Rio S. Boothello

Interaction of thrombin with sucrose octasulfate.

The serine protease thrombin plays multiple roles in many important physiological processes, especially coagulation, where it functions as both a pro- and anticoagulant. The polyanionic glycosaminoglycan heparin modulates thrombins activity through binding at exosite II. Sucrose octasulfate (SOS) is often used as a surrogate for heparin, but it is not known whether it is an effective heparin mimic in its interaction with thrombin. We have characterized the interaction of SOS with thrombin in solution and determined a crystal structure of their complex. SOS binds thrombin with a K(d) of ~1.4 μM, comparable to that of the much larger polymeric heparin measured under the same conditions. Nonionic (hydrogen bonding) interactions make a larger contribution to thrombin binding of SOS than to heparin. SOS binding to exosite II inhibits thrombins catalytic activity with high potency but with low efficacy. Analytical ultracentrifugation shows that bovine and human thrombins are monomers in solution in the presence of SOS, in contrast to their complexes with heparin, which are dimers. In the X-ray crystal structure, two molecules of SOS are bound nonequivalently to exosite II portions of a thrombin dimer, in contrast to the 1:2 stoichiometry of the heparin-thrombin complex, which has a different monomer association mode in the dimer. SOS and heparin binding to exosite II of thrombin differ on both chemical and structural levels and, perhaps most significantly, in thrombin inhibition. These differences may offer paths to the design of more potent exosite II binding, allosteric small molecules as modulators of thrombin function.

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.

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.

Glycosaminoglycan–Protein Interaction Studies Using Fluorescence Spectroscopy

Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG-protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein-GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG-protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

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.

Heparan sulfate hexasaccharide selectively inhibits cancer stem cells self-renewal by activating p38 MAP kinase

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.

A small group of sulfated benzofurans induces steady-state submaximal inhibition of thrombin

Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric inhibition of thrombin against fibrinogen (ΔY = 55-75%), the first time that a small molecule (MW  < 800) has been found to thwart macromolecular cleavage by a monomeric protease in a controlled manner. The work leads to the promising concept that it should be possible to develop allosteric inhibitors that reduce clotting, but do not completely eliminate it, thereby avoiding major bleeding complications that beset anticoagulants today.