Signal transducers and activators of transcription (Stats) mediate a variety of extracellular signals. In response to ligand binding to its receptor, these proteins become phosphorylated on an invariant tyrosine residue located near the C-terminus, either by the kinase activity of the receptor, or by Janus kinases (Jaks) recruited to the receptor. Upon tyrosine phosphorylation, Stat proteins dimerize via opposing SH2 domain of one monomer with the pTyr of the other monomer. They also become serine phosphorylated on a residue proximal to the tyrosine phosphorylation residue that can regulate its activity. The phosphorylated bi-dentate ligated dimer then translocates to the nucleus, binds specific DNA promoter sequences and initiates transcription of designated response elements. In normal cells Stats mediate signaling from growth factor receptors and immune stimuli. However in several cancer types, Stats are constitutively activated, including two highly homologous proteins Stat5a and Stat5b, which are present in many different cancers of breast, prostate, leukemias and lymphomas. We and others have shown that disruption of Stat5a/b activity can induce cell death in some tumor cell lines. It would therefore be advantageous to have a high throughput screen (HTS) to identify small molecule inhibitors of Stat5 to block their aberrant activation of this tumor pro-survival pathway. The objective of this project is to develop a HTS to identify small molecule inhibitors that block oligomerization of Stat5 and gene transcriptional activity. There has been little effort reported in the literature on the development of small molecule inhibitors of Stat5 function.
Our specific aims are as follows: 1. Discover and optimize small molecule inhibitors that will be derived from either a) Stat5 SH2 domain inhibitor combinatorial phosphopeptide library of known Stat5 docking sites on receptors and b) screening the NCI Diversity Set of small drug-like compounds. Both readouts will employ monitoring changes in fluorescence polarization. 2. Assay and confirm potential inhibitory compounds of Stat5 oligomerization and DNA binding/function by testing the most promising compounds in a confirmatory HTS cell based reporter assay to determine their effects on gene transcription. At the conclusion of this research we expect to have established a method to rapidly screen and identify small molecule inhibitors of Stat5. This screen should allow for the more rapid development of therapeutic agents to treat a variety of diseases. ? ?
