Rac activity in various blood cell lineages including neutrophils, (3) our lead compound, phox-I, binds to the Rac interactive pocket of p67phox and is effective in suppressing oxidase activity in primary neutrophils, and (4) a fMLP-induced lung inflammation mouse model is useful for screening novel compounds for treating both acute and chronic lung inflammatory disorders. Thus, the present Phase I STTR proposal focuses on utilizing our detailed understanding of the molecular regulation of NOX2 NADPH oxidase by Rac GTPases to develop first-in-class treatments for acute and chronic lung disorders. It will validate the target of NSC23766 and phox-I in: A) in vitro protein-protein interaction studies, B) mouse neutrophil studies and C) human neutrophil studies (Aim1); test toxicity and pharmacokinetics in vivo and determine the efficacy of NSC23766 and phox-I in acute lung inflammation mouse model (Aim2). The long term goal is to deliver a novel pharmaceutical target, the Rac-p67phox signaling axis, and suitable lead compounds targeting this key signaling site, for the treatment of acute lung inflammatory disorders ALI/ARDS. PUBLIC HEALTH RELEVANCE: Acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) are severe respiratory failure, arising after clinical events including major surgery, trauma, multiple transfusions and sepsis. In general, lung airway diseases are amongst the leading causes of morbidity and mortality in the United States of America. ALI/ARDS have an associated mortality of 40-80% and current pharmacological modalities have been unsuccessful in decreasing mortality. It is thus critically important to develop new therapies for the treatment of lung diseases. ALI and ARDS are associated with elevated neutrophil-related inflammation. The present Phase I STTR proposal will test the efficacy of two small molecule inhibitors of neutrophil functions that we have developed, on lung inflammatory disorders. Therefore, our study will help delivering a novel pharmaceutical target, and suitable lead compounds for the treatment of lung inflammation, such as ALI and ARDS.

描述（由申请方提供）：急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）的特征是在临床事件（包括大手术、创伤、多次输血和败血症）后出现快速和严重的呼吸衰竭。ALI/ARDS的相关死亡率为40-80%，目前的药理学方法在降低死亡率方面并不成功。ARDS、ALI（和其他气道疾病，包括慢性阻塞性肺病（COPD））与气道壁的嗜中性粒细胞浸润有关。大量的观察和实验数据支持中性粒细胞与上述气道疾病的严重程度和进展之间的密切相关性。该研究的目的是开发一种新型的抗炎药物，专门针对中性粒细胞功能，用于改善肺部疾病，包括急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）和慢性阻塞性肺疾病（COPD）。我们建议开发靶向Rac GT3和Rac-p67 phox相互作用抑制剂的小分子抑制剂，以下调肺部疾病中的中性粒细胞功能。 急性呼吸窘迫综合征（ARDS）和急性肺损伤（ALI）的主要临床表现是水肿，这是由于内皮功能障碍和微血管通透性增加所致。这是由自由基和活性氧（ROS）以及炎性细胞因子（例如，TNF-1）从活化的中性粒细胞浸润肺部。ROS的产生是由嗜中性粒细胞NADPH超氧化物复合物的活化引起的。NADPH超氧化物复合物由质膜结合的黄细胞色素b 558（cyt B）和称为p47 phox-p67 phox-p40 phox的调节复合物组成，其位于中性粒细胞的胞质溶胶中。p47 phox-p67 phox-p40 phox复合物响应于炎症信号引发的Rac GT3活化而易位至膜，以产生超氧阴离子（O2-）和其它有害的氧自由基。活化的Rac GT3和p67 phox之间的直接相互作用是该嗜中性粒细胞炎症过程中的重要步骤，并且是我们的小分子先导化合物的靶标：1）NSC 23766，一种选择性Rac GT3抑制剂，和2）phox-I，一种选择性p67 phox抑制剂phox-I。在初步研究中，我们已经确定：（1）Rac GTP酶Rac 1和Rac 2是基因靶向研究中中性粒细胞迁移和超氧化物产生的重要调节剂，（2）我们合理设计的Rac特异性小分子抑制剂NSC 23766在包括中性粒细胞在内的各种血细胞谱系中功能性抑制Rac活性，（3）我们的先导化合物phox-I，与p67 phox的Rac相互作用口袋结合，并有效抑制原发性嗜中性粒细胞中的氧化酶活性，和（4）fMLP诱导的肺部炎症小鼠模型可用于筛选治疗急性和慢性肺部炎症病症的新化合物。因此，目前的I期STTR提案侧重于利用我们对Rac GTP酶对NOX 2 NADPH氧化酶的分子调控的详细了解，以开发针对急性和慢性肺部疾病的一流治疗方法。将在以下研究中验证NSC 23766和phox-I的靶点：A）体外蛋白质-蛋白质相互作用研究，B）小鼠中性粒细胞研究和C）人中性粒细胞研究（Aim 1）;体内毒性和药代动力学测试，并确定NSC 23766和phox-I在急性肺部炎症小鼠模型（Aim 2）中的疗效。长期目标是提供一种新的药物靶点，Rac-p67 phox信号传导轴，以及靶向该关键信号传导位点的合适的先导化合物，用于治疗急性肺部炎症性疾病ALI/ARDS。 公共卫生相关性：急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）是严重的呼吸衰竭，在包括大手术、创伤、多次输血和脓毒症的临床事件后出现。一般而言，肺气道疾病是美利坚合众国发病率和死亡率的主要原因之一。ALI/ARDS的相关死亡率为40-80%，目前的药理学方法在降低死亡率方面并不成功。因此，开发用于治疗肺部疾病的新疗法至关重要。急性肺损伤和急性呼吸窘迫综合征与嗜中性粒细胞相关炎症升高有关。目前的I期STTR提案将测试我们开发的两种中性粒细胞功能小分子抑制剂对肺部炎症性疾病的疗效。因此，我们的研究将有助于提供一个新的药物靶点，以及合适的先导化合物用于治疗肺部炎症，如ALI和ARDS。