Glycosaminoglycans from Litopenaeus vannamei Inhibit the Alzheimer's Disease ß Secretase, BACE1.

Only palliative therapeutic options exist for the treatment of Alzheimer's Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer's Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer's disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal ß-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered.

Heparan Sulfate Proteoglycan Synthesis Is Dysregulated in Human Osteoarthritic Cartilage.

Osteoarthritis (OA) is a common degenerative joint disease, characterized by cartilage loss and subchondral bone remodeling in response to abnormal mechanical load. Heparan sulfate (HS) proteoglycans bind to many proteins that regulate cartilage homeostasis, including growth factors, morphogens, proteases, and their inhibitors, and modulate their localization, retention, and biological activity. Changes in HS expression and structure may thus have important consequences for joint health. We analyzed normal and osteoarthritic human knee cartilage, and found HS biosynthesis was markedly disrupted in OA, with 45% of the 38 genes analyzed differentially regulated in diseased cartilage. The expression of several HS core proteins, biosynthesis, and modification enzymes was increased in OA cartilage, whereas the expression of the HS proteoglycans syndecan 4 and betaglycan was reduced. The structure of HS was also altered, with increased levels of 6-O-sulfation in osteoarthritic samples, which correlated with increased expression of HS6ST1, a 6-O-sulfotransferase, and GLCE, an epimerase that promotes 6-O-sulfation. siRNA silencing of HS6ST1 expression in primary OA chondrocytes inhibited extracellular signal-regulated kinase phosphorylation in response to fibroblast growth factor 2, showing that changes in 6-O-sulfation impact a key cartilage signaling pathway. Given the broad range of homeostatic and repair pathways that HS regulates, these changes in proteoglycan expression and HS structure are likely to have significant effects on joint health and progression of OA.

A Glycosaminoglycan Extract from Portunus pelagicus Inhibits BACE1, the ß Secretase Implicated in Alzheimer's Disease.

Therapeutic options for Alzheimer's disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimer's disease-relevant ß-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimer's disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian-sourced heparins, primarily due to prion diseases and religious beliefs hinder the deployment of alternative heparin-based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract, isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 µg mL-1 (R2 = 0.94) and 2.43 µg mL-1 (R2 = 0.93), respectively), while possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

Sulfatase-mediated manipulation of the astrocyte-Schwann cell interface.

Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix more readily with astrocytes in culture and induce less astrogliosis. We previously demonstrated that OECs express higher levels of sulfatases, enzymes that remove 6-O-sulfate groups from heparan sulphate proteoglycans, than SCs and that RNAi knockdown of sulfatase prevented OEC-astrocyte mixing in vitro. As human OECs are difficult to culture in large numbers we have genetically engineered SCs using lentiviral vectors to express sulfatase 1 and 2 (SC-S1S2) and assessed their ability to interact with astrocytes. We demonstrate that SC-S1S2s have increased integrin-dependent motility in the presence of astrocytes via modulation of NRG and FGF receptor-linked PI3K/AKT intracellular signaling and do not form boundaries with astrocytes in culture. SC-astrocyte mixing is dependent on local NRG concentration and we propose that sulfatase enzymes influence the bioavailability of NRG ligand and thus influence SC behavior. We further demonstrate that injection of sulfatase expressing SCs into spinal cord white matter results in less glial reactivity than control SC injections comparable to that of OEC injections. Our data indicate that sulfatase-mediated modification of the extracellular matrix can influence glial interactions with astrocytes, and that SCs engineered to express sulfatase may be more OEC-like in character. This approach may be beneficial for cell transplant-mediated spinal cord repair. GLIA 2016 GLIA 2017;65:19-33.

Versatile Separation and Analysis of Heparan Sulfate Oligosaccharides Using Graphitized Carbon Liquid Chromatography and Electrospray Mass Spectrometry.

Heparin and heparan sulfate (HS) by nature contain multiple isomeric structures, which are fundamental for the regulation of biological processes. Here we report the use of a porous graphitized carbon (PGC) LC-MS method with effective separation and sensitivity to separate mixtures of digested HS oligosaccharides. Application of this method allowed the separation of oligosaccharide mixtures with various degree of polymerization (dp) ranging from dp4 to dp8, two dp4 isomers that were baseline resolved, four dp6 isomers, and the observation of a dp3 oligosaccharide. PGC LC-MS of complex mixtures demonstrated that compounds eluted from the column in decreasing order of hydrophilicity, with the more highly sulfated structures eluting first. Our data indicate that sulfation levels, chain length, and conformation all effect elution order. We found that PGC's resolving capabilities for the dp4 and dp6 isomeric structures makes this methodology particularly useful for the sequencing of HS saccharides, because the lack of contaminating isomeric structures provides unambiguous structural assignments from the MS/MS data. Collectively this work demonstrates that PGC column-based methods are powerful tools for enhanced separation and analysis of heterogeneous mixtures of HS saccharide species.

Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography.

The complexity of heparin and heparan sulfate saccharides makes their purification, including many isomeric structures, very challenging and is a bottleneck for structure-activity studies. High-resolution separations have been achieved by strong anion exchange (SAX) chromatography on Propac PA1 and cetyltrimethylammonium (CTA)-C18 silica columns; however, these entail subsequent desalting methodologies and consequent sample losses and are incompatible with orthogonal chromatography methodologies and, in particular, mass spectrometry. Here, we present the CTA-SAX purification of heparin oligosaccharides using volatile salt (VS) buffer. In VSCTA-SAX, the use of ammonium bicarbonate buffer for elution improves resolution through both weaker dissociation and conformational coordination of the ammonium across the sulfate groups. Using ion mobility mass spectrometry, we demonstrate that isomeric structures have different structural conformations, which makes chromatographic separation achievable. Resolution of such structures is improved compared to standard SAX methods, and in addition, VSCTA-SAX provides an orthogonal method to isolate saccharides with higher purity. Because ammonium bicarbonate is used, the samples can be evaporated rather than desalted, preventing substantial sample loss and allowing more effective subsequent analysis by electrospray mass spectrometry. We conclude that VSCTA-SAX is a powerful new tool that helps address the difficult challenge of heparin/heparan sulfate saccharide separation and will enhance structure-activity studies.

Heparan sulfate phage display antibodies recognise epitopes defined by a combination of sugar sequence and cation binding.

Phage display antibodies are widely used to follow heparan sulfate (HS) expression in tissues and cells. We demonstrate by ELISA, that cations alter phage display antibody binding profiles to HS and this is mediated by changes in polysaccharide conformation, demonstrated by circular dichroism spectroscopy. Native HS structures, expressed on the cell surfaces of neuroblastoma and fibroblast cells, also exhibited altered antibody binding profiles following exposure to low mM concentrations of these cations. Phage display antibodies recognise conformationally-defined HS epitopes, rather than sequence alone, as has been assumed, and resemble proteins in being sensitive to changes in both charge distribution and conformation following binding of cations to HS polysaccharides.

Single-entity heparan sulfate glycomimetic clusters for therapeutic applications.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan with a variety of critical functions in cell signaling and regulation. HS oligosaccharides can mimic or interfere with HS functions in biological systems; however, their exploitation has been hindered by the complexity of their synthesis. Polyvalent displays of small specific HS structures on dendritic cores offer more accessible constructs with potential advantages as therapeutics, but the synthesis of single-entity HS polyvalent compounds has not previously been described. Herein we report the synthesis of a novel targeted library of single-entity glycomimetic clusters capped with varied HS saccharides. They have the ability to mimic longer natural HS saccharides in their inhibition of the Alzheimer's disease (AD) protease BACE-1. We have identified several single-entity HS clusters with IC50 values in the low-nanomolar range. These HS clusters are drug leads for AD and offer a novel framework for the manipulation of heparan sulfate-protein interactions in general.

Differential sulfation remodelling of heparan sulfate by extracellular 6-O-sulfatases regulates fibroblast growth factor-induced boundary formation by glial cells: implications for glial cell transplantation.

Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HSs) and fibroblast growth factors (FGFs) 1 and 9 as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC-conditioned and rat OEC-conditioned media showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semisynthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared with OECs, and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs, compared with SCs, to suppress boundary formation. Furthermore, specific anti-FGF1 and anti-FGF9 antibodies disrupted SC-astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signaling mechanism via FGF receptors on astrocytes. We propose a model in which FGF1 and FGF9 signaling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGF receptor 3-IIIb-mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair.

Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

A sulphated glycosaminoglycan extract from Placopecten magellanicus inhibits the Alzheimer's disease ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1).

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marine-derived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC50 = 4.8 µg.mL-1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate.

High sensitivity (zeptomole) detection of BODIPY-labelled heparan sulfate (HS) disaccharides by ion-paired RP-HPLC and LIF detection enables analysis of HS from mosquito midguts.

