Glycosaminoglycan Mimetics Research Articles

On the Process of Discovering Leads That Target the Heparin-Binding Site of Neutrophil Elastase in the Sputum of Cystic Fibrosis Patients.

Cystic fibrosis (CF) is a disease of dysregulated salt and fluid homeostasis that results in the massive accumulation of neutrophil elastase, resulting in lung degradation and death. The current CF therapy relies on inhaled deoxyribonuclease and hypertonic saline but does not address the elastolytic degradation of the lung. We reasoned that allosteric agents targeting the heparin-binding site of neutrophil elastase would offer a therapeutic paradigm. Screening a library of 60 nonsaccharide glycosaminoglycan mimetics (NSGMs) led to the discovery of 23 hits against neutrophil elastase. To identify a lead NSGM that works in sync with the current CF-relieving agents, we developed a rigorous protocol based on fundamental computational, biochemical, mechanistic, and adverse effect studies. The lead NSGM so identified neutralized neutrophil elastase present in the sputum of CF patients in the presence of deoxyribonuclease and high-salt conditions. Our work presents the process for discovering potent, small, synthetic, allosteric, anti-CF agents, while also identifying a novel lead for further studies in animal models of CF.

Glycosaminoglycans and Glycosaminoglycan Mimetics in Cancer and Inflammation.

Glycosaminoglycans (GAGs) are a class of biomolecules expressed virtually on all mammalian cells and usually covalently attached to proteins, forming proteoglycans. They are present not only on the cell surface, but also in the intracellular milieu and extracellular matrix. GAGs interact with multiple ligands, both soluble and insoluble, and modulate an important role in various physiological and pathological processes including cancer, bacterial and viral infections, inflammation, Alzheimer’s disease, and many more. Considering their involvement in multiple diseases, their use in the development of drugs has been of significant interest in both academia and industry. Many GAG-based drugs are being developed with encouraging results in animal models and clinical trials, showcasing their potential for development as therapeutics. In this review, the role GAGs play in both the development and inhibition of cancer and inflammation is presented. Further, advancements in the development of GAGs and their mimetics as anti-cancer and anti-inflammatory agents are discussed.

Design and Synthesis of Photoaffinity Probe for Identification of Protein Targets of Glycosaminoglycan Mimetics

Glycosaminoglycans (GAGs) are complex biomacromolecules ubiquitously distributed in the animal kingdom where they play major roles in the large number of physiological and pathological processes. Identification of protein targets of GAGs and GAG mimetics has been a major challenge because of the size of the library of GAG‐binding proteins. We reasoned that photoaffinity probes of bioactive GAG mimetics would serve as a useful method for identification of protein targets. This work presents synthesis and mass spectrometric characterization of novel photoactivatable analogs of a lead anti‐cancer agent for identification of its plausible protein targets. Photoactivatable analog of the lead GAG mimetic G2.2 containing diazirine and alkyne groups G2PA was synthesized through a series of 10 steps. Structural characterization was performed using 1H/13C NMR and HRMS analysis. A test case of photoactivation was performed by first incubating insulin‐like growth factor‐1 with mimetic G2PA at 4 °C and then photo‐irradiated for 5 min at 368 nm. The sample was reduced using DTT, alkylated with iodoacetamide in dark, digested with trypsin and cleaned up using C18 StageTip. Proteomics analysis of the photoactivated IGF‐1–G2PA complex was performed on nLC‐Q Exactive (Thermo) in a top 12 data dependent acquisition. Proteomic analysis of the IGF‐1–G2PA complex gave a mass peak at 2688 m/z corresponding to the peptide APQTGIVDEccFR covalently linked to the photoactivated G2PA. MS/MS analysis of this fragment showed peaks 1) at 2391 m/z corresponding to TGIVDEccFR+G2PA; 2) 1098 m/z corresponding IVDEccFR; and 3) 297 m/z corresponding APQ. Proteomic analysis indicated G2PA covalently modified the thyronine residue present in APQTGIVDEccFR sequence of IGF‐1. Thus, the diazirine‐based photo‐activation approach for the test case of IGF‐1 identified the site of covalent conjugation and validated the approach. This approach, in principle, could be more generally used to identify protein targets of GAGs and GAG mimetics.Support or Funding InformationThis work is supported by HL107152, HL090586 from National Heart, Lung and Blood Institute to URDThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Inhibition of a Panel of Cancer Stem Cell Lines by Novel Glycosaminoglycan Mimetics

