A high throughput platform for nuclear receptor ligand and drug discovery

描述（由申请人提供）：斑马鱼是最强大和通用的系统，用于高通量筛选具有药物潜力的小分子。我们利用这些众多的优势来开发转基因鱼种，这些鱼种对针对人类转录因子家族的激素和药物有反应，这些转录因子家族被称为核受体（NRs）。NRs，以及控制其活动的小亲脂激素和药物，是当今绝大多数最严重和最昂贵的疾病的潜在监管者。我们开发的鱼线对人类NR激素和药物会发出绿色荧光。强大的亲和力标签也允许分离和鉴定结合的激素或药物。利用其中一种转基因系，我们已经进行了几次小分子筛选，并取得了巨大的成功，鉴定出了一种新的内源性激素调节因子。在发现的几十种活性化合物中，有一种看起来是治疗糖尿病和其他肥胖相关疾病的绝佳候选药物。因此，在拟建的研究项目中产生的约50个转基因鱼种，每个代表一个人类NR基因，将具有巨大的潜力，可以理解和预防许多代谢紊乱和相关疾病，如癌症、抑郁症、记忆和免疫紊乱。新的药物和激素也将具有令人难以置信的潜力来阐明由nr控制的遗传和分子途径。虽然我们的筛选系统的上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，我们需要替换系统中利用酵母转录因子GAL4的部分。GAL4的DNA结合位点极其“G/C”丰富，这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取代我们每个细胞系中的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。然而，许多nr兼任阻遏者，四分之一的nr仅担任阻遏者。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，以便在抑制存在的情况下看到荧光。设计了两种新的载体来做到这一点。本研究产生的约50个新的转基因品系将立即提供给斑马鱼群落。这些细胞系，以及在随后的筛选中发现的激素/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.