Finding a Needle in a Haystack: Development of a Combinatorial Virtual Screening Approach for Identifying High Specificity Heparin/Heparan Sulfate Sequence(s)

We describe a combinatorial virtual screening approach for predicting high specificity heparin/heparan sulfate
sequences using the well-studied antithrombin−heparin interaction as a test case. Heparan sulfate
hexasaccharides were simulated in the ‘average backbone' conformation, wherein the inter-glycosidic bond
angles were held constant at the mean of the known solution values, irrespective of their sequence. Molecular
docking utilized GOLD with restrained inter-glycosidic torsions and intra-ring conformations, but flexible
substituents at the 2-, 3-, and 6-positions and explicit incorporation of conformational variability of the
iduronate residues. The approach reproduces the binding geometry of the sequence-specific heparin
pentasaccharide to within 2.5 Å. Screening of a combinatorial virtual library of 6859 heparin hexasaccharides
using a dual filter strategy, in which predicted antithrombin affinity was the first filter and self-consistency
of docking was the second, resulted in only 10 sequences. Of these, nine were found to bind antithrombin
in a manner identical to the natural pentasaccharide, while a novel hexasaccharide bound the inhibitor in a
unique but dramatically different geometry and orientation. This work presents the first approach on
combinatorial library screening for heparin/heparan sulfate GAGs to determine high specificity sequences
and opens up huge opportunities to investigate numerous other physiologically relevant GAG−protein
interactions.