Axial Symmetry Directs Ligand Orientation in Galectin-3

Programmatic control over bound ligand poses remains a significant challenge in drug design due to the lack of universal design principles that could be harnessed to direct the ligand orientation in the binding site. Here, we demonstrate that molecular symmetry, combined with the high specificity of lectin-carbohydrate recognition, can be utilized to control the binding of small molecules to galectin-3. We synthesized a series of galectin-3 ligands that differ in the C₂-rotational symmetry features of their saccharide cores, imposing direct control over their binding behavior. Using a combination of ligand-observed and protein-observed NMR spectroscopy, complemented by free energy calculations, we were able to comprehensively describe the symmetry-resolved binding poses within this ligand series as well as the underlying thermodynamics. Our work offers new strategies for designing galectin-3 inhibitors with predictable binding behavior and the delivery of substituents to defined locations within the binding pocket.