The contribution of cyclic imide stereoisomers on cereblon-dependent activity

Thalidomide, lenalidomide, and their derivatives mimic glutarimide and aspartimide protein modifications that give rise to a motif recognized by the E3 ligase substrate adapter cereblon (CRBN). These cyclic imides have a chiral center that, given the biological significance of chirality, may influence CRBN's function and therapeutic applications. Here, we systematically examine cyclic imides in small molecules, peptides, and proteins to assess their racemization, CRBN engagement, ternary complex formation <i>in vitro</i>, and resulting degradation outcomes in cells. While the thalidomide-binding domain of CRBN consistently favors the (<i>S</i>)-stereoisomer across all cyclic imide small molecule ligands and engineered proteins, we find that, in some cases, the (<i>R</i>)-stereoisomer can bind to CRBN, either enhancing or hindering the eventual target engagement and degradation. Lenalidomide and its derivatives racemize more rapidly (<i>t</i> _50%ee = 4-5 h) than the C-terminal cyclic imide under non-enzymatic conditions. These findings highlight that although the (<i>S</i>)-stereoisomer of the cyclic imide is the primary ligand for the thalidomide-binding domain of CRBN, the (<i>R</i>)-stereoisomer, if present, has the potential to contribute to CRBN-dependent cellular activity.