Abstract
Purpose :
Galectin-3 is a β-galactoside-binding protein, whose inhibition has been shown to induce therapeutic effects in experimental models of ocular diseases. Although the binding site of galectin-3 has already been solved, pharmacological studies often neglect its unique polymerization pattern during drug discovery. In this study, we conducted a series of in-silico assays to derive novel insights regarding real-time behavior of galectin-3 polymerization.
Methods :
Homology model of mouse galectin-3 was prepared using human galectin-3 (PDB: 6RZJ) as template structure (sequence identity: 77.4 %). Structures of lactose, NA2, NA3, and NA4 were generated using GLYCAM and docked using AutoDock Vina (Scripps Research, USA). The docked complexes were then subjected to 100 ns of molecular dynamics (MD) simulations, and the key binding interactions were analyzed using Maestro (Schrödinger, Inc., USA). Finally, molecular mechanics with generalized Born and surface area solvation (MM/GBSA) analyses were employed to derive the binding free energy of each protein-ligand complex.
Results :
NA2, NA3, and NA4 formed stable complexes with tetrameric galectin-3 with docking scores of -10.2, -7.2, and -9.2 kcal/mol, respectively. They formed weaker interactions with monomeric, dimeric, and trimeric galectin-3 likely because of the decreased size of the binding pocket. Meanwhile, lactose favored monomeric galectin-3 with a docking score of -5.5 kcal/mol. MD simulations also revealed N-glycans forming the most stable complexes with tetrameric galectin-3, as indicated by MM/GBSA binding free energies of -40.99, -79.07, and -93.46 kcal/mol for NA2, NA3, and NA4, respectively. Curiously, as the number of sugar branches increased from NA2 to NA4, the binding free energies became more negative. Key binding residues discovered were ASN174, TRP181, and GLU184 that are part of the canonical Asn-Trp-Gly-Arg sugar binding sequence of galectin-3.
Conclusions :
Our results indicate that N-glycans form the most stable interactions with tetrameric galectin-3. As the number of sugar branches increased from NA2 to NA4, the binding free energies decreased, suggesting a correlation between higher order branch pattern and stability of ligand binding. Together, our results provide novel perspectives on the binding site of galectin-3 and recommend future pharmacological studies to consider its tetrameric state.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.