PROJECT SUMMARY This is a renewal application of R37 GM058867 which has supported our foundational efforts in chemical glycobiology tool development since 1999. In the next granting period we will focus our efforts on a new chemical biology platform for targeted degradation of extracellular proteins. Targeted protein degradation platforms such as proteolysis targeting chimeras (PROTACs) are now well-established as powerful strategies to address canonically “undruggable” proteins. However, canonical PROTAC approaches involve manipulation of a cytosolic protein degradation machinery and therefore are fundamentally limited to targets with ligandable cytosolic domains. This requirement excludes most secreted and cell-surface membrane-associated proteins, which are estimated to comprise 40% of protein-encoding genes and are key agents in cancer, aging-related diseases, and autoimmune disorders. Thus, there has been a recent surge of interest in new approaches for targeted degradation of extracellular proteins, with a particular focus on harnessing the endosome-lysosome pathway. The work proposed herein focuses on what we believe to be a leading technology in this space. We developed “lysosome targeting chimeras” (LYTACs) that direct proteins of interest to lysosomes via engagement of the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs comprise a binding element (e.g., an antibody or small molecule ligand) specific to the extracellular target protein, conjugated to mannose-6-phosphate (M6P) analogs that engage CI-M6PR. The receptor endogenously transports lysosomal enzymes marked with M6P caps on N-glycans residues to their destination organelle by cycling continuously between endosomes, the cell surface, and the Golgi complex. CI-M6PR has been exploited to deliver therapeutic enzymes for treatment of lysosomal storage disorders. However, prior to our work, this lysosome delivery system had not been contemplated as a vehicle for targeted degradation. In preliminary work we used bioorthogonal chemistries to conjugate ligands or antibodies that bind a protein of interest to synthetic CI-M6PR engagers. We demonstrated that both soluble extracellular proteins and membrane-bound cell-surface proteins can be targeted for degradation by LYTACs. These preliminary studies set the stage for expansion of the program to include fundamental studies of LYTAC scope and mechanism as well as translational therapeutic applications. The Specific Aims of this project are to (1) synthesize homogeneous LYTACs and optimize structures for in vitro and in vivo applications, (2) characterize the LYTACable proteome, and (3) apply LYTACs in therapeutic models that involve soluble and cell-surface membrane-bound targets.