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: 8442908
• 负责人: SHUJI KISHI
• 金额: $ 35.92万
• 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/8442908
• 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; 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; screening; 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.

描述（由申请人提供）：该提案解决的重要生物医学科学问题是需要新药治疗与氧化应激有关的代谢和退化性疾病。这种多种慢性疾病的范围与对环境和内源性压力源不断挑战的许多组织和器官的氧化损害有关。细胞通过响应氧化应激而激活抗氧化剂基因来保护自己免受氧化损伤，但是在衰老过程以及在某些情况下，在压力过高的某些情况下，保护性反应受到损害或不足。但是，由于这种损害的基本缺乏治疗剂。因此，选择性抑制对特定组织和器官的氧化损伤的药物具有很大的治疗价值。生理抗氧化剂反应基本上涉及通过转录因子NF-E2相关因子2（NRF2）的结合，由抗氧化反应调节元件（IS）驱动的一组基因的转录激活。 NRF2/是调节大多数抗氧化剂酶的表达，包括PI类谷胱甘肽S-转移酶（GSTP1）；实际上，当动物用电力处理时，脊椎动物的NRF2激活后，GSTP1强烈诱导。该提案描述了通过使用基于细胞的和基于动物的高通量屏幕的组合顺序方法的发展，这些屏幕在佛罗里达州Scripps唯一可行。在AIM 1中，除了传统的基于CEL的响应性记者基因方法外，还将采用新的基于细胞的NRF2降解的记者策略，以识别促进对氧化应激的内源性反应的药物铅，从而防止氧化损伤。这些基于细胞的测定方法可以通过对大约100万种化合物组成的化学文库进行超高吞吐量筛选，并具有已知具有适合药物开发的特性的化学库来有效地追求。在AIM 2中，我们将使用一种新的转基因斑马鱼表达GSTP1启动子驱动的增强的绿色荧光蛋白（GFP）报道器系统的大量全体生物屏幕，我们将在实时生物体中的二级筛选策略中使用象征性的化合物1。 同时地。可以进一步评估斑马鱼胚胎和幼虫筛查中经过验证的化学命中，以确定器官特异性，随着鱼的生长和预防氧化损伤的功效。 AIM 3旨在了解这些选择的小分子如何在体内起作用，以便可以根据抗氧化剂反应的激活机理设计其他药物。将利用化学遗传方法，例如针对合理的信号通路以及基于形态的遗传操纵的化学抑制剂，以确定激活剂和/或在筛选分析中鉴定出的抗氧化信号通路的哪些成分。