A Hexasaccharide Containing Rare 2-O-Sulfate-Glucuronic Acid Residues Selectively Activates Heparin Cofactor II

Glycosaminoglycan (GAG) sequences that selectively target heparin cofactor II (HCII), a key serpin present in human plasma, remain unknown. Using a computational strategy on a library of 46 656 heparan sulfate hexasaccharides we identified a rare sequence consisting of consecutive glucuronic acid 2-O-sulfate residues as selectively targeting HCII. This and four other unique hexasaccharides were chemically synthesized. The designed sequence was found to activate HCII ca. 250-fold, while leaving aside antithrombin, a closely related serpin, essentially unactivated. This group of rare designed hexasaccharides will help understand HCII function. More importantly, our results show for the first time that rigorous use of computational techniques can lead to discovery of unique GAG sequences that can selectively target GAG-binding protein(s), which may lead to chemical biology or drug discovery tools. Heparin cofactor II (HCII) is a serine protease inhibitor (serpin) that circulates in human plasma at high levels. Although it has been known to selectively inhibit thrombin for several decades, its true physiologic function remains to be understood. HCII is known to bind to glycosaminoglycans (GAGs) such as dermatan sulfate (DS) and heparan sulfate (HS), which help mediate its inhibition of thrombin. One of the key reasons for the inability to identify HCIIQs biologic role is the lack of knowledge on the specificity of HCII-GAG interaction. HCII is an interesting serpin. It bears considerable similarity to antithrombin (AT), another plasma serpin that mediates the anticoagulant action of heparin and fondaparinux, 6] two clinically used drugs. AT and HCII are homologous in primary, secondary and tertiary structure (Figure S1 in the Supporting Information). Yet, whereas AT binds specifically to fondaparinux sequence in heparin, HCII is considered to bind non-specifically to heparin and HS. Further, although both serpins display a two-step, inducedfit, allosteric activation mechanism in inhibiting their target enzymes, no HS oligosaccharide has been discovered to induce robust activation of HCII. Identifying GAG sequences that selectively target proteins is extremely challenging. A key reason for this is their structural complexity. HS, a highly anionic polymer containing variably sulfated, acetylated and epimerized residues, presents enormous structural diversity that makes comprehensive analysis of all possible sequences difficult. Thus, identifying key “needles” in this haystack, especially with synthesis 12] or isolation of oligosaccharides from nature nearly impossible. We had earlier developed a dual-filter computational algorithm, called combinatorial virtual library screening (CVLS) strategy, to rapidly sort HS sequences into “specific” and “non-specific” bins. 15] We wondered whether this tool could pinpoint HS sequences that preferentially activate HCII for inhibition of thrombin. Further, we posited that such a sequence, if any, would be different from the pentasaccharide sequence (i.e., fondaparinux) that selectively activates AT. Herein, we show that application of the CVLS strategy on a library of all-possible natural HS hexasaccharides led to the identification of five distinct sequences that were predicted to differentially target the two homologous serpins. We developed a novel multi-step synthesis of these five HS sequences and studied each for activation of the two serpins. The results led to the discovery that a unique hexasaccharide that contains two rare 2-O-sulfate glucuronic acid (GlcA2S) residues in tandem selectively activates HCII nearly 250fold, which is equivalent to AT activation induced by fondaparinux. To identify HCII-specific sequences, we compared the overall tertiary structures of HCII and AT. 9] The HS binding site in AT is formed by helices A (hA) and D (hD), which contains R46, R47, K114, K125, R129, R132 and K133 that define the specificity of HS recognition. The corresponding [*] Dr. N. V. Sankaranarayanan, Dr. R. S. Boothello, Dr. A. Raghuraman, Dr. P. D. Mosier, Prof. Dr. U. R. Desai Department of Medicinal Chemistry and Institute of Structural Biology, Drug Discovery and Development Virginia Commonwealth University