Recruitment to\nthe Proteasome Is Necessary but Not\nSufficient for Chemically Induced, Ubiquitin-Independent Degradation\nof Native Proteins

Targeted\nprotein degradation (TPD) is a promising strategy\nfor\ndrug development. Most degraders function by forcing the association\nof the target protein (TP) with an E3 Ubiquitin (Ub) ligase, which,\nin favorable cases, results in the polyubiquitylation of the TP and\nits subsequent degradation by the 26S proteasome. An alternative strategy\nwould be to create chemical dimerizers that bypass the requirement\nfor polyubiquitylation by recruiting the target protein directly to\nthe proteasome. Direct-to-proteasome degraders (DPDs) may exhibit\ndifferent characteristics than ubiquitin-dependent degraders, but\nfew studies of this type of TPD have been published, largely due to\nthe dearth of suitable proteasome ligands. To facilitate studies of\nDPDs, we report here a mammalian cell line in which the HaloTag protein\nis fused to the proteasome via Rpn13, one of the ubiquitin receptors.\nIn these cells, a chloroalkane serves as a covalent proteasome ligand\nsurrogate. We show that chimeric molecules comprised of a chloroalkane\nlinked to a ligand for the BET family of proteins or the Cdk2/7/9\nfamily of kinases result in ubiquitin-independent degradation of some\nof these target proteins. We use this system, the first that allows\nfacile degradation of native proteins in a ubiquitin-independent fashion,\nto probe two issues: the effect of varying the length of the linker\nconnecting the chloroalkane and the target ligand and the selectivity\nof degradation within the protein families engaged by the target ligand.