PROJECT SUMMARY/ABSTRACT Targeting of oncogenic kinases with small molecule inhibitors is ow a well validated paradigm for treatment of particular cancer types. Unfortunately, resistance to ATP-competitive inhibitors typically develops due to the selection for tumor cells that harbor new mutations that induce resistance to kinase inhibitors or due to activation of by-pass signaling pathways. In addition, many tumors depend on the scaffolding function of a kinase and not on enzymatic kinase activity, thereby rendering inhibitors ineffective. For example, the pseudokinase HER3 is an obligate heterodimerization partner with EGFR and HER2 but its kinase activity is not required. In the first funding cycle, we successfully explored a fundamentally different approach to abrogate kinase signaling by developing novel small molecules that induce kinase degradation via the ubiquitin-proteasome pathway and systematically explored the degradable kinome. Here we propose to expand on this extensive fundamental work to increase coverage of the degradable kinome, further investigate the mechanism of small molecule-mediated protein degradation and expand probe development to non-degrading molecular glues. In particular, we will increase the chemical space of potential degraders to not-yet explored kinase ligand scaffolds as well as recently reported E3 ligase ligands including ligands for ligases such as IAP, MDM2, KEAP1, DCAF11, DCAF15, DCAF16, AHR, FEM1B, RNF4 and RNF114 and novel ligase attachment geometries for our previously characterized E3 ligases CRBN and VHL, and will also include the development of trivalent degrader molecules as a novel compound design approach. We have also developed new and improved global proteomics workflows to enable increased proteomic profiling of degraders in a panel of up to five different cell lineages, which will increase identified kinases from ~400 to almost 600 per compound treatment. In addition, we have developed new chemoproteomics workflows to determine ternary complex formation of glue molecules and are able to determine ubiquitination sites and profiles of degrader molecules. This extended degrader characterization data will enable us to further expand our open-source accessible degradable kinome database and will deepen our understanding of E3 ligase-dependent degradation mechanisms. We will further broaden our probe development efforts to HER3-targeting non-degrading glue molecules to investigate alternative modes of action for development of novel therapeutics. The goal of this grant application is to expand the degradable kinome database by covering an extended chemical space and expanding cellular treatment conditions, interrogate structural mechanisms of targeted protein degradation and non-degrading complexes, and assemble a HER3-targeted compound library for the development of non-degrading glue molecules as a novel therapeutic strategy to modulate HER3 activity in cancer models.