Systematic Virtual Screening of Heparan Sulfate Oligosaccharides and Mechanistic Insights into Heparanase Inhibition

Abstract

Heparanase (Hpse) is an endo-β-glucuronidase that specifically cleaves heparan sulfate (HS) and plays critical roles in the pathogenesis of multiple diseases, making it a well-validated therapeutic target. To identify novel Hpse inhibitors, we developed a rule-based computational pipeline to construct a rigorously curated database of HS oligosaccharides ranging from disaccharides to hexasaccharides. This database systematically encompasses all possible sulfation patterns and stereochemical configurations of the three monosaccharide species in HS. Using a multistage virtual screening strategy against Hpse, we prioritized high-affinity HS oligosaccharides for detailed analysis. Structural motif profiling of the top-ranking candidates revealed that even-numbered oligosaccharides preferentially retain the iduronic acid (IdoA)-N-acetyl-3-O-sulfated glucosamine (GlcNAc3S) motif, whereas odd-numbered species are enriched with IdoA2S and 3,6-O-sulfated glucosamine (GlcN3S6S) motifs. Systematic desulfation of the lead compound, HP-dp5R1, found that removal of 2-O- or 3-O-sulfate groups resulted in substantially larger penalties to binding-free energy compared with removal of 6-O- or N-sulfates, indicating a dominant role for 2-O/3-O sulfation in Hpse recognition. Molecular dynamics simulations and binding-free energy calculations further revealed that longer oligosaccharides form extensive interactions with the heparin-binding domains (HBD1 and HBD2) flanking the catalytic cleft, leading to reduced conformational fluctuation. Together with preliminary experimental results using selected octasaccharides that resemble the structural features of the top-hit oligosaccharides identified from virtual screening, we demonstrate that the integrated computational platform provides a robust foundation for the rational design of HS-based Hpse inhibitors. More broadly, the virtual HS oligosaccharide database described here is expected to serve as a powerful resource for screening other HS-binding proteins and for the discovery of novel agonists or antagonists.