Nehru Viji Sankaranarayanan

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

Abstract 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 interaction with a receptor on hyperglycemic dividing cells prevents intracellular hyaluronan synthesis and autophagy responses in models of type 1 diabetes.

Previous studies and ongoing research indicate the importance of an interaction between a putative receptor on dividing cells in hyperglycemia and the non-reducing end motifs of heparin stored in mast cell secretory granules and how this interaction prevents activation of hyaluronan synthesis in intracellular compartments and subsequent autophagy. This suggests a new role for endosomal heparanase in exposing this cryptic motif present in the initial large heparin chains on serglycin and in the highly sulfated (NS) domains of heparan sulfate.

Chemoenzymatically prepared heparan sulfate containing rare 2-O-sulfonated glucuronic acid residues.

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcAp2S). Genetic algorithm-based computational docking and filtering suggested that GlcAp2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcAp2S and 2-N-sulfonated glucosamine residues, labeled as HS2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcAp2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-O-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences.

Designing “High-Affinity, High-Specificity” Glycosaminoglycan Sequences Through Computerized Modeling

The prediction of high-affinity and/or high-specificity protein-glycosaminoglycan (GAG) interactions is an inherently difficult task, due to several factors including the shallow nature of the typical GAG-binding site and the inherent size, flexibility, diversity, and polydisperse nature of the GAG molecules. Here, we present a generally applicable methodology termed Combinatorial Library Virtual Screening (CVLS) that can identify potential high-affinity, high-specificity protein-GAG interactions from very large GAG combinatorial libraries and a suitable GAG-binding protein. We describe the CVLS approach along with the rationale behind it and provide validation for the method using the well-known antithrombin-thrombin-heparin system.

Solution structure of CXCL13 and heparan sulfate binding show that GAG binding site and cellular signalling rely on distinct domains

Chemokines promote directional cell migration through binding to G-protein-coupled receptors, and as such are involved in a large array of developmental, homeostatic and pathological processes. They also interact with heparan sulfate (HS), the functional consequences of which depend on the respective location of the receptor- and the HS-binding sites, a detail that remains elusive for most chemokines. Here, to set up a biochemical framework to investigate how HS can regulate CXCL13 activity, we solved the solution structure of CXCL13. We showed that it comprises an unusually long and disordered C-terminal domain, appended to a classical chemokine-like structure. Using three independent experimental approaches, we found that it displays a unique association mode to HS, involving two clusters located in the α-helix and the C-terminal domain. Computational approaches were used to analyse the HS sequences preferentially recognized by the protein and gain atomic-level understanding of the CXCL13 dimerization induced upon HS binding. Starting with four sets of 254 HS tetrasaccharides, we identified 25 sequences that bind to CXCL13 monomer, among which a single one bound to CXCL13 dimer with high consistency. Importantly, we found that CXCL13 can be functionally presented to its receptor in a HS-bound form, suggesting that it can promote adhesion-dependent cell migration. Consistently, we designed CXCL13 mutations that preclude interaction with HS without affecting CXCR5-dependent cell signalling, opening the possibility to unambiguously demonstrate the role of HS in the biological function of this chemokine.

A molecular dynamics-based algorithm for evaluating the glycosaminoglycan mimicking potential of synthetic, homogenous, sulfated small molecules

Glycosaminoglycans (GAGs) are key natural biopolymers that exhibit a range of biological functions including growth and differentiation. Despite this multiplicity of function, natural GAG sequences have not yielded drugs because of problems of heterogeneity and synthesis. Recently, several homogenous non-saccharide glycosaminoglycan mimetics (NSGMs) have been reported as agents displaying major therapeutic promise. Yet, it remains unclear whether sulfated NSGMs structurally mimic sulfated GAGs. To address this, we developed a three-step molecular dynamics (MD)-based algorithm to compare sulfated NSGMs with GAGs. In the first step of this algorithm, parameters related to the range of conformations sampled by the two highly sulfated molecules as free entities in water were compared. The second step compared identity of binding site geometries and the final step evaluated comparable dynamics and interactions in the protein-bound state. Using a test case of interactions with fibroblast growth factor-related proteins, we show that this three-step algorithm effectively predicts the GAG structure mimicking property of NSGMs. Specifically, we show that two unique dimeric NSGMs mimic hexameric GAG sequences in the protein-bound state. In contrast, closely related monomeric and trimeric NSGMs do not mimic GAG in either the free or bound states. These results correspond well with the functional properties of NSGMs. The results show for the first time that appropriately designed sulfated NSGMs can be good structural mimetics of GAGs and the incorporation of a MD-based strategy at the NSGM library screening stage can identify promising mimetics of targeted GAG sequences.

