Antiviral Activities of Heparan Sulfate Mimetic RAFT Polymers Against Mosquito-borne Viruses.

Mosquito-borne viruses are a major worldwide health problem associated with high morbidity and mortality rates and significant impacts on national healthcare budgets. The development of antiviral drugs for both the treatment and prophylaxis of these diseases is thus of considerable importance. To address the need for therapeutics with antiviral activity, a library of heparan sulfate mimetic polymers was screened against dengue virus (DENV), Yellow fever virus (YFV), Zika virus (ZIKV), and Ross River virus (RRV). The polymers were prepared by RAFT polymerization of various acidic monomers with a target MW of 20 kDa (average Mn ∼ 27 kDa by GPC). Among the polymers, poly(SS), a homopolymer of sodium styrenesulfonate, was identified as a broad spectrum antiviral with activity against all the tested viruses and particularly potent inhibition of YFV (IC50 = 310 pM). Our results further uncovered that poly(SS) exhibited a robust inhibition of ZIKV infection in both mosquito and human cell lines, which points out the potential functions of poly(SS) in preventing mosquito-borne viruses associated diseases by blocking viral transmission in their mosquito vectors and mitigating viral infection in patients.

Identification of a Pentasaccharide Lead Compound with High Affinity to the SARS-CoV-2 Spike Protein via In Silico Screening.

The spike (S) protein on the surface of the SARS-CoV-2 virus is critical to mediate fusion with the host cell membrane through interaction with angiotensin-converting enzyme 2 (ACE2). Additionally, heparan sulfate (HS) on the host cell surface acts as an attachment factor to facilitate the binding of the S receptor binding domain (RBD) to the ACE2 receptor. Aiming at interfering with the HS-RBD interaction to protect against SARS-CoV-2 infection, we have established a pentasaccharide library composed of 14,112 compounds covering the possible sulfate substitutions on the three sugar units (GlcA, IdoA, and GlcN) of HS. The library was used for virtual screening against RBD domains of SARS-CoV-2. Molecular modeling was carried out to evaluate the potential antiviral properties of the top-hit pentasaccharide focusing on the interactive regions around the interface of RBD-HS-ACE2. The lead pentasaccharide with the highest affinity for RBD was analyzed via drug-likeness calculations, showing better predicted druggable profiles than those currently reported for RBD-binding HS mimetics. The results provide significant information for the development of HS-mimetics as anti-SARS-CoV-2 agents.

EUSEDcollab: a network of data from European catchments to monitor net soil erosion by water.

As a network of researchers we release an open-access database (EUSEDcollab) of water discharge and suspended sediment yield time series records collected in small to medium sized catchments in Europe. EUSEDcollab is compiled to overcome the scarcity of open-access data at relevant spatial scales for studies on runoff, soil loss by water erosion and sediment delivery. Multi-source measurement data from numerous researchers and institutions were harmonised into a common time series and metadata structure. Data reuse is facilitated through accompanying metadata descriptors providing background technical information for each monitoring station setup. Across ten European countries, EUSEDcollab covers over 1600 catchment years of data from 245 catchments at event (11 catchments), daily (22 catchments) and monthly (212 catchments) temporal resolution, and is unique in its focus on small to medium catchment drainage areas (median = 43 km2, min = 0.04 km2, max = 817 km2) with applicability for soil erosion research. We release this database with the aim of uniting people, knowledge and data through the European Union Soil Observatory (EUSO).

Siderophore conjugates to combat antibiotic-resistant bacteria.

