https://escholarship.org/content/qt232927v1/qt232927v1.pdf?t=qmgs8s

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2.

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition.

Dear Editor, COVID-19, caused by the SARS-CoV-2 virus, has now spread worldwide with catastrophic human and economic impacts and currently has infected over 10 million people and killed over 500,000. In an effort to mitigate disease symptoms and impede viral spread, efforts in vaccine development and drug discovery are being conducted at a rapid pace. Recently, we showed that the well-known anticoagulant heparin has exceptional binding affinity to the spike protein (S-protein) of SARS-CoV-2. The Sprotein of SARS-CoV-2 bound more tightly to immobilized heparin (KD= ~10 −11 M) than the S-proteins of either SARS-CoV (KD= ~10 −7 M) or MERS-CoV (KD= ~10 M). However, it is not known whether the tight binding of heparin to the SARS-CoV-2 S-protein translates into potent antiviral activity. In the current study, we evaluated the in vitro antiviral properties of heparin and other closely related polysaccharides to assess the relevance of heparin-related GAGs and other sulfated polysaccharides as part of the pharmacopeia of potential therapeutics that target SARS-CoV-2. Vero-CCL81, which expresses both ACE2 and TMPRSS2, were used for viral replication at high titer for use in antiviral assays. Heparin, heparan sulfates, other glycosaminoglycans (GAGs), and fucoidan and other highly sulfated polysaccharides were screened using surface plasmon resonance (SPR) to measure binding affinity to the SARSCoV-2 S-protein (Fig. 1a). Briefly, solution competition studies between surface immobilized heparin and other sulfated polysaccharides were evaluated by injecting SARS-CoV-2 S-protein (50 nM) alone or mixed with 1 μM of an indicated polysaccharide in SPR buffer at a flow rate of 30 μL/min. After each run, dissociation and regeneration were performed. For each set of competition experiments, a control experiment (S-protein without polysaccharide) was performed to ensure the surface was fully regenerated. Among the tested polysaccharides, RPI27 and RPI-28, complex sulfated polysaccharides (fucoidans) extracted from the seaweed Saccharina japonica, chemo-enzymatically synthesized trisulfated (TriS) heparin, and unfractionated USP-heparin itself were able to compete with heparin for S-protein binding. We selected these compounds along with a non-anticoagulant low molecular weight heparin (NACH) for further study (Fig. 1b). The other GAGs including heparan sulfate, the chondroitin sulfates, and keratan sulfate show no competitive binding when compared to the control. Standard assays were performed to quantify potential cytotoxicity and antiviral activity. Cytotoxicity determination of the polysaccharides was performed using Vero cells and the standard water-soluble tetrazolium salt-1 (WST-1) assay (Takara Bio Inc., Japan). None of the tested polysaccharides showed toxicity even at the highest concentrations tested. Vero cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 2.5 × 10 with varying dosages of polysaccharide to confirm antiviral activity. A focus reduction assay was performed 48 h post infection to determine efficacy. Antiviral activities correlated with the SPR results. The most potent compound tested, RPI-27, is a high molecular weight, branched polysaccharide related to the known compound fucoidan, and had an EC50 of 8.3 ± 4.6 μg/mL, which corresponds to ~83 nM (Fig. 1c, d and Supplementary Table S1). This is substantially more potent than

/pdf/sulfated-polysaccharides-effectively-inhibit-sars-cov-2-in-2tb0bb3rd1.pdf

Sulfated polysaccharides effectively inhibit SARS-CoV-2 in vitro

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.

/pdf/heparin-inhibits-cellular-invasion-by-sars-cov-2-structural-ez1me1kq3u.pdf

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

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has caused a pandemic of historic proportions and continues to spread globally, with enormous consequences to human health. Currently there is no vaccine, effective therapeutic, or prophylactic. As with other betacoronaviruses, attachment and entry of SARS-CoV-2 are mediated by the spike glycoprotein (SGP). In addition to its well-documented interaction with its receptor, human angiotensin-converting enzyme 2 (hACE2), SGP has been found to bind to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we pseudotyped SARS-CoV-2 SGP on a third-generation lentiviral (pLV) vector and tested the impact of various sulfated polysaccharides on transduction efficiency in mammalian cells. The pLV vector pseudotyped SGP efficiently and produced high titers on HEK293T cells. Various sulfated polysaccharides potently neutralized pLV-S pseudotyped virus with clear structure-based differences in antiviral activity and affinity to SGP. Concentration-response curves showed that pLV-S particles were efficiently neutralized by a range of concentrations of unfractionated heparin (UFH), enoxaparin, 6-O-desulfated UFH, and 6-O-desulfated enoxaparin with 50% inhibitory concentrations (IC50s) of 5.99 µg/liter, 1.08 mg/liter, 1.77 µg/liter, and 5.86 mg/liter, respectively. In summary, several sulfated polysaccharides show potent anti-SARS-CoV-2 activity and can be developed for prophylactic as well as therapeutic purposes.IMPORTANCE The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, in late 2019 and its subsequent spread to the rest of the world has created a pandemic situation unprecedented in modern history. While ACE2 has been identified as the viral receptor, cellular polysaccharides have also been implicated in virus entry. The SARS-CoV-2 spike glycoprotein (SGP) binds to glycosaminoglycans like heparan sulfate, which is found on the surface of virtually all mammalian cells. Here, we report structure-based differences in antiviral activity and affinity to SGP for several sulfated polysaccharides, including both well-characterized FDA-approved drugs and novel marine sulfated polysaccharides, which can be developed for prophylactic as well as therapeutic purposes.

Effective Inhibition of SARS-CoV-2 Entry by Heparin and Enoxaparin Derivatives.

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681-686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453-459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.

https://biorxiv.org/content/10.1101/2020.04.14.041459v1.full.pdf

David W. Montgomery

Papers

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions.

Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry

Comparison of cellooligosaccharide conformations in complexes with proteins with energy maps for cellobiose.

Tools to Find Glycoproteins in the Protein Data Bank and Generate Realistic 3D Structures for Them