Indirect ubiquitination independent of endogenous ubiquitination machinery for targeted protein degradation

Heterobifunctional small degrader molecules that hijack endogenous E3 ubiquitin ligases have attracted attention for the rapid and irreversible knock-down of target proteins via ubiquitination. However, the formation of appropriately oriented E3 ligase-target complexes is required for efficient ubiquitination of the target, which complicates the molecular optimization and leads to acquired drug resistance caused by the loss of E3 ligase activity and mutations at the E3-target interfaces. Here, we report on indirect ubiquitination as a chemical strategy for E3-indepedent ubiquitin-tethering to the target substrate. Comprising a ligand molecule and a ubiquitin moiety, the designed chimeric molecule enables the ubiquitination of the target proteins via non-covalent interactions, which lead to the proteasomal degradation of recombinant Bcl-2 and NF-κB p50, and intracellular endogenous Bcl-2. Indirect ubiquitination offers a design platform for non-covalent tethering of a ubiquitin-based proteolytic modifier to be added in the molecular toolbox for the targeted protein degradation.