Functional Assays for Stress Biosensor Probes in the Nrf2-ARE System

Nrf2-ARE 系统中应激生物传感器探针的功能测定

• 批准号: 8276407
• 负责人: SHUJI KISHI
• 金额: $ 37.22万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8276407
• 关键词: Adaptor Signaling Protein; Address; Aging-Related Process; Agonist; Animal Model; Animals; Antioxidants; Aryl Hydrocarbon Receptor; Binding; Biological Assay; Biological Availability; Biosensor; Cell Line; Cells; Chemicals; Chimeric Proteins; Chronic; Chronic Disease; Chronic Obstructive Airway Disease; Complement 3; Complex; Cyclic AMP-Dependent Protein Kinases; Cysteine; Degenerative Disorder; Detection; Developing Countries; Development; Diabetes Mellitus; Diabetic Neuropathies; Diet; Disease; Embryo; Enzymes; Fatty acid glycerol esters; Fishes; Florida; Fluorescence; Galactosidase; Generations; Genes; Glutathione S-Transferase; Goals; Homeostasis; Huntington Disease; Image; Impairment; LacZ Genes; Lactamase; Life; Ligands; Ligase; Literature; Mammalian Cell; Measurement; Measures; Mediating; Metabolic; Metabolic Diseases; Monitor; Morphologic artifacts; N-terminal; NF-E2-related factor 2; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Organ; Organ Specificity; Organism; Oxidative Stress; Parkinson Disease; Pharmaceutical Preparations; Pharmacology; Physiological; Prevalence; Prevention; Property; Protein Kinase C; Proteins; Receptor Signaling; Regulation; Regulatory Element; Reporter; Reporter Genes; Resistance; Response Elements; Screening procedure; Second Messenger Systems; Series; Signal Pathway; Signal Transduction; Societies; Stress; Stress Response Signaling; Structure; System; Testing; Therapeutic; Therapeutic Agents; Tissues; Toxic effect; Transcriptional Activation; Transgenic Organisms; Triage; Vertebrates; Whole Organism; Work; Zebrafish; base; biological adaptation to stress; blood glucose regulation; chemical genetics; citrate carrier; combinatorial; cullin-3; design; diethyl maleate; drug candidate; drug development; genetic manipulation; ghrelin; glucagon-like peptide; glutathione S-transferase pi; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; lipid biosynthesis; novel; overexpression; oxidative damage; prevent; promoter; receptor; response; second messenger; small molecule; small molecule libraries; stressor; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): The important biomedical scientific problem addressed by this proposal is the need for new drugs to treat metabolic and degenerative disorders related to oxidative stress. A spectrum of such multiple chronic disorders is associated with oxidative damage to many tissues and organs that are constantly challenged by environmental and endogenous stressors. Cells protect themselves from oxidative damage by activation of antioxidant genes in response to oxidative stress, but during the aging process as well as under certain situations where stress is excessive, the protective response is impaired or inadequate. However, there is a fundamental lack of therapeutic agents against such damage. Therefore, drugs that selectively inhibit oxidative damage to specific tissues and organs would be of great therapeutic value. The physiological antioxidant response essentially involves transcriptional activation of a battery of genes driven by an antioxidant response regulatory element (ARE) through binding of the transcription factor NF-E2-related factor 2 (Nrf2). Nrf2/ARE regulates expression of most antioxidant enzymes including the Pi class glutathione S-transferases (GSTP1s); indeed, gstp1 is strongly induced upon Nrf2 activation in vertebrates when the animals are treated with electrophiles. This proposal describes the development of a combinatorial sequential approach by using both cell-based and animal-based high-throughput screens that are uniquely feasible at Scripps Florida. In Aim 1, in addition to the traditional cel-based ARE-responsive reporter gene approach, a new cell-based Nrf2 degron- fused reporter strategy will be employed in order to identify drug leads that promote an endogenous response to oxidative stress and thereby prevent oxidative damage. These cell-based assays can be efficiently pursued by complimenting ultra-high throughput screening of a chemical library comprised of approximately 1 million compounds with structures known to have properties suitable for drug development. In Aim 2, a high-volume whole-organism screen, which we would have developed, using a new transgenic zebrafish expressing gstp1 promoter-driven enhanced green fluorescence protein (GFP) reporter system will be used in a live organism- oriented secondary screening strategy for pre-selected chemical compounds in Aim 1. This allows us to validate developmental toxicity and bioavailability of drug candidates to be addressed simultaneously. Validated chemical hits in the zebrafish embryo and larval screen can be further evaluated to determine organ specificity as the fish grow and for efficacy in preventing oxidative damage. Aim 3 is designed to understand how these selected small molecules work in vivo so that additional drugs can be designed based on the mechanism of activation of the antioxidant response. Chemical genetic approaches, such as selective chemical inhibitors against plausible signaling pathways as well as morpholino-based genetic manipulations in zebrafish will be exploited to establish which components of the antioxidant signaling pathway are modified by activators and/or potentiators identified in the screening assays. PUBLIC HEALTH RELEVANCE: There is an urgent unmet need to develop novel drugs for the treatment and/or prevention of metabolic and degenerative disorders and to elucidate the underlying causes of these disorders. In such disorders, loss of optimal functioning is associated with increased oxidative damage to cells and impairment of the cellular mechanisms and homeostasis that normally protect cells from oxidative stress. The objectives of this proposal are to 1) exploit new cell-based high- throughput screening assays to identify drug leads that enhance resistance to oxidative damage; 2) evaluate the efficacy of such compounds in physiologically relevant novel animals models; and 3) understand how these compounds act, to further develop and characterize a novel series of drugs for treating metabolic and degenerative disorders.

