The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 μg.ml ⁻¹ , which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae .

The evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed. The HP-SEC/TDA technique permits the evaluation of polymeric samples by exploiting the combined and simultaneous action of three on-line detectors: light scattering detectors (LALLS/RALLS); refractometer and viscometer. Three commercial LMWH samples, enoxaparin, tinzaparin and dalteparin, a γ-ray depolymerized heparin (γ-Hep) and its chromatographic fractions, and a synthetic pentasaccharide were analysed by HP-SEC/TDA. The same samples were analysed also with a conventional HP-SEC method employing refractive index (RI) and UV detectors and two different chromatographic column set, silica gel and polymeric gel columns. In both chromatographic systems, two different calibration curves were built up by using (i) γ-Hep chromatographic fractions and the corresponding Mw parameters obtained via HP-SEC/TDA; (ii) the whole γ-Hep preparation with broad Mw dispersion and the corresponding cumulative distribution function calculated via HP-SEC/TDA. In addition, also a chromatographic column calibration according to European Pharmacopoeia indication was built up. By comparing all the obtained results, some important differences among Mw and size distribution values of the three LMWHs were found with the five different calibration methods and with HP-SEC/TDA method. In particular, the detection of the lower molecular weight components turned out to be the most critical aspect. Whereas HP-SEC/TDA may underestimate species under 2 KDa when present in low concentration, other methods appeared to emphasize their content.

Heparanase is an endo-β-d-glucuronidase that cleaves the heparan sulfate chains of heparan sulfate proteoglycans and is implicated in angiogenesis and metastasis. With the aim of establishing a simple and reliable method for studying the susceptibility of heparin/heparan sulfate oligosaccharides to be cleaved by heparanase, an on-line ion pair reversed-phase high-performance liquid chromatographic/electrospray ionization mass spectrometric method was set up. The method works in the micromolar range of concentration and does not require derivatization of the substrate or of the products. It is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal heparin disaccharide standard. Substrates were (1) the synthetic pentasaccharides GlcN_NS,6S - GlcA - GlcN_NS,3S,6S - IdoA_2S - GlcN_NS,6S - OMe (AGA*IA_M) and GlcN_NS,6S - GlcA - GlcN_NS,6S - IdoA_2S - GlcN_NS,6S - OMe (AGAIA_M), corresponding to the heparin/heparan sulfate active site for antithrombin, and to the same sequence devoid of the 3-O-sulfate group in the central glucosamine, respectively; and (2) two natural heparin octasaccharides containing the AGA*IA sequence in different locations along the chain. The two pentasaccharides exhibited a higher susceptibility to heparanase cleavage with respect to the octasaccharides. The commercial availability of AGA*IA_M makes it an ideal substrate to determine the specific activity of heparanase preparations. The present method could also be used for rapid screening of potential heparanase inhibitors.

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.

A number of low molecular weight heparin (LMWH) products are available for clinical use and although all share a similar mechanism of action, they are classified as distinct drugs because of the different depolymerisation processes of the native heparin resulting in substantial pharmacokinetic and pharmacodynamics differences. While enoxaparin has been extensively investigated, little information is available regarding the LMWH dalteparin. The present study is focused on the detailed structural characterization of Fragmin^® by LC-MS and NMR applied both to the whole drug and to its enzymatic products. For a more in-depth approach, size homogeneous octasaccharide and decasaccharide components together with their fractions endowed with high or no affinity toward antithrombin were also isolated and their structural profiles characterized. The combination of different analytical strategies here described represents a useful tool for the assessment of batch-to-batch structural variability and for comparative evaluation of structural features of biosimilar products.