Antimicrobial resistance (AMR) is a global threat to society due to the increasing emergence of multi-drug resistant bacteria that are not susceptible to our last line of defence antibiotics. Exacerbating this issue is a severe gap in antibiotic development, with no new clinically relevant classes of antibiotics developed in the last two decades. The combination of the rapidly increasing emergence of resistance and scarcity of new antibiotics in the clinical pipeline means there is an urgent need for new efficacious treatment strategies. One promising solution, known as the 'Trojan horse' approach, hijacks the iron transport system of bacteria to deliver antibiotics directly into cells - effectively tricking bacteria into killing themselves. This transport system uses natively produced siderophores, which are small molecules with a high affinity for iron. By linking antibiotics to siderophores, to make siderophore antibiotic conjugates, the activity of existing antibiotics can potentially be reinvigorated. The success of this strategy was recently exemplified with the clinical release of cefiderocol, a cephalosporin-siderophore conjugate with potent antibacterial activity against carbapenem-resistant and multi-drug resistant Gram-negative bacilli. This review discusses the recent advancements in siderophore antibiotic conjugates and the challenges associated with the design of these compounds that need to be overcome to deliver more efficacious therapeutics. Potential strategies have also been suggested for new generations of siderophore-antibiotics with enhanced activity.

An Enzymatic Activity Assay for Heparanase That Is Useful for Evaluating Clinically Relevant Inhibitors and Studying Kinetics.

The enzyme heparanase cleaves heparan sulfate and is involved in a range of human diseases including cancer, inflammation, diabetes, and viral infection. There is a need for a simple and reliable enzymatic assay to allow for the screening of compounds to find inhibitors of heparanase. We have developed an assay that uses the heparinoid fondaparinux as enzyme substrate and detects one of the products of catalysis, which contains a newly formed reducing terminus, with the tetrazolium salt WST-1. Due to the homogenous substrate and single point of cleavage therein, this assay allows for more systematic kinetic analysis of heparanase inhibitors. Here, we provide a detailed method for conducting this assay and also provide information to assist researchers in evaluating whether the assay is performing properly in their laboratories.

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, ß-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited ß-glucuronidase (ß-GLU). The d-GlcA-configured 9 was a potent inhibitor of ß-GLU and a moderate inhibitor of the endo-ß-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

Evidence for Multiple Binding Modes in the Initial Contact Between SARS-CoV-2 Spike S1 Protein and Cell Surface Glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

An Improved Protocol for the Stereoselective Synthesis of ß-d-Glycosyl Fluorides from 2-O-Acyl Thioglycosides.

A safe and operationally simple protocol for the preparation of ß-d-glycosyl fluorides is presented. We demonstrate that a precise combination of XtalFluor-M, N-bromosuccinimide, and Et3N·3HF can mediate facile, high-yielding, and diastereoselective conversions of 2-O-acyl thioglycosides to ß-d- and other 1,2-trans glycosyl fluorides. The key roles of these reagents are dissected in this work, as is the impact of their interplay on the fluorination stereoselectivity.

SARS-CoV-2 triggers complement activation through interactions with heparan sulfate.

Objectives: To determine whether SARS-CoV-2 can trigger complement activation, the pathways that are involved and the functional significance of the resultant effect. Methods: SARS-CoV-2 was inoculated into a human lepirudin-anticoagulated whole blood model, which contains a full repertoire of complement factors and leukocytes that express complement receptors. Complement activation was determined by measuring C5a production with an ELISA, and pretreatment with specific inhibitors was used to identify the pathways involved. The functional significance of this was then assessed by measuring markers of C5a signalling including leukocyte C5aR1 internalisation and CD11b upregulation with flow cytometry. Results: SARS-CoV-2 inoculation in this whole blood model caused progressive C5a production over 24 h, which was significantly reduced by inhibitors for factor B, C3, C5 and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface interactions with heparan sulfate. Functional analysis of this phenomenon revealed that C5aR1 signalling and CD11b upregulation in granulocytes and monocytes was delayed and only occurred after 24 h. Conclusion: SARS-CoV-2 is a noncanonical alternative pathway activator that progressively triggers complement activation through interactions with heparan sulfate.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 by Disrupting the Spike-ACE2 Interaction.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin.

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase, a Key Enzyme in Cancer and Viral Infections.

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-ß-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS-CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end ß-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.

Development of Improved Synthetic Routes to Pixatimod (PG545), a Sulfated Oligosaccharide-Steroid Conjugate.