描述（由申请人提供）：本提案解决的重要生物医学科学问题是需要新的药物来治疗与氧化应激相关的代谢和退行性疾病。这种多种慢性疾病的频谱与许多组织和器官的氧化损伤有关，这些组织和器官经常受到环境和内源性应激源的挑战。细胞通过激活抗氧化基因来应对氧化应激，保护自己免受氧化损伤，但在衰老过程中以及在压力过大的某些情况下，保护反应受损或不足。然而，从根本上缺乏治疗这种损伤的药物。因此，选择性抑制特定组织和器官氧化损伤的药物将具有很大的治疗价值。生理抗氧化反应本质上涉及由抗氧化反应调控元件（ARE）通过结合转录因子nf - e2相关因子2 （Nrf2）驱动的一系列基因的转录激活。Nrf2/ARE调节大多数抗氧化酶的表达，包括Pi类谷胱甘肽s -转移酶（GSTP1s）；事实上，在脊椎动物中，当动物被亲电试剂处理时，gstp1强烈地诱导Nrf2激活。该提案描述了一种组合序列方法的发展，通过使用基于细胞和基于动物的高通量筛选，这在Scripps Florida是唯一可行的。在Aim 1中，除了传统的基于细胞的ARE-responsive报告基因方法外，将采用一种新的基于细胞的Nrf2 degron融合报告基因策略，以确定促进氧化应激内源性反应的药物线索，从而防止氧化损伤。这些基于细胞的分析可以通过对化学文库的超高通量筛选来有效地进行，化学文库由大约100万种已知结构具有适合药物开发的特性的化合物组成。在Aim 2中，我们将使用一种表达gstp1启动子驱动的增强绿色荧光蛋白（GFP）报告系统的新型转基因斑马鱼，开发出一种大容量的全生物筛选方法，用于Aim 1中预先选择的化合物的活体定向二级筛选策略。这使我们能够同时验证候选药物的发育毒性和生物利用度。在斑马鱼胚胎和幼虫筛选中验证的化学物质可以进一步评估，以确定鱼类生长过程中的器官特异性和防止氧化损伤的功效。目的3旨在了解这些选定的小分子如何在体内起作用，以便根据激活抗氧化反应的机制设计其他药物。化学遗传学方法，如选择性化学抑制剂对可能的信号通路，以及斑马鱼基于morpholino的遗传操作，将被用来确定抗氧化信号通路的哪些成分被筛选试验中确定的激活剂和/或增强剂修饰。