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.
The proposed project will generate approximately 50 new transgenic fish lines, each representing a different drug discovery platform for one of the most important family of human drug targets - the nuclear receptors. Each of these lines is expected to produce novel green fluorescent protein expression patterns over the course of development, which can be used to visualize, probe and understand developmental and physiological programs in live animals. Hormones and drugs discovered using these fish will also serve as powerful tools for the understanding of metabolic and endocrine based disorders such as diabetes, Alzheimer's and cancer.