PurposeTo characterize the anti‐cancer stem cell (CSC) properties of non‐saccharide glycosaminoglycan mimetics (NSGMs) and their lipid‐modified analogs in a panel of colon cancer cells.MethodFour novel NSGMs were screened for their ability to inhibit the formation of 1° spheroids in 12 intestinal tumor cells belonging to one of four consensus molecular subtypes (CMS) for intestinal cancers and were chosen to be representative of common genetic variants in colon cancer. Spheroid were grown by suspending monolayer cancer cells in stem cell media in low‐adhesion plates and the number of spheroids was measured with light microscopy. Spheroid forming cell frequency (SFF) was determined in the 1° spheroids by plating them at varying concentration 1→124 cells/well and recording 2° spheroids (present or absent) in each well using http://bioinf.wehi.edu.au/software/elda web tool. Dose‐response curves were generated by examining 1° spheroid formation following treatment with increasing concentration of NSGMs (0→5000 μM). The IC50 values for each of the NSGMs were determined using the standard sigmoidal regression equation. Inhibition of monolayers of corresponding cancer cells by NSGMs was assessed through a colorimetric MTT assay.ResultsThe MTT assay in monolayer conditions revealed no or minimal cytotoxicity with all the NSGMs at concentrations >300μM, suggesting selective targeting of CSCs. Screening for primary spheroid inhibition revealed three distinct responses concerning the lead NSGM G2.2. G2.2 showed a mean IC50 of 225 μM across all cell lines (range 134 – 252 μM). Intriguingly, CMS1, CMS2, and neuroendocrine cells were either moderately resistant or highly resistant. On the other hand, none of the CMS3 and CMS4 cell lines were highly resistant, and a majority (4/8) were sensitive to G2.2. Intriguingly, we observed a significant inverse correlation (r = −0.56, p= 0.06) between SFF and IC50 for G2.2, suggesting that the level of pre‐existing CSCs could determine G2.2's efficacy. Interestingly, the lipid‐modified analogs showed higher potency in comparison with the parent molecule G2.2. Of the three lipid‐modified analogs, G8C was the most potent inhibitor of CSC's (with a mean IC50 of 11 μM) against intestinal cancer spheroids. In fact, G8C displayed sub‐micromolar inhibition (IC50 = 0.73 μM) against the most sensitive colorectal cell line HT‐29 (CMS3). Except for one case, the resistant cell lines turned sensitive or less resistant to treatment with the three lipid analogs.ConclusionsG2.2 (and related NSGMs) selectively inhibit CSCs and its efficacy strongly correlates with the levels of pre‐existing CSCs and/or CMS subtypes 3 (KRAs activation) and 4 (TGF‐β pathway activation) providing valuable clues about their mechanism. Lipid‐modified NSGMs represent potent and selective anti‐CSCs agents with paradigm‐shifting implications for colorectal cancer treatment.Support or Funding InformationGrant support from the following sources:1P01 HL1071521R25 HL1286391R01 HL090586This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A small, synthetic glycosaminoglycan mimetic as an inhibitor of human heparanase