The fine structure of heparan sulfate (HS), the glycosaminoglycan polysaccharide component of cell surface and extracellular matrix HS proteoglycans, coordinates the complex cell signalling processes that control homeostasis and drive development in multicellular animals. In addition, HS is involved in the infection of mammals by viruses, bacteria and parasites. The current detection limit for fluorescently labelled HS disaccharides (low femtomole; 10-15 mol), has effectively hampered investigations of HS composition in small, functionally-relevant populations of cells and tissues that may illuminate the structural requirements for infection and other biochemical processes. Here, an ultra-high sensitivity method is described that utilises a combination of reverse-phase HPLC, with tetraoctylammonium bromide (TOAB) as the ion-pairing reagent and laser-induced fluorescence detection of BODIPY-FL-labelled disaccharides. The method provides an unparalleled increase in the sensitivity of detection by ∼six orders of magnitude, enabling detection in the zeptomolar range (∼10-21 moles; <1000 labelled molecules). This facilitates determination of HS disaccharide compositional analysis from minute samples of selected tissues, as demonstrated by analysis of HS isolated from the midguts of Anopheles gambiae mosquitoes that was achieved without approaching the limit of detection.

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.

The Hyperlipidaemic Drug Fenofibrate Significantly Reduces Infection by SARS-CoV-2 in Cell Culture Models.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has caused a significant number of fatalities and worldwide disruption. To identify drugs to repurpose to treat SARS-CoV-2 infections, we established a screen to measure the dimerization of angiotensin-converting enzyme 2 (ACE2), the primary receptor for the virus. This screen identified fenofibric acid, the active metabolite of fenofibrate. Fenofibric acid also destabilized the receptor-binding domain (RBD) of the viral spike protein and inhibited RBD binding to ACE2 in enzyme-linked immunosorbent assay (ELISA) and whole cell-binding assays. Fenofibrate and fenofibric acid were tested by two independent laboratories measuring infection of cultured Vero cells using two different SARS-CoV-2 isolates. In both settings at drug concentrations, which are clinically achievable, fenofibrate and fenofibric acid reduced viral infection by up to 70%. Together with its extensive history of clinical use and its relatively good safety profile, this study identifies fenofibrate as a potential therapeutic agent requiring an urgent clinical evaluation to treat SARS-CoV-2 infection.

Novel mechanisms of fibroblast growth factor receptor 1 regulation by extracellular matrix protein anosmin-1.

Activation of fibroblast growth factor (FGF) signaling is initiated by a multiprotein complex formation between FGF, FGF receptor (FGFR), and heparan sulfate proteoglycan on the cell membrane. Cross-talk with other factors could affect this complex assembly and modulate the biological response of cells to FGF. We have previously demonstrated that anosmin-1, a glycosylated extracellular matrix protein, interacts with the FGFR1 signaling complex and enhances its activity in an IIIc isoform-specific and HS-dependent manner. The molecular mechanism of anosmin-1 action on FGFR1 signaling, however, remains unknown. Here, we show that anosmin-1 directly binds to FGFR1 with high affinity. This interaction involves domains in the N terminus of anosmin-1 (cysteine-rich region, whey acidic protein-like domain and the first fibronectin type III domain) and the D2-D3 extracellular domains of FGFR1. In contrast, anosmin-1 binds to FGFR2IIIc with much lower affinity and displays negligible binding to FGFR3IIIc. We also show that FGFR1-bound anosmin-1, although capable of binding to FGF2 alone, cannot bind to a FGF2.heparin complex, thus preventing FGFR1.FGF2.heparin complex formation. By contrast, heparin-bound anosmin-1 binds to pre-formed FGF2.FGFR1 complex, generating an anosmin-1.FGFR1.FGF2.heparin complex. Furthermore, a functional interaction between anosmin-1 and the FGFR1 signaling complex is demonstrated by immunofluorescence co-localization and Transwell migration assays where anosmin-1 was shown to induce opposing effects during chemotaxis of human neuronal cells. Our study provides molecular and cellular evidence for a modulatory action of anosmin-1 on FGFR1 signaling, whereby binding of anosmin-1 to FGFR1 and heparin can play a dual role in assembly and activity of the ternary FGFR1.FGF2.heparin complex.

Rapid purification and high sensitivity analysis of heparan sulfate from cells and tissues: toward glycomics profiling.

