| Protein kinases are an important class of enzymes that catalyze the transfer of the gamma phosphate of adenosine triphosphate (ATP) on to a serine, threonine, or tyrosine residue of their protein substrate. Because these phosphorylation events are critical to intracellular signaling, protein kinase activity is highly regulated. Not surprisingly, misregulation of protein kinases has been linked to a number of disease states, including cancer, diabetes, and inflammation. For this reason protein kinases have become an attractive target for the development of therapeutics. Unfortunately, little is known about the cellular function of most kinases (based on genetic studies there is believed to be 518 protein kinases in humans). In particular, very little is known about the spatial and temporal regulation of protein kinases. In order to better understand the cellular role of protein kinases, new chemical tools must be developed. For this reason we set two goals, first, to develop a general method to generate a potent and selective bivalent inhibitor of any protein kinase, and second, to develop inhibitors that can be controlled both spatially and temporally in living cells.;The work herein encompasses utilizing the self-labeling protein O6-alkylguanine-DNA-alkyltransferase to display two ligands, one being an ATP-competitive small molecule and the other being a genetically encoded ligand which targets SRC homology 3 (SH3) domains. Through this method we were able to generate potent and selective bivalent inhibitors of the highly homologous non-receptor tyrosine kinases SRC and ABL. By altering the small-molecule inhibitor used as well as the genetically encoded ligand, we were also able to expand this method to signaling interactions beyond the SH3 domain and generate potent and selective inhibitors of the structurally divergent kinases Pim1, p38alpha, and EGFR. We show that these bivalent inhibitors can be assembled in situ in living cells. Lastly, using light-addressable nanocapsules we have been able to release our most potent bivalent inhibitor of ABL in Interleukin-3 (IL-3) independent, BCR-ABL transformed, Ba/F3 cells. Intracellular release of the bivalent ABL inhibitor conferred IL-3 dependence and in the absence of IL-3 resulted in apoptosis, indicating that this bivalent inhibitor can alter biological processes in living cells. |