The heparan sulfate (HS) mimetic pixatimod (PG545) is a highly potent inhibitor of angiogenesis, tumor growth, and metastasis currently in clinical trials for cancer. PG545 has also demonstrated potent antiviral activity against numerous HS-dependent viruses, including SARS-CoV-2, and shows promise as an antiviral drug for the treatment of COVID-19. Structurally, PG545 consists of a fully sulfated tetrasaccharide conjugated to the steroid 5α-cholestan-3ß-ol. The reported synthesis of PG545 suffers from a low yield and poor selectivity in the critical glycosylation step. Given its clinical importance, new efficient routes for the synthesis of PG545 and analogues were developed. Particular attention was given to improving the key glycosylation step by using more stable protecting groups and optimized glycosyl donors.

From Cancer to COVID-19: A Perspective on Targeting Heparan Sulfate-Protein Interactions.

Heparan sulfate (HS) is a complex, polyanionic polysaccharide ubiquitously expressed on cell surfaces and in the extracellular matrix. HS interacts with numerous proteins to mediate a vast array of biological and pathological processes. Inhibition of HS-protein interactions is thus an attractive approach for new therapeutic development for cancer and infectious diseases, including COVID-19; however, synthesis of well-defined native HS oligosaccharides remains challenging. This has aroused significant interest in the development of HS mimetics which are more synthetically tractable and have fewer side effects, such as undesired anticoagulant activity. This account provides a perspective on the design and synthesis of different classes of HS mimetics with useful properties, and the development of various assays and molecular modelling tools to progress our understanding of their interactions with HS-binding proteins.

Evidence of a putative glycosaminoglycan binding site on the glycosylated SARS-CoV-2 spike protein N-terminal domain.

SARS-CoV-2 has rapidly spread throughout the world's population since its initial discovery in 2019. The virus infects cells via a glycosylated spike protein located on its surface. The protein primarily binds to the angiotensin-converting enzyme-2 (ACE2) receptor, using glycosaminoglycans (GAGs) as co-receptors. Here, we performed bioinformatics and molecular dynamics simulations of the spike protein to investigate the existence of additional GAG binding sites on the receptor-binding domain (RBD), separate from previously reported heparin-binding sites. A putative GAG binding site in the N-terminal domain (NTD) of the protein was identified, encompassing residues 245-246. We hypothesized that GAGs of a sufficient length might bridge the gap between this site and the PRRARS furin cleavage site, including the mutation S247R. Docking studies using GlycoTorch Vina and subsequent MD simulations of the spike trimer in the presence of dodecasaccharides of the GAGs heparin and heparan sulfate supported this possibility. The heparan sulfate chain bridged the gap, binding the furin cleavage site and S247R. In contrast, the heparin chain bound the furin cleavage site and surrounding glycosylation structures, but not S247R. These findings identify a site in the spike protein that favors heparan sulfate binding that may be particularly pertinent for a better understanding of the recent UK and South African strains. This will also assist in future targeted therapy programs that could include repurposing clinical heparan sulfate mimetics.

Corrigendum: Heparanase Promotes Syndecan-1 Expression to Mediate Fibrillar Collagen and Mammographic Density in Human Breast Tissue Cultured ex vivo.

[This corrects the article DOI: 10.3389/fcell.2020.00599.].

Inhibition of Tumor-Host Cell Interactions Using Synthetic Heparin Mimetics.

