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

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 neo‐substrates 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 crosslinking 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 neo‐substrates 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 anti‐proliferative effects on cancer cells, and application of our method identified neo‐substrates, revealing a previously unrecognized target landscape and advancing our understanding of E3 ligase–neo‐substrate interactions. Overall, our study provides a powerful tool for neo‐substrate 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.