Site-Directed Covalent DNA-Encoded Library Selection for Targeted Protein Degradation

Targeted protein degradation (TPD) via the ubiquitin-proteasome system (UPS) has emerged as a powerful therapeutic strategy with the potential to tackle “undruggable” proteins by surpassing occupancy-driven inhibition. Degraders are mainly classified into molecular glue degraders (MGDs) and proteolysis-targeting chimeras (PROTACs). Despite the clinical success of MGDs such as thalidomide and the progress of over 50 degrader candidates into clinical trials, current approaches rely heavily on a small subset of E3 ligases, particularly VHL and CRBN. This leads to resistance and limits tissue specificity, underscoring the need to expand the E3 ligase repertoire and develop novel degraders. A key barrier is that PROTACable sites, where E3 ligases induce TPD, remain largely undefined, and scalable strategies for discovering new E3 ligase ligands are lacking. This proposal addresses these challenges by developing degraders from novel covalent E3 ligase ligands discovered through site-specific ligand incorporation-induced proximity (SLIP) combined with covalent DNA-encoded library (CoDEL) selection. The core of this approach is the use of activity-based protein profiling (ABPP) of hydroxy O-nucleophilic residues to guide SLIP validation, revealing new PROTACable sites on E3 ligases, followed by CoDEL selection to discover covalent ligands that engage these sites. The platform, SdCoDEL-TPD, will apply newly identified covalent ligands in PROTAC design and evaluate their efficacy for targets challenging to degrade through CRBN- or VHL- based PROTACs, such as EML4-ALK and the Bcr-AblT315I mutant. The potential of these covalent ligands to function as MGDs will also be explored. By mapping PROTACable sites and enabling the discovery of covalent ligands across previously inaccessible E3 ligases, SdCoDEL-TPD will generate unprecedented E3 ligase resources, advance rational design of degraders, expand the scope of TPD applications, and potentially deliver new therapeutic insights.