From Docking and Molecular Dynamics to Experimental Discovery: Exploring the Hydrophobic Landscapes of Heparanase to Design Potent Inhibitors

Heparanase (HPSE), a glycoside hydrolase that cleaves heparan sulfate chains, plays a crucial role in cancer progression by remodeling the extracellular matrix and facilitating tumor metastasis. This study employed a computational design approach to develop novel HPSE inhibitors using aminoglycoside paromomycin and neomycin analogs. These analogs feature a defined <i>N</i>-sulfation sequence combined with either charged or hydrophobic groups. Initial docking screenings indicated that hydrophobic-capped ligands exhibit binding energies comparable to the free hydroxyl ligands, despite displaying lower overall binding efficiencies. Molecular dynamics simulations revealed that these hydrophobic-capped ligands adopt a folded conformation, with the saccharide moiety anchored in the enzyme's active site and the hydrophobic aromatic groups stabilizing the interaction. This conformation exposes the hydrophobic groups to the solvent, potentially enhancing inhibitory potency by increasing ligand retention within the active site. Further analysis revealed that the hydrophobic capped ligands exhibited a higher ligand binding stability as shown by a lower RMSD during the MD simulation. Experimental validation corroborated the computational findings, demonstrating that the introduction of hydrophobic aromatic groups led to a >100-fold increase in inhibitory potency, with IC₅₀ values in the low nanomolar range. These results suggest that simultaneously targeting the charged and hydrophobic pockets of HPSE could yield more potent inhibitors, offering a promising strategy for future cancer therapeutics.