A high throughput platform for nuclear receptor ligand and drug discovery

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。我们已经利用这些众多的优势来开发转基因鱼类，这些鱼类对靶向称为核受体（NR）的人类转录因子家族的激素和药物有反应。核受体，以及控制其活性的小的亲脂性激素和药物，是当今绝大多数最严重和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对人类NR激素和药物做出反应。强大的亲和标签还允许分离和鉴定结合的激素或药物。使用这些转基因品系之一，我们已经进行了几次小分子筛选，取得了很大的成功，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，有一种看起来是治疗糖尿病和其他肥胖相关疾病的优秀候选药物。因此，在拟议的研究项目中产生的约50个转基因鱼系，每个代表一个人类NR基因，将具有巨大的潜力，用于理解和预防许多代谢疾病和相关疾病，如癌症，抑郁症，记忆和免疫疾病。新的药物和激素也将具有令人难以置信的潜力，用于阐明由NR控制的遗传和分子途径。虽然我们的筛选系统的前一个版本已经验证了这个平台的潜力，但我们需要添加额外的功能，使其功能齐全，易于共享。首先，我们需要替换一部分利用酵母转录因子GAL 4的系统。GAL 4的DNA结合位点非常富含“G/C”，这导致我们的荧光报告基因立即或最终沉默。我们正在解决这个问题，用细菌转录因子莱克萨（LexA）的结合位点取代了我们每个细胞系中的GAL 4组分，其结合位点为34% G/C，而GAL 4的结合位点为68% G/C。我们最初的系统的第二个问题是，它是为能够激活靶基因转录的NR设计的。然而，许多NR部分时间作为阻遏物，四分之一的NR仅作为阻遏物。因此，为了观察影响这些NR活动的激素和药物，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。两个新的载体已经被设计来做到这一点。在这项研究中产生的约50个新的转基因品系将立即提供给斑马鱼社区。这些细胞系，以及在后续筛选中发现的激素/药物，将为监测、控制、干扰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。

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.