Project Summary: Proteolysis Targeting Chimeras (PROTACs) have introduced a new pharmacological paradigm, event-driven pharmacology. PROTACs are bifunctional small-molecules that simultaneously engage an E3 ubiquitin ligase and a protein of interest (POI). Ternary formation induced by PROTAC binding results in ubiquitination of the POI by the E3 ligase and subsequent degradation of the POI by the 26S proteasome. Most research efforts have focused on increasing POI diversity. However, identification and development of E3 ligase recruiting elements (E3REs) has lagged. The next innovation for PROTAC technology is the induction of tumor-specific protein degradation. PROTACs that induce degradation only in tumor cells are likely to have decreased off-target cytotoxicity, thereby improving their therapeutic utility. However, the E3 ligases most commonly recruited, Von Hippel-lindau, Cereblon, and Mouse double minute 2 homolog, are expressed in both cancerous and untransformed tissues. New E3REs must be developed that engage E3 ligases with tumor specific expression to impart tumor-specificity. Type I Melanoma Antigen Gene (MAGE) family proteins are cancer testis antigens, whose expression is restricted to the male germ line, but can be re-expressed in cancers. MAGE-A3 binds TRIM28, a ubiquitously expressed protein with E3 ligase activity, to form an oncogenic tumor-specific E3 ligase complex. A PROTAC harboring a MAGE-A3 E3RE may be able to recruit MAGE-A3/TRIM28 and induce tumor-specific degradation. MAGE-A3 has been found to exist as a dimer in solution. A fluorescent peptide that mimics key residues involved in this dimerization will be used to develop a fluorescence polarization assay (FP). The FP assay will then be used to identify a small-molecule ligand of MAGE-A3. Orthogonal biophysical assays, like thermal shift, intrinsic Trp fluorescence, and isothermal calorimetry will then be used to confirm binding. Once identified, the MAGE-A3 ligand will be used as an E3RE in the synthesis of a MAGE-A3 based PROTAC. Cellular experiments using the HaloTag7-GFP reporter system developed in the Crews Lab will then be used to test the activity of MAGE-A3 based PROTACs. This project will determine if a MAGE-A3 ERE3 can be used to recruit the MAGE-A3/TRIM28 E3 ligase complex to induce tumor-specific protein degradation. PROTACs created during this project may serve as the starting point for the future development of a tumor-specific therapy.