Human heparanase (HPSE) is an endo‐b‐D‐glucuronidase that cleaves glycosaminoglycan (GAG) chains of heparan sulfate proteoglycans (HSPGs) present on cell surfaces and in the extracellular matrix (ECM). HPSE is upregulated in many types of cancers and promotes an aggressive tumor growth leading to metastasis. HPSE is also involved in various other pathologies such as inflammation, thrombosis, atherosclerosis and kidney dysfunction. Because of these important roles, HPSE is a promising target for the development of a novel therapeutic. In fact, several HS mimetics including PI‐88, SST000, PG545, and M402 are being pursued in clinical trials. Yet, each of these is saccharide mimetics of HS, which are either polymeric, and therefore heterogeneous, or are very difficult to prepare. We have pursued the idea that small, synthetic agents called Non‐Saccharide Glycosaminoglycan Mimetics (NSGMs) would modulate pathological responses such cancer, coagulation, and inflammation in which GAGs play important roles. We hypothesized that an optimal small, synthetic NSGM that potently binds to HPSE would inhibit its enzymatic activity and lead to a novel, anti‐angiogenesis agent. Therefore, we screened an in‐house library 70 NSGMs and identified 35 agents that displayed reasonable HPSE inhibition. Detailed studies using a one‐step FRET assay as well as UPLC‐MS/MS assay showed that an NSGM labeled as G2.2 exhibited high anti‐HPSE potency of 3.2±0.3 μM. G2.2 is synthesized in five simple steps in high overall yields (>30%) from readily available natural product quercetin. G2.2 significantly blocked the migration and invasion of HT‐29 spheroid cells compared to vehicle‐treated control cells. The highly promising anti‐HPSE agent is currently being investigated in animal models of angiogenesis.Support or Funding InformationP‐01 HL107152This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycosaminoglycans and Glycosaminoglycan Mimetics as Human Neutrophil Elastase Inhibitors for Cystic Fibrosis Management

Cystic fibrosis (CF), a genetic disease, affects multiple organs, especially the lungs. Human neutrophil elastase (HNE), a serine protease vital for host defense mechanism, is markedly elevated in CF and plays a key role in disease progression. In fact, it is a biomarker of disease progression. Yet, there are no FDA‐approved small molecule inhibitors for HNE. The current CF therapy employs a host of symptom‐alleviating agents including DNAse to lyse mucous, hypertonic saline to aid mucous clearance, and antibiotics to combat infection. CF therapy would benefit greatly from HNE inhibitors.Previous work has shown that HNE is potently inhibited by negatively charged molecules such as heparin. Yet, heparin's powerful anticoagulant property limits its usefulness in CF. To identify selective glycosaminoglycan (GAG)‐based inhibitors of HNE, we studied the anti‐HNE activity of various GAG species of different sulfation patters, molecular weights and chain lengths. Our studies indicate that GAG sulfation patterns different from those required for anti‐coagulant property drive HNE inhibition. The results also show that sequences longer than hexasaccharides are required for anti‐HNE activity. Michaelis‐Menten kinetics suggests an allosteric mechanism of inhibition, which provides an advantage for the development of selective agents. Additionally, salt dependence studies of HNE inhibition points to the involvement of both ionic and non‐ionic interactions for HNE inhibition.Based on these results, we screened a library of 60 diverse, non‐saccharide GAG mimetics (NSGMs) against HNE, of which 10 molecules were found to be nanomolar inhibitors. Since hypertonic saline is an integral part of current CF therapy, we studied the effect of high salt levels on these inhibitors and identified six NSGMs that retained excellent potency, probably arising from considerable specific, non‐ionic interactions in binding to HNE. Studies on inhibition of HNE degradation of macromolecular substrate elastin led to further winnowing of inhibitors to two promising NSGMs. Finally, studies on a panel of serine proteases and coagulation assays led to the selection of a tetrasulfated diflavonoid NSGM for further testing in sputum from CF patients. This agent showed a dose‐dependent decrease in HNE activity of CF sputum when used in combination with DNAse. These studies indicate that GAG‐based inhibition of HNE is a promising avenue for developing anti‐CF agents. More specifically, the results suggest that a tetra‐sulfated diflavonoid NSGM could add value to the trio of DNAse, hypertonic saline and antibiotics used currently in the clinic.Support or Funding InformationThis work is funded by NHLBI grant 1P01HL107152 to Dr. Umesh R. Desai.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cross-Species Analysis of Glycosaminoglycan Binding Proteins Reveals Some Animal Models Are "More Equal" than Others.

