Molecular mechanism of interspecies differences in the binding affinity of TD139 to Galectin-3.

Galectin-3 (Gal-3), a β-galactoside binding lectin, has been implicated in a plethora of pathological disorders including fibrosis, inflammation, cancer, and metabolic diseases. TD139 - a thio-digalactoside inhibitor developed by Galecto Biotech as a potential therapeutic for idiopathic pulmonary fibrosis, is the most advanced small-molecule galectin-3 inhibitor in clinical studies. It binds to human Gal-3 with high affinity but has lower affinity towards mouse and rat homologs, which is also manifested in the differential inhibition of Gal-3 function. Using biophysical methods and high resolution X-ray co-crystal structures of TD139 and Gal-3 proteins, we demonstrate that a single amino acid change corresponding to A146 in human Gal-3 is sufficient for the observed reduction in the binding affinity of TD139 in rodents. Site-directed mutagenesis of A146V (in human Gal-3) & V160A (in mouse Gal-3) was sufficient to interchange the affinities, mainly by affecting the off rates of the inhibitor binding. Additionally, molecular dynamics simulations of both wild type and mutant structures revealed the sustained favorable non-covalent interactions between the fluorophenyl ring and the active site A146 (human Gal-3 and mouse V160A) that corroborate the finding from biophysical studies. Current findings have ramifications in the context of optimization of drug candidates against Gal-3.