posted on 2022-01-03, 19:39authored byZhi Lin, Yuka Amako, Farah Kabir, Hope A. Flaxman, Bogdan Budnik, Christina M. Woo
The thalidomide analogue lenalidomide
(Len) is a clinical therapeutic
that alters the substrate engagement of cereblon (CRBN), a substrate
receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development
of photolenalidomide (pLen), a Len probe with a photoaffinity label
and enrichment handle, designed for target identification by chemical
proteomics. pLen preserves the substrate degradation profile, phenotypic
antiproliferative and immunomodulatory properties of Len, and enhances
interactions with the thalidomide-binding domain of CRBN, as revealed
by binding site mapping and molecular modeling. Using pLen, we captured
the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells
and further identified a new target, eukaryotic translation initiation
factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly
labeled by pLen and forms a ternary complex with CRBN in the presence
of Len across several epithelial cell lines but is itself not ubiquitylated
or degraded. These data point to the existence of a broader array
of targets induced by ligands to CRBN that may or may not be degraded,
which can be identified by the highly translatable application of
pLen to additional biological systems.