Binding Modes of Thalidomide Derivatives in Cereblon-Neosubstrate Complexes Revealed by Molecular Dynamics and Free Energy Calculations.

Immunomodulatory drugs such as thalidomide and its derivatives act as molecular glues by binding to the E3 ligase substrate receptor cereblon (CRBN) and promoting the selective recruitment and degradation of specific neosubstrates. Despite extensive structural and experimental characterization, the molecular origin of ligand-induced neosubstrate selectivity remains incompletely understood. Here, we present a comparative computational study of ternary CRBN-ligand-neosubstrate complexes involving two biologically relevant neosubstrates, IKZF1 and SALL4, and five thalidomide derivatives displaying distinct experimental selectivity profiles. Using molecular dynamics simulations and thermodynamic integration calculations, we analyze ligand binding poses, CRBN-ligand interactions, and protein-protein contacts within the ternary complexes. Our results show that all ligands bind CRBN through a conserved interaction network within the thalidomide-binding domain, while direct and persistent ligand-neosubstrate contacts are not observed for the most stable binding modes. Moreover, the CRBN-neosubstrate interaction patterns remain largely unchanged across ligands, and calculated relative binding free energies do not reproduce experimentally observed selectivity trends. These findings suggest that ligand-induced selectivity cannot be explained solely by static interaction patterns in the ternary complex and point to the importance of additional factors, such as CRBN conformational dynamics and kinetic effects, in controlling neosubstrate recruitment.