Cross‐Linking Profiling of Molecular Glue Degrader‐Induced E3 Ligase Interactome to Expand Target Space

Abstract Molecular glue (MG) degraders, small molecules with significant therapeutic potential for targeting undruggable proteins, are emerging as new modality in drug discovery. Profiling the E3 ligase interactome induced by MG degraders provides insights into their mechanism of action and identifies clinically relevant neosubstrates for degradation, thereby offering new therapeutic opportunities. However, established methods face significant challenges in comprehensive and accurate profiling of MG degrader‐induced E3 ligase interactome. Herein, we introduce the concept of globally cross‐linking profiling of the MG degrader‐induced E3 ligase interactome in living cells, achieved by integrating genetic code expansion technology with mass spectrometry‐based proteomics. Our approach presents an efficient and robust strategy for identifying neosubstrates recruited to cereblon E3 ligase by the known degraders CC‐885 and DKY709, offering valuable insights for clinical evaluation and significantly expanding their target space. Moreover, we developed two novel MG degraders with potent antiproliferative effects on cancer cells, and application of our method identified neosubstrates, revealing a previously unrecognized target landscape and advancing our understanding of E3 ligase–neosubstrate interactions. Overall, our study provides a powerful tool for neosubstrate identification and expanding target space of E3 ligase, opening new opportunities for developing next‐generation MG degraders to address the clinical challenge of undruggable targets.