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Unbiased mapping of cereblon neosubstrate landscape by high-throughput proteomics

ABSTRACT: Molecular glue degraders (MGDs) are small molecules that harness the ubiquitin-proteasome system to induce degradation of target proteins, including those lacking conventional druggable pockets. Given the challenges in their rational design, MGD discovery predominantly relies on screening-based approaches, such as cell viability assays. However, one potential limitation of such screening methods is the risk of overlooking non-essential neosubstrates of potential therapeutic value. To address this concern, we present a high-throughput proteome-wide MGD screening platform utilizing label-free, data-independent acquisition mass spectrometry (DIA-MS) for integrated proteomics and ubiquitinomics analysis. Processing a diverse set of 100 CRBN-ligands across two cancer cell lines reveals a broad array of neosubstrates, including 50 novel candidates validated by MS-based ubiquitinomics. These findings considerably expand the current landscape of CRBN-mediated neosubstrates. Comprehensive hit validation and structure-degradation relationship analyses guided by global proteomics, identifies highly selective and potent phenyl glutarimide-based degraders of novel neosubstrates, including KDM4B, G3BP2 and VCL, none of which contain the classical CRBN degron motif. This study demonstrates that comprehensive, high-throughput proteomic screening offers new opportunities in MGD drug discovery.

INSTRUMENT(S): timsTOF Pro, timsTOF HT, timsTOF Ultra 2

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Zoran Rankovic

PROVIDER: MSV000096721 | MassIVE | Fri Dec 20 23:41:00 GMT 2024

SECONDARY ACCESSION(S): PXD059111

REPOSITORIES: MassIVE

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