Studies on glycosaminoglycans and proteoglycans (PGs) have been hampered by difficulties in isolation and analysis by traditional methods that are laborious and lack sensitivity and throughput. Here we demonstrate a simple method for rapid isolation of proteoglycans (RIP) employing phenol/guanidine/chloroform reagent to purify heparan sulfate (HS) PGs quantitatively from various tissues and cells. We further show that this generic purification methodology, when applied in concert with a BODIPY fluorescent label, permits structural analyses on RIP-purified HS at approximately 1,000-fold higher sensitivity than standard UV detection methods and approximately 10-100-fold higher sensitivity than previous fluorescence detection methods. The utility of RIP-BODIPY methodology was demonstrated by rapid profiling of HS structural composition from small tissue samples, multiple mouse organs, and as little as a few thousand cultured cells. It was also used to generate novel insights into in vivo structural changes in HS from Sulf1 knock-out mice for the first time that differed significantly from previous observations limited to tissue culture experiments. RIP was also applied to purify HS for bioassay testing, exemplified by cell assays of fibroblast growth factor signaling activation; this generated data from 2-O-sulfotransferase knock-out mice and revealed an unexpected deficiency in fibroblast growth factor activation by HS from heterozygous mice. These data demonstrate that RIP will underpin emerging efforts to develop glycomics profiling strategies for HS and other glycosaminoglycans to explore their structure-function relationships in complex biological systems.

Comparable stabilisation, structural changes and activities can be induced in FGF by a variety of HS and non-GAG analogues: implications for sequence-activity relationships.

The activities of heparan sulfate (HS) and heparin do not correlate simply with sulfation levels or sequence. The alternative hypothesis, that appropriate charge and conformational characteristics for protein binding and activity can be provided by other sequences in heparan sulfate and, possibly, also in unrelated sulfated polysaccharides, is explored. Differential scanning fluorimetry was used to measure the thermostabilisation bestowed by modified heparin polysaccharides (proxies for heparan sulfate) on fibroblast growth factor-1 (FGF-1) and fibroblast growth factor-2 (FGF-2), prototypical heparan sulfate-binding proteins, revealing varied abilities and primary sequence-activity redundancy. The effect of substitution pattern on the heparin/heparan sulfate backbone was explored using principal component analysis of (13)C NMR chemical shift data for homogeneously modified heparin polysaccharides revealing complex conformational effects. No simple relationship emerged between these polysaccharides, with their distinct charge distributions and geometries, and their ability to signal. Other, structurally unrelated sulfated polysaccharides were also able to support signalling. These influenced FGF stabilisation in a similar manner to the HS analogues and provided analogous cell signalling activity. For FGF-1, but not FGF-2, signaling correlated strongly with protein stabilisation and circular dichroism spectroscopy demonstrated that some non-HS polysaccharides invoked comparable secondary structural changes to those induced by heparin. Active conformations can readily be found in several heparin derivatives, as well as among non-HS polysaccharides, which comprise unrelated primary sequences, confirming the hypothesis and implying that the level of unique information contained in HS sequences may be much lower than previously thought.

Inhibition of BACE1, the ß-secretase implicated in Alzheimer's disease, by a chondroitin sulfate extract from Sardina pilchardus.

The pharmaceutical and anticoagulant agent heparin, a member of the glycosaminoglycan family of carbohydrates, has previously been identified as a potent inhibitor of a key Alzheimer's disease drug target, the primary neuronal ß-secretase, ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). The anticoagulant activity of heparin has, however, precluded the repurposing of this widely used pharmaceutical as an Alzheimer's disease therapeutic. Here, a glycosaminoglycan extract, composed predominantly of 4-sulfated chondroitin sulfate, has been isolated from Sardina pilchardus, which possess the ability to inhibit BACE1 (IC50 [half maximal inhibitory concentration] = 4.8 µg/mL), while displaying highly attenuated anticoagulant activities (activated partial thromboplastin time EC50 [median effective concentration] = 403.8 µg/mL, prothrombin time EC50 = 1.3 mg/mL). The marine-derived, chondroitin sulfate extract destabilizes BACE1, determined via differential scanning fluorimetry (ΔTm -5°C), to a similar extent as heparin, suggesting that BACE1 inhibition by glycosaminoglycans may occur through a common mode of action, which may assist in the screening of glycan-based BACE1 inhibitors for Alzheimer's disease.

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides.

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a "Shotgun" Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation.

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.