Development of Photolenalidomide for Cellular Target Identification

Abstract

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.

Molecular and structural characterization of lenalidomide-mediated sequestration of eIF3i

“…To study the effect of lenalidomide on the eIF3 complex, we initially examined the protein associations of endogenous eIF3i after coimmunoprecipitation from HEK293T cells stably expressing CRBN (HEK293T-CRBN) with or without lenalidomide at 50 µM, 9 a concentration relevant to the anti-inflammatory and antiangiogenetic uses of lenalidomide. 18-20 Given the direct interactions of eIF3i with eIF3b and eIF3g elucidated in the cryo-EM and crystal structure of the eIF3 subcomplex (b:g:i) in the eIF3 complex, 14, 21, 22 we found that eIF3i coimmunoprecipitated with eIF3b and eIF3g, as expected ( Figure 1b ).…”

“…Taken together, these data indicate that eIF3i is a direct target of CRBN and lenalidomide in MM.1S and MOLM13 cells, and that this interaction becomes observable when other targets such as IKZF1 are degraded ( Figure 5c ). Although the degradation of IKZF1 is almost complete at 8 h in both MM.1S and MOLM13 ( Figure S11, S13 ), 5-day incubation with lenalidomide was reported to be needed for antiproliferative effects in MM.1S and MOLM13, 2, 9, 30 which indicates that additional factors may contribute to the cytotoxicity of lenalidomide.…”

“…Previously, we observed the sequestration of eIF3i in epithelial cell lines, but not in multiple myeloma MM.1S cells, potentially due to the stronger recruitment of IKZF1 to CRBN by lenalidomide. 9 However, as cytotoxicity starts at 5 d incubation of lenalidomide while IKZF1 is long depleted by the CRBNlenalidomide complex, 2,9,30 we hypothesized that the proteome without lenalidomide target IKZF1 can serve as a new proteome, where subsequent complexation with eIF3i may occur. We first monitored the protein levels of IKZF1, IKZF3 and eIF3i in MM.1S cells over time (Figure S11).…”

“…6,7,8 In our pursuit to discover additional targets of lenalidomide, we recently reported the eukaryotic translation initiation factor 3 subunit i (eIF3i) as a novel target for lenalidomide in epithelial cells using a photoaffinity labeling (PAL) and chemical proteomics strategy. 9 eIF3i is a subunit of the eIF3 complex, which is composed of 13 subunits and involved in the protein translation initiation process, including regulation of ribosomal complexes such as 40S and 60S prior to initiation, and 43S pre-initiation complex in mRNA scanning and recognition. [10][11][12] We previously found that eIF3i is stabilized in a complex with CRBN and lenalidomide, and unlike other known targets of lenalidomide, is not degraded upon recruitment to CRBN.…”

Cereblon covalent modulation through structure-based design of histidine targeting chemical probes

“…A lenalidomide derivative bearing a diazirine photoaffinity handle was recently shown to label His353 in CRBN following UV irradiation, 47 through a mechanism that may involve nucleophilic attack of the imidazole residue on the diazo intermediate. 48 This study and ours reveal the surprisingly high reactivity of His353 in CRBN (a residue that is highly conserved across species) and we thus speculate that this may reflect its possible role in the endogenous function of CRBN.…”