Illuminating the "dark" kinases - effects on proteome translocation and chromatin binding

Technologies for DNA sequencing and protein analysis have accelerated so rapidly that generation of infor- mation now outpaces our ability to completely understand it. As a result, many novel genes and proteins have been discovered, but their functions remain unknown. The intent of the current proposal is to illuminate the function of one group of these uncharacterized (“dark”) proteins, the kinases, which are likely to have critical activities. Human cells have over 600 kinases, but the role played by a third of them is unknown. Kinases regulate all aspects of intracellular signaling, affecting everything from growth and differentiation to protein localization and DNA repair. One key activity of kinases is to activate or inhibit transcription factors (TFs), which regulate the activity of genes in the nucleus, thereby controlling the fate and activity of the cell. Many kinases therefore are targets of pharmaceutical drug development, particularly as cancer therapeutics, due to their role in regulating TFs and other key molecules. Talus has developed the ChESS-DIA technology to follow the localization of the entire population of proteins (the “proteome”) in the nucleus that are either unbound or bound to chromatin. In the current proposal, the ChESS-DIA method will be adapted to measure the effect of specific “dark” kinases on the nuclear and genomic localization of the proteome. In Aim 1 we will chemically deplete known kinases by targeted protein degradation and use ChESS-DIA to observe the effects on all TFs. The method will be optimized to ensure the results are consistent with known effects of these kinases on TFs. We will then use the optimized ChESS-DIA protocol to analyze the effect of dark kinases on TF localization. This information is critically valuable for developing novel drugs that target cellular activities of each affected TF. In Aim 2 will analyze the effects of dark kinases in more detail, by determining the exact amino acid sites on TFs that are recognized and modified (i.e., phosphorylated) by dark kinases. As in Aim 1, the ChESS-DIA technology will be tested and optimized first with known kinases, and the subsequently used to analyze understudied dark kinases. Drugs against kinases are relatively straightforward to develop, because the protein structure is well defined and accessible for interaction with small molecule drugs. In contrast, the structures of most TFs generally are difficult targets for drug development. The information obtained through this project will not only show which dark kinases are likely to be strong drug targets, but it will also be the first identification of the specific protein sequences on TFs that represent valuable drug targets. We anticipate partnering with academic and pharmaceutical partners to develop novel therapeutics to treat a variety of diseases for which there are no effective therapies.