The goal of this project is to establish a cell-based high-throughput screening assay involving two molecular reporters to identify compounds that selectively modulate the activity of the transforming growth factor beta signaling pathway in humans. The TGF-beta pathway has a key role in the control of growth and development. In cancer, depending on context and mutational status, TGF-beta can contribute positively or negatively to tumor development. Modulating the activity of the TGF-beta pathway using small molecules may become part of successful cancer therapy. Screening efforts have so far focused on inhibiting the TGF-beta receptor. However, finding novel specific modulators (activators and inhibitors) of TGF-beta signaling downstream of the receptor, targeting components of the intracellular signaling pathway, would open new therapeutic opportunities. The reporters under development are involved in two key processes downstream of TGF-beta, nuclear translocation and transcription. We have developed a cell-based fluorescence assay to measure the cytosolic to nuclear translocation of SMAD2, a transcription factor, which is phosphorylated by the activated TGF-beta receptor complex. As a transcriptional reporter, we propose to develop a TGF-beta-inducible nitroreductase assay. Both reporters can be combined in each cell, resulting in a highly versatile, informative and robust cell-based screening system for small compound and siRNA library screening. Applications of the proposed assay may in the future contribute significantly to the understanding and treatment of cancers involving TGF-beta. Our own screen - and subsequent screens by others using our assay - may yield lead compounds for drug development. Identification of the targets of the compounds may contribute to a better understanding of the underlying biological processes of TGF-beta signaling. Under this R21 proposal, the transcriptional reporter is to be developed and the combination of both reporters in each cell is to be adapted to HTS format. Results will be shared openly with the scientific community and the assay made available to other investigators upon request. The TGF-beta pathway has a key role in the control of growth and development. In cancer, depending on context and mutational status, TGF-beta can contribute positively or negatively to tumor development. The goal of this project is to establish a high- throughput screening assay to identify chemical compounds that selectively modulate the activity of the transforming growth factor beta signaling pathway in humans. Modulating the activity of the TGF-beta pathway using small molecules may become part of successful cancer therapy. ? ? ?