Low-molecular-weight heparin (LMWH) is the guideline-based drug for antithrombotic treatment of cancer patients, while its direct antitumor effects are a matter of ongoing debate. Although therapeutically established for decades, LMWH has several drawbacks mainly associated with its origin from animal sources. Aiming to overcome these limitations, a library of synthetic heparin mimetic polymers consisting of homo- and copolymers of sulfonated and carboxylated noncarbohydrate monomers has recently been synthesized via reversible addition-fragmentation chain transfer polymerization. These heparin mimetics were investigated for their capacities to interfere with simulated steps of tumor cell metastasis. Among them, homo- and copolymers from sodium 4-styrenesulfonate (poly(SSS)) with acrylic acid (poly(SSS-co-AA)) with an MW between 5 and 50 kDa efficiently attenuated cancer cell-induced coagulation and thus platelet activation and degranulation similar to or even better than LMWH. Furthermore, independent of anticoagulant activities, these polymers affected other metastasis-relevant targets with impressive affinities. Hence, they blocked heparanase enzymatic activity outmatching commercial heparins or a glycosidic drug candidate. Furthermore, these polymers bind P-selectin and the integrin VLA-4 similar to or even better than heparin, indicated by a biosensor approach and thus efficiently blocked melanoma cell binding to endothelium under blood flow conditions. This is the first report on the prospects of synthetic heparin mimetics as promising nontoxic compounds in oncology to potentially substitute heparin as an anticoagulant and to better understand its role as an antimetastatic drug.

Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin.

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

GlycoTorch Vina: Docking Designed and Tested for Glycosaminoglycans.

Glycosaminoglycans (GAGs) are a family of anionic carbohydrates that play an essential role in the physiology and pathology of all eukaryotic life forms. Experimental determination of GAG-protein complexes is challenging due to their difficult isolation from biological sources, natural heterogeneity, and conformational flexibility-including possible ring puckering of sulfated iduronic acid from 1C4 to 2SO conformation. To overcome these challenges, we present GlycoTorch Vina (GTV), a molecular docking tool based on the carbohydrate docking program VinaCarb (VC). Our program is unique in that it contains parameters to model 2SO sugars while also supporting glycosidic linkages specific to GAGs. We discuss how crystallographic models of carbohydrates can be biased by the choice of refinement software and structural dictionaries. To overcome these variations, we carefully curated 12 of the best available GAG and GAG-like crystal structures (ranging from tetra- to octasaccharides or longer) obtained from the PDB-REDO server and refined using the same protocol. Both GTV and VC produced pose predictions with a mean root-mean-square deviation (RMSD) of 3.1 Å from the native crystal structure-a statistically significant improvement when compared to AutoDock Vina (4.5 Å) and the commercial software Glide (5.9 Å). Examples of how real-space correlation coefficients can be used to better assess the accuracy of docking pose predictions are given. Comparisons between statistical distributions of empirical "salt bridge" interactions, relevant to GAGs, were compared to density functional theory (DFT) studies of model salt bridges, and water-mediated salt bridges; however, there was generally a poor agreement between these data. Water bridges appear to play an important, yet poorly understood, role in the structures of GAG-protein complexes. To aid in the rapid prototyping of future pose scoring functions, we include a module that allows users to include their own torsional and nonbonded parameters.

Mucopolysaccharidosis type II (Hunter syndrome): Clinical and biochemical aspects of the disease and approaches to its diagnosis and treatment.

Mucopolysaccharidosis type II (MPS II, Hunter syndrome) is a rare X-linked lysosomal storage disease caused by mutations of the gene encoding the lysosomal enzyme iduronate-2-sulfatase (IDS), the role of which is to hydrolytically remove O-linked sulfates from the two glycosaminoglycans (GAGs) heparan sulfate (HS) and dermatan sulfate (DS). HS and DS are linear, heterogeneous polysaccharides composed of repeating disaccharide subunits of l-iduronic acid (IdoA) or d-glucuronic acid, (1→4)-linked to d-glucosamine (for HS), or (1→3)-linked to 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) (for DS). In healthy cells, IDS cleaves the sulfo group found at the C-2 position of terminal non-reducing end IdoA residues in HS and DS. The loss of IDS enzyme activity leads to progressive lysosomal storage of HS and DS in tissues and organs such as the brain, liver, spleen, heart, bone, joints and airways. Consequently, this leads to the phenotypic features characteristic of the disease. This review provides an overview of the disease profile and clinical manifestation, with a particular focus on the biochemical basis of the disease and chemical approaches to the development of new diagnostics, as well as discussing current treatment options and emerging new therapies.