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: 8841343
• 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/8841343
• 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; 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; animal imaging; base; cofactor; design; drug candidate; drug discovery; high throughput screening; imaging platform; novel; programs; promoter; protein expression; 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）：斑马鱼是用于高通量筛选具有药物潜力的小分子的最强大和通用的系统。我们利用这些众多优势来开发转基因鱼品系，这些鱼品系能够对针对人类转录因子家族（称为核受体（NR））的激素和药物做出反应。 NR 以及控制其活性的小型亲脂性激素和药物，是当今绝大多数最严重、最昂贵的疾病的潜在调节剂。我们开发的鱼线通过发出绿色荧光来响应人类 NR 激素和药物。强大的亲和标签还可以分离和识别结合的激素或药物。利用这些转基因品系之一，我们已经进行了几次小分子筛选，取得了巨大成功，并鉴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，其中一种看起来是治疗糖尿病和其他肥胖相关疾病的极好的候选药物。因此，在拟议的研究项目中产生的约 50 个转基因鱼系，每个代表人类 NR 基因之一，将在理解和预防众多代谢紊乱和相关疾病（如癌症、抑郁症、记忆和免疫紊乱）方面具有巨大的潜力。新药物和激素在阐明 NR 控制的遗传和分子途径方面也具有令人难以置信的潜力。虽然我们的筛选系统的先前版本已经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，我们需要替换系统中使用酵母转录因子 GAL4 的部分。 GAL4 的 DNA 结合位点极其丰富，这会导致我们的荧光报告基因立即或最终沉默。我们通过用细菌转录因子 LexA 替换每个品系中的 GAL4 成分来解决这个问题，LexA 的结合位点为 34% G/C，而 GAL4 的结合位点为 68% G/C。我们最初的系统的第二个问题是它是为可以激活目标基因转录的 NR 设计的。然而，许多 NR 兼职充当阻遏蛋白，四分之一的 NR 仅充当阻遏蛋白。因此，为了观察影响这些 NR 活性的激素和药物，我们需要重新设计我们的系统，以便在存在抑制的情况下看到荧光。两个新的载体被设计来做到这一点。本研究中产生的约 50 个新转基因品系将立即提供给斑马鱼群体。这些细胞系以及随后筛选中发现的激素/药物将为监测、控制、破坏和理解发育过程中发育、生理、代谢和行为的相关方面提供强大的试剂。