Glycosaminoglycan (GAG) mimetics are synthetic or semi-synthetic analogues of heparin or heparan sulfate, which are designed to interact with GAG binding sites on proteins. The preclinical stages of drug development rely on efficacy and toxicity assessment in animals and aim to apply these findings to clinical studies. However, such data may not always reflect the human situation possibly because the GAG binding site on the protein ligand in animals and humans could differ. Possible inter-species differences in the GAG-binding sites on antithrombin III, heparanase, and chemokines of the CCL and CXCL families were examined by sequence alignments, molecular modelling and assessment of surface electrostatic potentials to determine if one species of laboratory animal is likely to result in more clinically relevant data than another. For each protein, current understanding of GAG binding is reviewed from a protein structure and function perspective. This combinatorial analysis shows chemokine dimers and oligomers can present different GAG binding surfaces for the same target protein, whereas a cleft-like GAG binding site will differently influence the types of GAG structures that bind and the species preferable for preclinical work. Such analyses will allow an informed choice of animal(s) for preclinical studies of GAG mimetic drugs.

Directing GDNF-mediated neuronal signaling with proactively programmable cell-surface saccharide-free glycosaminoglycan mimetics.

A significant barrier to harnessing the power of cell-surface glycosaminoglycans (GAGs) to modulate glial cell-line-derived neurotrophic factor (GDNF) signaling is the difficulty in accessing key GAG structures involved. Here, we report tailored GDNF signaling using synthetic polyproline-based GAG mimetics (PGMs). PGMs deliver the much needed proactive programmability for GDNF recognition and effectively modulate GDNF-mediated neuronal processes in a cellular context.

A synthetic glycosaminoglycan mimetic blocks HSV-1 infection in human iris stromal cells

Herpes simplex virus type-1 (HSV-1) is a significant pathogen that affects vision by targeting multiple regions in the human eye including iris. Using a focused library of synthetic non-saccharide glycosaminoglycan mimetics (NSGMs), we identified sulfated pentagalloylglucoside (SPGG) as a potent inhibitor of HSV-1 entry and cell-to-cell spread in the primary cultures of human iris stromal (HIS) cells isolated from eye donors. Using in vitro β-galactosidase reporter assay and plaque reduction assay, SPGG was found to inhibit HSV-1 entry in a dosage-dependent manner (IC50 ∼6.0 μM). Interestingly, a pronounced inhibition in HSV-1 entry and spread was observed in HIS cells, or a cell line expressing specific gD-receptor, when virions were pre-treated with mimetics suggesting a possible interaction between SPGG and the HSV-1 glycoprotein. To examine the significance of gD-SPGG interaction, HIS cells were pretreated with SPGG, which showed a significant reduction in gD binding. Taken together, our results provide strong evidence of SPGG being a novel viral entry inhibitor against ocular HSV infection.

Inhibition of Herpes Simplex Virus-1 Entry into Human Cells by Nonsaccharide 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.

Structure-Activity Relationships of GAG Mimetic-Functionalized Mesoporous Silica Nanoparticles and Evaluation of Acyclovir-Loaded Antiviral Nanoparticles with Dual Mechanisms of Action.

Mesoporous silica nanoparticles (MSNs) are drug delivery agents that are able to incorporate drugs within their pores. Furthermore, MSNs can be functionalized by attachment of bioactive ligands on their surface to enhance their activity, and nanoparticles modified with glycosaminoglycan (GAG) mimetics inhibit the entry of herpes simplex virus (HSV) into cells. In this study, structure–activity relationships of GAGs attached to MSNs were investigated in relation to HSV-1 and HSV-2, and acyclovir was loaded into the pores of MSNs. The sulfonate group was demonstrated to be essential for antiviral activity, which was enhanced by incorporating a benzene group within the ligand. Loading acyclovir into GAG mimetic-functionalized MSNs reduced the viral infection, resulting in nanoparticles that simultaneously target two distinct viral pathways, namely, inhibition of viral entry and inhibition of DNA replication.

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1 → 4)-Glc-β(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC50 of 270 nM and 2.4 µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.

Facile saccharide-free mimetics that recapitulate key features of glycosaminoglycan sulfation patterns.

