A high throughput platform for nuclear receptor ligand and drug discovery

用于核受体配体和药物发现的高通量平台

• 批准号: 8725654
• 负责人: Henry M. Krause
• 金额: $ 23.26万
• 依托单位: UNIVERSITY OF TORONTO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-26 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725654
• 关键词: Affect; Affinity; Agonist; Alzheimer' s Disease; Animals; Atlases; Bacteriophage lambda; Behavior; Binding; Binding Sites; Bypass; Chimeric Proteins; Chromatin; Communities; DNA Binding; DNA Binding Domain; Detection; Development; Developmental Process; Diabetes Mellitus; Disease; Drug Controls; Drug Targeting; Elements; Endocrine; Engineering; Family; Fishes; Fluorescence; Funding; Gene Expression; Gene Expression Profile; Genetic Transcription; Genomics; Green Fluorescent Proteins; Health; Hormonal; Hormones; Human; Image; Imagery; Immune System Diseases; Institutes; Life; Ligands; Malignant Neoplasms; Memory Disorders; Mental Depression; Metabolic; Metabolic Diseases; Metabolism; Molecular Genetics; Monitor; Nuclear Hormone Receptors; Nuclear Receptor Gene; Nuclear Receptors; Obesity; Obesity associated disease; Pathway interactions; Pattern; Pharmaceutical Preparations; Physiological; Physiology; Prevention; Protein Binding; Reagent; Reporter; Repression; Research; Research Project Grants; Response Elements; Site; Staging; System; Time; Tissues; Transcription Coactivator; Transgenic Organisms; Work; Yeasts; Zebrafish; base; cofactor; design; drug candidate; drug discovery; high throughput screening; novel; programs; promoter; protein expression; public health relevance; receptor function; research study; screening; small molecule; success; tool; transcription factor; vector

DESCRIPTION (provided by applicant): Zebrafish are the most powerful and versatile system for the high throughput screening of small molecules with drug potential. We have taken advantage of these numerous advantages to develop transgenic fish lines that respond to hormones and drugs that target the human family of transcription factors called Nuclear Receptors (NRs). NRs, and the small lipophilic hormones and drugs that control their activity, are potential regulators of the vast majority of today's most crippling and costly diseases. The fish lines that we have developed respond to human NR hormones and drugs by fluorescing green. Powerful affinity tags also allow isolation and identification of bound hormones or drugs. Using one of these transgenic lines, we have already carried several small molecule screens, with excellent success, and identified a new endogenous hormonal regulator. Of the dozens of active compounds discovered, one looks to be an excellent candidate drug for the treatment of diabetes and other obesity related diseases. Thus, the ~50 transgenic fish lines generated in the proposed research project, each representing one of the human NR genes, will have enormous potential for the understanding and prevention of numerous metabolic disorders and related diseases such as cancer, depression, memory and immune disorders. New drugs and hormones will also have incredible potential for the elucidation of the genetic and molecular pathways controlled by NRs. While a previous version of our screening system has validated the potential of this platform, we need to add additional features to make it fully functional and readily sharable. First, we need to replace a portion of the system that makes use of the yeast transcription factor GAL4. The DNA binding sites for GAL4 are extremely 'G/C' rich, which leads to immediate or eventual silencing of our fluorescent reporters. We are fixing this by replacing the GAL4 component in each of our lines with that of a bacterial transcription factor, LexA, whose binding sites are 34% G/C, as compared to 68% G/C for GAL4. A second problem with our initial system was that it was designed for NRs that can activate target gene transcription. However, many NRs act part time as repressors, and a quarter of NRs only work as repressors. Hence, to observe hormones and drugs that affect these NR activities, we need to re-engineer our system so that we see fluorescence in the presence of repression. Two new vectors have been designed to do this. The ~ 50 new transgenic lines generated in this study will be made immediately available to the zebrafish community. These lines, and the hormones/drugs discovered in subsequent screens, will provide powerful reagents for monitoring, controlling, disrupting and understanding related aspects of development, physiology, metabolism and behavior over the course of development.

描述（由申请人提供）：斑马鱼是对具有药物潜力的小分子进行高吞吐量筛选的最强大和通用的系统。我们利用了这些众多优势来开发转基因鱼类品系，以响应针对人类的转录因子核受体（NRS）的激素和药物。 NRS以及控制其活性的小型亲脂激素和药物是当今绝大多数最残酷和昂贵的疾病的潜在调节剂。我们已经开发出的鱼类通过荧光绿色对人类NR激素和药物的反应。强大的亲和力标签还允许隔离和鉴定结合的激素或药物。使用这些转基因线之一，我们已经携带了几个小分子筛选，并取得了出色的成功，并确定了一种新的内源性激素调节剂。在发现的数十种活性化合物中，人们看起来是一种用于治疗糖尿病和其他肥胖相关疾病的候选药物。因此，拟议的研究项目中产生的约50种转基因鱼类线（每个代表人类NR基因之一）将具有理解和预防多种代谢性疾病和相关疾病（例如癌症，抑郁，记忆和免疫疾病）的巨大潜力。新药和激素也将具有阐明由NRS控制的遗传和分子途径的难以置信的潜力。虽然我们的筛选系统的先前版本验证了该平台的潜力，但我们需要添加其他功能，以使其功能齐全且易于共享。首先，我们需要替换使用酵母转录因子GAL4的系统的一部分。 GAL4的DNA结合位点非常富含“ G/C”，这会导致我们的荧光记者立即或最终沉默。我们通过用细菌转录因子Lexa替换每条线的GAL4分量来解决此问题，Lexa的结合位点为34％g/c，而GAL4的结合位点为68％g/c。我们初始系统的第二个问题是它是为可以激活靶基因转录的NR设计的。但是，许多NRS兼职作为阻遏物，而四分之一的NRS仅作为压制器工作。因此，要观察影响这些NR活性的激素和药物，我们需要重新设计系统，以便在抑制存在下看到荧光。已经设计了两个新的向量来做到这一点。本研究中产生的约50条新的转基因线将立即向斑马鱼社区提供。这些线以及在随后的屏幕中发现的激素/药物将提供强大的试剂，以监视，控制，破坏和理解发展过程中发展，生理，代谢和行为的相关方面。