NMR-based Analysis of the Interaction between Pomalidomide and the C-terminal Domain of Cereblon

초록

Cereblon (CRBN) is a substrate receptor of the CRL4 E3 ubiquitin ligase complex and a key component in the mechanism of action of immunomodulatory drugs (IMiDs) such as pomalidomide. In this study, we investigated the molecular interaction between pomalidomide and the C-terminal thalidomide-binding domain of CRBN (CdCRBN) using solution NMR spectroscopy, isothermal titration calorimetry (ITC), and fluorescence-based competition assays. Two glycine residues, G354 and G383, located near the ligand-binding site, were employed as markers to monitor binding-induced chemical shift perturbations. The 1H, 15N-HSQC titration revealed concentration-dependent chemical shift changes, indicating fast exchange kinetics. The dissociation constant (Kd) calculated by NMR was 55 ± 1.8 μM, while ITC analysis showed a stronger binding affinity (Kd = 12.5 μM), suggesting an enthalpy-driven interaction. A fluorescence resonance energy transfer (FRET)-based competition assay using MANT-uracil further supported the interaction, yielding a Ki of 2.1 μM. These results confirm that pomalidomide binds to the canonical thalidomide-binding pocket of CdCRBN, providing a structural and thermodynamic basis for its function as a molecular glue.

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