Exploring the selectivity of molecules targeting the carbohydrate binding site in galectins

Oligo- and polysaccharides encode biological information through the “sugar code” interpreted by galectins (Gals), a family of β-galactoside–binding lectins involved in key physiological and pathological processes, making them attractive therapeutic targets. This study aimed to elucidate the molecular basis of galectin–ligand recognition to support the rational design of selective inhibitors. Using high-resolution X-ray crystallography, isothermal titration calorimetry, and site-directed mutagenesis, we investigated the interactions of novel carbohydrate-based ligands with human Gal-1, Gal-3, and Gal-7. Guided by the reference structure of benzyl 3,3′-seleno-digalactoside bound to Gal-3, we designed and synthesized a new series of monosaccharide-based ligands that exploit a Gal-3–specific cation–π interaction between the ligand aromatic moiety and residue R144 within the carbohydrate-binding site. While R144 has previously been recognized as an important determinant of ligand affinity, our results demonstrate that it also plays a crucial role in conferring selectivity over other galectins. Consistently, mutation of R144 to serine significantly reduced ligand affinity and abolished the selectivity advantage, highlighting the dual contribution of this residue to both binding and selectivity. These findings identify R144 as a key structural determinant for the rational design of selective Gal-3 inhibitors.