Lenalidomide as a molecular glue: Modulating human serum albumin self-interactions and aggregation

Abstract

Lenalidomide (LNA), a potent immunomodulatory drug, acts as a molecular glue that enhances drug-protein interaction. This study investigates the interaction between LNA and human serum albumin (HSA), focusing on structural modifications and protein aggregation. Employing a combination of spectroscopic techniques, including intrinsic fluorescence, temperature scanning, and circular dichroism (CD), alongside molecular docking and molecular dynamics (MD) simulations, we explored the drug–protein interaction dynamics and thermodynamic parameters. Our findings reveal that LNA induces both dynamic and static quenching mechanisms, primarily driven by hydrophobic interactions. Temperature scanning fluorescence showed no significant change in HSA’s melting temperature (T_m) with LNA. CD analysis indicated an increase in beta-sheet content, suggesting enhanced protein aggregation. Additionally, our analyses confirmed the increased size and stability of HSA aggregates in the presence of LNA. Docking simulations identified subdomain IIA as the primary binding site for the HSA-LNA complex, with a binding energy of −7.8 kcal·mol⁻¹. Key interactions, including hydrogen bonding and van der Waals forces, were driven by residues such as His 146, Asp 108, and Ala 194, ensuring stable and specific ligand binding. Molecular dynamics (MD) simulations revealed the temperature-dependent dynamics of HSA and its ligand-bound complex. Analyses of root mean square deviation (RMSD) and root mean square fluctuation (RMSF) indicated minor structural changes and variations in flexibility at both 298 and 310 K, with ligand binding providing slight stabilization to the protein at lower temperatures. Additional studies are warranted to fully elucidate the conformational effects induced by drug binding. Radius of gyration analysis suggested a subtle expansion of the protein upon ligand binding. Hydrogen bond analysis demonstrated stable interactions, averaging 3.0 bonds at 298 K and 3.4 bonds at 310 K, underscoring their significance in complex stabilization. Solvent-accessible surface area (SASA) analysis indicated that HSA-ligand complexes possess less dense microstructures compared to free HSA, with increased solvent accessibility at both temperatures. Interaction energy calculations and MM-PBSA studies revealed stronger binding affinities at 310 K, primarily driven by van der Waals forces, which is consistent with experimental findings. These insights into the molecular interactions between LNA and HSA provide valuable implications for drug development and therapeutic applications, highlighting the potential of LNA as a molecular glue in modulating protein interactions.