Site-resolved assessment of targeted protein degradation

Induced proximity using small molecules, exemplified by targeted protein degradation (TPD), represents a highly promising therapeutic strategy with significant untapped potential. However, evaluating an induced proximity event that accurately reflects drug binding typically requires the challenging and costly development of specific ligands, which limits the advancement of medicines based on this modality. To overcome this bottleneck, we combine genetic code expansion with ultra-fast bioorthogonal chemistry to sensitize specific protein sites at single-residue resolution to a generic bioorthogonal proximity inducer (BPI) molecule. Mammalian cells expressing sensitized mutants of the ubiquitin E3 ligases VHL and CRBN exhibit neosubstrate degradation in the presence of a BPI equipped with a ligand targeting bromodomain and extraterminal (BET) proteins. Furthermore, we demonstrate E3-independent degradation through recruitment of an upstream E2 conjugating enzyme. We anticipate that this approach will have broad applicability, enabling comprehensive assessment of the scope of induced proximity.