2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

&NA; High mobility group box 1 (HMGB1) is an alarmin released from macrophages after infection or inflammation and is a biomarker of lung disease progression in patients with cystic fibrosis. We reported that 2‐O, 3‐O desulfated heparin (ODSH) inhibits the release of HMGB1 from murine macrophages triggered by neutrophil elastase both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this study, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase that has been linked to HMGB1 acetylation and release. ODSH inhibited both neutrophil elastase and LPS‐triggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration‐dependent manner. Fluorescein‐labeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus, suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro, resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognizes a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (dissociation constant ˜1 nM) in a highly cooperative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.

Transforming growth factor-β2 is sequestered in preterm human milk by chondroitin sulfate proteoglycans

Human milk contains biologically important amounts of transforming growth factor-β2 isoform (TGF-β2), which is presumed to protect against inflammatory gut mucosal injury in the neonate. In preclinical models, enterally administered TGF-β2 can protect against experimental necrotizing enterocolitis, an inflammatory bowel necrosis of premature infants. In this study, we investigated whether TGF-β bioactivity in human preterm milk could be enhanced for therapeutic purposes by adding recombinant TGF-β2 (rTGF-β2) to milk prior to feeding. Milk-borne TGF-β bioactivity was measured by established luciferase reporter assays. Molecular interactions of TGF-β2 were investigated by nondenaturing gel electrophoresis and immunoblots, computational molecular modeling, and affinity capillary electrophoresis. Addition of rTGF-β2 (20-40 nM) to human preterm milk samples failed to increase TGF-β bioactivity in milk. Milk-borne TGF-β2 was bound to chondroitin sulfate (CS) containing proteoglycan(s) such as biglycan, which are expressed in high concentrations in milk. Chondroitinase treatment of milk increased the bioactivity of both endogenous and rTGF-β2, and consequently, enhanced the ability of preterm milk to suppress LPS-induced NF-κB activation in macrophages. These findings provide a mechanism for the normally low bioavailability of milk-borne TGF-β2 and identify chondroitinase digestion of milk as a potential therapeutic strategy to enhance the anti-inflammatory effects of preterm milk.

Potent, Selective, Allosteric Inhibition of Human Plasmin by Sulfated Non-Saccharide Glycosaminoglycan Mimetics

Although plasmin inhibitors could be used in multiple disorders, their use has been restricted to preventing blood loss in hemostatic dysregulation because of poor efficacy and adverse effects of current agents. We reasoned that a new class of direct inhibitors that offer better efficacy, selectivity, and safety could be discovered by exploiting allosterism in plasmin, a protease homologous to other allosteric serine proteases. We report on the synthesis, biological activity, and mechanism of action of a group of small molecules, called non-saccharide glycosaminoglycan mimetics (NSGMs), as direct allosteric plasmin inhibitors. Our results show that distinct NSGMs selectively inhibit human full-length plasmin. The molecule inhibited clot lysis, alluding to its promise as an allosteric regulator of plasmin. We show that direct allosteric inhibition of plasmin could led to new antifibrinolytic agent(s) that may exhibit better efficacy, potency, selectivity, and safety in comparison to current therapy.

Inhibition of Herpes Simplex Virus-1 Entry into Human Cells by Non-Saccharide Glycosaminoglycan Mimetics

Although heparan sulfate (HS) has been implicated in facilitating entry of enveloped viruses including herpes simplex virus (HSV), small molecules that effectively compete with this abundant, cell surface macromolecule remain unknown. We reasoned that entry of HSV-1 involving its glycoprotein D (gD) binding to HS could be competitively targeted through small, synthetic, nonsaccharide glycosaminoglycan mimetics (NSGMs). Screening a library of NSGMs identified a small, distinct group that bound gD with affinities of 8-120 nM. Studies on HSV-1 entry into HeLa, HFF-1, and VK2/E6E7 cells identified inhibitors with potencies in the range of 0.4-1.0 μM. These synthetic NSGMs are likely to offer promising chemical biology probes and/or antiviral drug discovery opportunities.