Development of photo-lenalidomide for cellular target identification

Abstract

The thalidomide analog lenalidomide 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 photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, 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 photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/452075v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1619364org.highwire.dtl.DTLVardef@113a07corg.highwire.dtl.DTLVardef@18d36acorg.highwire.dtl.DTLVardef@8e64_HPS_FORMAT_FIGEXP M_FIG C_FIG