Understanding Dermatan Sulfate−Heparin Cofactor II Interaction through Virtual Library Screening

Raghuraman, Arjun; Mosier, Philip D.; Desai, Umesh R.

doi:10.1021/ml100048y

Cited by 38 publications

(51 citation statements)

References 35 publications

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“…This in silico molecular modeling approach has been used extensively in the literature for studying interactions of GAGs with proteins of diverse types (22,23,29,30). The structure of p300 shows several regions that are rich in basic amino acids.…”

Section: Odsh Interaction With P300 Using In Silico Molecular Modelingmentioning

confidence: 99%

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

Zheng

Kummarapurugu

Afosah

et al. 2017

Am J Respir Cell Mol Biol

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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 LPStriggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration-dependent manner. Fluoresceinlabeled 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 z1 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.Keywords: p300; heparin; high mobility group box 1 Clinical RelevanceWe report that a modified heparin, 2-O, 3-O desulfated heparin (ODSH), inhibits high mobility group box 1 release from macrophages via inhibition of a histone acetyltransferase, p300, required for acetylation. This is a novel mechanism by which ODSH blocks inflammation, and this finding supports the development of ODSH as an antiinflammatory therapy for cystic fibrosis.High mobility group box 1 (HMGB1) is found at high concentrations in the airways of patients with chronic inflammatory lung diseases. Increased airway HMGB1 is associated with bronchopulmonary dysplasia in preterm infants (1), severe asthma in children (2), and chronic obstructive pulmonary disease in adults (3, 4). HMGB1 is a biomarker for lung disease progression in patients with cystic fibrosis (CF) (4, 5). Furthermore, a polymorphism in the gene Ager, which encodes the HMGB1 receptor, receptor for advanced glycation end products (RAGE), results in increased RAGE expression

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Section: Odsh Interaction With P300 Using In Silico Molecular Modelingmentioning

confidence: 99%

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

Zheng

Kummarapurugu

Afosah

et al. 2017

Am J Respir Cell Mol Biol

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“…17–22 We have previously explored both SBVS and LBVS, individually, to discover sulfated small molecules and understand their interaction with proteins. 11,12,23,24 Whereas SBVS of sulfated small molecules has proved to be challenging because of lack of robust scoring functions used in the docking, LBVS has been found to be beset with inability to consider the shape of the binding site. 25 LBVS is useful when applied to large libraries but such libraries are not available for sulfated small molecules.…”

mentioning

confidence: 99%

On scaffold hopping: Challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

Sidhu

Mosier

Zhou

et al. 2013

Bioorganic & Medicinal Chemistry Letters

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The design of sulfated, small, non-saccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing non-saccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS) and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfated benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation enzyme. Screening the ZINC database containing nearly 1 million non-sulfated small molecules using a pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting the possibility of this approach for scaffold hopping. Michaelis-Menten kinetics showed allosteric inhibition mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for identification of sulfated small molecules hits as modulators of glycosaminoglycan–protein interactions.

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“…[6F]FFR-T also reported a 15% fluorescence quench for binding of high- M r dermatan sulfate (DS) to exosite II [16], and its combination with [4′F]FPR-T may be equally useful for studying the assembly of thrombin and HCII in ternary and higher order complexes with DS. Heparin and DS bind different subsites in exosite II [56], and the geometry of DS bridging of thrombin and HCII may be different from that of heparin bridging, a hypothesis that was confirmed by a recently published template model for DS binding [57]. These findings are consistent with previous studies of selective exosite II mutagenesis and exosite II ligand binding showing decreased efficiency of the heparin-catalyzed thrombin inactivation by HCII but lacking an effect on thrombin inactivation in the presence of DS [16,39,58].…”

Section: Discussionmentioning

confidence: 99%

Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex

Verhamme

2012

Analytical Biochemistry

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Thrombin inactivation by heparin cofactor II (HCII) is accelerated by ternary complex formation with heparin. The novel active-site-labeled thrombins, [4′F]FPR-T and [6F]FFR-T, and the exosite I probe, Hir-(54–65)( SO3−), characterized thrombin exosite I and II interactions with HCII and heparin in the complex. HCII binding to exosite I of heparin-bound [4′F]FPR-T caused a saturable fluorescence increase, absent with antithrombin. Heparin binding to exosite II and a second weaker site caused fluorescence quenching of [6F]-FFR-T, attenuated by simultaneous Hir-(54–65)( SO3−) binding. Stopped-flow analysis demonstrated ordered assembly of HCII and the [6F]FFR-T·heparin complex, in agreement with tighter heparin binding to thrombin than to HCII. Saturating HCII dependences and bell-shaped heparin dependences of the fluorescence change reported ternary complex formation, consistent with a template mechanism in which the thrombin-heparin complex binds HCII and allowing for interaction of thrombin·(heparin)2 complexes with HCII. Hir-(54–65)( SO3−) displacement in reactions with FPR-blocked and active thrombin indicated a concerted action of the active site and exosite I during ternary complex formation. These studies demonstrate that binding of HCII to the thrombin·heparin complex is dramatically enhanced compared with heparin binding alone and that exosite I is still available for ligand or HCII binding when both heparin binding sites on thrombin are saturated.

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Understanding Dermatan Sulfate−Heparin Cofactor II Interaction through Virtual Library Screening

Cited by 38 publications

References 35 publications

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

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

On scaffold hopping: Challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex

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