The thalidomide analog lenalidomide is a clinical therapeutic that alters substrate binding to cereblon (CRBN), an E3 ubiquitin ligase factor. However, substrate discovery largely relies on the indirect measurement of depleted substrates by global proteomics, which overlooks sequestered substrates that are not degraded. Here, we report a chemoproteomic strategy for direct target identification using photo-lenalidomide as a probe. Photo-lenalidomide labels and degrades known targets of lenalidomide and possesses similar anti-angiogenetic and anti-inflammatory properties. Using photo-lenalidomide, we identified eukaryotic translation initiation factor 3 subunit i (eIF3i) as a non-degraded substrate of the lenalidomide–CRBN complex. Lenalidomide stabilizes eIF3i in a complex with CRBN, which sequesters eIF3i from the eIF3 complex and inhibits angiogenesis in endothelial cells. Thus, lenalidomide promotes ternary complexes with CRBN that extend beyond protein degradation to targeted protein sequestration, which are readily discovered via the highly translatable strategy for direct target identification with photolenalidomide.

Lenalidomide promotes a stabilized complex with CRBN and eIF3i revealed by chemical proteomics

ACS Fall 2021

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