No abstract

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 A. Screening of a combinatorial virtual library of 6,859 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.

Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS5 containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS5 activated the inhibitor nearly 30-fold, nearly 2 to 3-fold higher than our first generation flavan-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.

In our previous work, we discovered potent HSV-1 inhibitory activity arising from sulfated form of lignin, a highly networked natural biopolymer composed of substituted phenylpropanoid monomers (Raghuraman et al. Biomacromolecules 2005, 6, 2822). We present here detailed characterization of the viral inhibitory properties of this interesting macromolecule. The inhibition was proportional to the average molecular weight of the lignin sulfate preparation with IC 50 values in the range of 17 nM to 5 μM against HSV-1 and HSV-2, and 29 nM to 763 nM against HIV-1. Cytotoxicity studies displayed selectivity indices in the range of 14 to 31 suggesting reasonably good difference between activity and toxicity for polymeric preparations. Comparative molecular modeling studies suggest that lignin sulfate may contain certain structural features that mimic the three-dimensional organization of sulfate groups in heparan sulfate, thereby providing a plausible basis for its anti-viral activity. The combination of strongly hydrophobic (-Ar) and strongly hydrophilic (-OSO 3 −) groups in lignin sulfate makes this chemically modified biopolymer an interesting molecule for further work.