Atomic-Level Insights into the Molecular Recognition of Anticancer Naphthoquinones by Human Serum Albumin: The Role of Apolar Side Chains in Binding Stability

Naphthoquinone derivatives, specifically 01 (lawsone; 2-hydroxynaphthalene-1,4-dione), 02 (lapachol; 2-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,4-dione), and 03 (2-hydroxy-3-styrylnaphthalene-1,4-dione), represent a versatile class of natural and synthetic molecules. These compounds hold significant potential as ligands for the development of novel anticancer metal complexes. In this study, we investigated the intermolecular interactions between these ligands and Human Serum Albumin (HSA), the primary protein responsible for drug transport in the human bloodstream. To achieve this, a synergistic approach was employed, combining NMR binding-target techniques, such as Saturation Transfer Difference (STD-NMR) and 2D-NOESY, with computational tools including molecular docking, molecular dynamics (MD) simulations, and binding affinity predictions. These methods allowed for a detailed characterization of the binding interface at the atomic level, defined as Group Epitope Mapping (GEM), while also enabling the estimation of the dissociation constants (K _D​ ) for the resulting adducts. The results demonstrate that the presence of an unsaturated lateral chain significantly contributes to the stabilization of the supramolecular arrangement. Specifically, the experimental K _D​ values, 7.00 mM for 01, 1.40 mM for 02, and 1.13 mM for 03, indicate that increasing the size and apolarity of the substituent leads to more efficient HSA interaction. Furthermore, 2D-NOESY experiments suggest that these naphthoquinones are spatially directed toward peripheral aliphatic domains (alanine, leucine, and valine). In agreement with these findings, the calculated docking scores follow a consistent trend (01 < 02 < 03), with derivative 03 approaching the binding affinity of the reference ligand, warfarin. Moreover, GNINA-based affinity predictions and CNN_VS scores further corroborate that derivatives bearing apolar lateral chains exhibit superior interaction profiles. Taken together, these unprecedented results establish that incorporating apolar substituents is a robust strategy for enhancing HSA binding. Ultimately, this study provides a valuable guideline for the rational design of naphthoquinone derivatives with optimized drug-delivery properties.