Unbiased mapping of cereblon neosubstrate landscape by high-throughput proteomics.

Molecular glue degraders (MGDs) are small molecules that co-opt the ubiquitin-proteasome system to induce degradation of target proteins, including those considered undruggable. Their discovery remains challenging due to the lack of rational design strategies and limited throughput of unbiased proteome-wide screening approaches. To address this gap, we develop a high-throughput proteomics platform based on label-free, data-independent acquisition mass spectrometry (DIA-MS), enabling integrated proteomics and ubiquitinomics profiling. Screening a diverse set of 100 cereblon (CRBN)-recruiting ligands on this platform leads to identification of a broad array of novel degraders and neosubstrates. Subsequent hit validation and structure-degradation relationship analyses guided by global proteomics reveal highly selective and potent phenyl glutarimide-based degraders targeting previously uncharacterized neosubstrates such as KDM4B, G3BP2 and VCL; none of which contain the classical CRBN β-hairpin degron. These findings underscore the power of unbiased high-throughput proteomics in MGD drug discovery and reveal a substantially expanded CRBN neosubstrate landscape beyond that defined by classical immunomodulatory imid drugs (IMiDs).