Controlling glycosaminoglycan (GAG) activity to exploit its immense potential in biology ultimately requires facile manipulation of sulfation patterns associated with GAGs. However, satisfying this requirement in full remains challenging, given that synthesis of GAGs is technically arduous while convenient GAG mimetics often produce sulfation patterns that are uncharacteristic of GAGs. To overcome this, we develop saccharide-free polyproline-based GAG mimetics (PGMs) that can be facilely assembled via amide coupling chemistry. Molecular dynamics simulations show that PGMs recapitulate key GAG structural features (i.e. ∼9 Å-sized repeating units, periodicity and helicity) and as with GAGs, can be tuned to introduce systematic variations in sulfate clustering and spacing. Functionally, a variety of PGMs control various GAG activities (concerning P-selectin, neurotrophic factors and heparinase) and exhibit GAG-like characteristics such as progressive modulation, comparable effectiveness with heparins, need for different sequences to suit different activities and the presence of a "minimal bioactive length". Furthermore, PGMs produce consistent effects in vivo and successfully provide therapeutic benefits over cancer metastasis. Taken together with their high level of biosafety, PGMs answer the long-standing need for an effective and practicable strategy to manipulate GAG-appropriate sulfation patterns and exploit GAG activity in medicine and biotechnology.

Heparin-fibronectin interactions in the development of extracellular matrix insolubility

During extracellular matrix (ECM) assembly, fibronectin (FN) fibrils are irreversibly converted into a detergent-insoluble form which, through FN's multi-domain structure, can interact with collagens, matricellular proteins, and growth factors to build a definitive matrix. FN also has heparin/heparan sulfate (HS) binding sites. Using HS-deficient CHO cells, we show that the addition of soluble heparin significantly increased the amount of FN matrix that these cells assemble. Sulfated HS glycosaminoglycan (GAG) mimetics similarly increased FN assembly and demonstrated a dependence on GAG sulfation. The length of the heparin chains also plays a role in assembly. Chains of sufficient length to bind to two FN molecules gave maximal stimulation of assembly whereas shorter heparin had less of an effect. Using a decellularized fibroblast matrix for proteolysis, detergent fractionation, and mass spectrometry, we found that the predominant domain within insoluble fibril fragments is FN's major heparin-binding domain HepII (modules III12–14). Multiple HepII domains bind simultaneously to a single heparin chain in size exclusion chromatography analyses. We propose a model in which heparin/HS binding to the HepII domain connects multiple FNs together to facilitate the formation of protein interactions for insoluble fibril assembly.

Circulating endothelial progenitors in vascular repair.

Endothelial Colony Forming Cells (ECFCs) are obtained in culture from Circulating Endothelial Progenitor Cells. They display all characteristics of endothelial cells and they display stem cells features. Cord blood-derived ECFCs (CB-ECFCs) have a high clonogenic and proliferative potentials, and exhibit vascular repair capabilities useful for the treatment of ischemic diseases. However, the link between immaturity and functional properties of CB-ECFCs is still poorly defined. We showed that these cells have a high clonogenic potential and are capable to be efficiently reprogrammed into induced pluripotent stem cells. Moreover, we analyzed the expression of a broad panel of genes involved in embryonic stem cell properties. We define a novel stem cell transcriptional signature for CB-ECFCs fora better characterization and stratification according to their stem cell profile. We then improved the yield of CB-ECFC production for obtaining cells more functional in fewer passages. We used Glycosaminoglycans (GAG), components from the extracellular matrix which potentiate heparin binding growth factor activities. GAG mimetics were designed, having the capacity to increase the yield of ECFC during the isolation process, to increase the number of colonies, improve adhesion, proliferation, migration and self-renewal. GAG mimetics have thus great interest for vascular regeneration in combination with ECFC. Our results show that CB-ECFC are immature cells harboring specific functions such as formation of colonies, proliferation and formation of vascular structures in vitro and in vivo.

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.

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.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

Factor XIIIa (FXIIIa) is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa’s active site by using sulfated glycosaminoglycans (GAGs) or non-saccharide GAG mimetics (NSGMs) would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%). Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71%) and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles.

Herpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo- and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo- and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses.