Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin
Courtney J Mycroft-West, Dunhao Su, Isabel Pagani, Timothy R Rudd, Stefano Elli, Neha S Gandhi, Scott E Guimond, Gavin J Miller, Maria C Z Meneghetti, Helena B Nader, Yong Li, Quentin M Nunes, Patricia Procter, Nicasio Mancini, Massimo Clementi, Antonella Bisio, Nicholas R Forsyth, Vito Ferro, Jeremy E Turnbull, Marco Guerrini, David G Fernig, Elisa Vicenzi, Edwin A Yates, Marcelo A Lima, Mark A Skidmore, Courtney J Mycroft-West, Dunhao Su, Isabel Pagani, Timothy R Rudd, Stefano Elli, Neha S Gandhi, Scott E Guimond, Gavin J Miller, Maria C Z Meneghetti, Helena B Nader, Yong Li, Quentin M Nunes, Patricia Procter, Nicasio Mancini, Massimo Clementi, Antonella Bisio, Nicholas R Forsyth, Vito Ferro, Jeremy E Turnbull, Marco Guerrini, David G Fernig, Elisa Vicenzi, Edwin A Yates, Marcelo A Lima, Mark A Skidmore
Abstract
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.
Conflict of interest statement
None declared.
Thieme. All rights reserved.
Figures
References
- Woznica A, Gerdt J P, Hulett R E, Clardy J, King N. Mating in the closest living relatives of animals is induced by a bacterial chondroitinase. Cell. 2017;170(06):1.175E14–1.183E14.
- Ori A, Wilkinson M C, Fernig D G. A systems biology approach for the investigation of the heparin/heparan sulfate interactome. J Biol Chem. 2011;286(22):19892–19904.
- Ireland E V, Woznica A, King N. Synergistic cues from diverse bacteria enhance multicellular development in a choanoflagellate. Appl Environ Microbiol. 2020;86(11):e02920-19.
- Ori A, Wilkinson M C, Fernig D G. The heparanome and regulation of cell function: structures, functions and challenges. Front Biosci. 2008;13:4309–4338.
- Nunes Q M, Su D, Brownridge P J. The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis. PLoS One. 2019;14(06):e0217633.
- Rudd T R, Preston M D, Yates E A. The nature of the conserved basic amino acid sequences found among 437 heparin binding proteins determined by network analysis. Mol Biosyst. 2017;13(05):852–865.
- Meneghetti M CZ, Hughes A J, Rudd T R. Heparan sulfate and heparin interactions with proteins. J R Soc Interface. 2015;12(110):589.
- Cagno V, Tseligka E D, Jones S T, Tapparel C. Heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias? Viruses. 2019;11(07):596.
- Chandra N, Liu Y, Liu J X. Sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37. Viruses. 2019;11(03):247.
- Xu D, Esko J D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem. 2014;83:129–157.
- Vicenzi E, Canducci F, Pinna D. Coronaviridae and SARS-associated coronavirus strain HSR1. Emerg Infect Dis. 2004;10(03):413–418.
- Vicenzi E, Pagani I, Ghezzi S. Subverting the mechanisms of cell death: flavivirus manipulation of host cell responses to infection. Biochem Soc Trans. 2018;46(03):609–617.
- Ghezzi S, Cooper L, Rubio A. Heparin prevents Zika virus induced-cytopathic effects in human neural progenitor cells. Antiviral Res. 2017;140:13–17.
- WuDunn D, Spear P G. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J Virol. 1989;63(01):52–58.
- Skidmore M A, Kajaste-Rudnitski A, Wells N M. Inhibition of influenza H5N1 invasion by modified heparin derivatives. MedChemComm. 2015;6:640–646.
- Rusnati M, Coltrini D, Oreste P. Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size. J Biol Chem. 1997;272(17):11313–11320.
- Harrop H A, Rider C C. Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4. Glycobiology. 1998;8(02):131–137.
- Clementi N, Criscuolo E, Diotti R A.Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro bioRxiv 202010.1101/2020.03.29.014407. Accessed November 1, 2020 at:
- Yates E A, Santini F, Guerrini M, Naggi A, Torri G, Casu B. 1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives. Carbohydr Res. 1996;294:15–27.
- Micsonai A, Wien F, Kernya L. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A. 2015;112(24):E3095–E3103.
- Rudd T R, Skidmore M A, Guimond S E. The potential for circular dichroism as an additional facile and sensitive method of monitoring low-molecular-weight heparins and heparinoids. Thromb Haemost. 2009;102(05):874–878.
- Rudd T R, Guimond S E, Skidmore M A. Influence of substitution pattern and cation binding on conformation and activity in heparin derivatives. Glycobiology. 2007;17(09):983–993.
- Duchesne L, Gentili D, Comes-Franchini M, Fernig D G. Robust ligand shells for biological applications of gold nanoparticles. Langmuir. 2008;24(23):13572–13580.
- Thakar D, Migliorini E, Coche-Guerente L. A quartz crystal microbalance method to study the terminal functionalization of glycosaminoglycans. Chem Commun (Camb) 2014;50(96):15148–15151.
- Migliorini E, Thakar D, Sadir R. Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels. Biomaterials. 2014;35(32):8903–8915.
- Wang Q, Zhang Y, Wu L. Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell. 2020;181(04):8.94E11–9.04E11.
- Lan J, Ge J, Yu J.Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Nature 2020581(7807):215–220.
- Boittier E, Burns J M, Gandhi N S.GlycoTorch vina: improved docking of sulfated sugars using QM-derived scoring functions 2020. Accessed November 1, 2020 at:
- Best R B, Hummer G. Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. J Phys Chem B. 2009;113(26):9004–9015.
- Kirschner K N, Yongye A B, Tschampel S M. GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J Comput Chem. 2008;29(04):622–655.
- Jorgensen W L, Chandrasekhar J, Madura J D. Comparison of simple potential functions for simulating liquid water. J Chem Phys. 1983;79:926–935.
- Case D A, Cerutti D S, Cheatham T EI.Amber 2017 Reference Manual Univ. California; San Francisco: 2017. Accessed November 1, 2020 at:
- Pettersen E F, Goddard T D, Huang C C. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–1612.
- Dolinsky T J, Nielsen J E, McCammon J A, Baker N A.PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations Nucleic Acids Res 200432(Web Server issue):W665-7.
- Onufriev A, Bashford D, Case D A. Exploring protein native states and large-scale conformational changes with a modified generalized born model. Proteins. 2004;55(02):383–394.
- Weiser J, Shenkin P S, Still W C. Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO) J Comput Chem. 1999;20:217–230.
- Genheden S, Ryde U. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities. Expert Opin Drug Discov. 2015;10(05):449–461.
- Dixon B, Smith R, Artigas A.Can nebulised heparin reduce time to extubation in SARS CoV 2 the CHARTER study protocol medRxiv 202010.1101/2020.04.28.20082552. Accessed November 1, 2020 at:
- Schmith V D, Zhou J J, Lohmer L RL. The approved dose of ivermectin alone is not the ideal dose for the treatment of COVID-19. Clin Pharmacol Ther. 2020;108(04):762–765.
- Duchesne L, Octeau V, Bearon R N. Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate. PLoS Biol. 2012;10(07):e1001361.
- Schuck P, Zhao H. The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing. Methods Mol Biol. 2010;627:15–54.
- Sadir R, Forest E, Lortat-Jacob H. The heparan sulfate binding sequence of interferon-γ increased the on rate of the interferon-γ-interferon-γ receptor complex formation. J Biol Chem. 1998;273(18):10919–10925.
- Yates E A, Santini F, De Cristofano B. Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1J(CH) coupling constants in heparin derivatives. Carbohydr Res. 2000;329(01):239–247.
- Rudd T R, Yates E A. Conformational degeneracy restricts the effective information content of heparan sulfate. Mol Biosyst. 2010;6(05):902–908.
- Lima M A, Hughes A J, Veraldi N. Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity. MedChemComm. 2013;4:870–873.
- Chang Y C, Wang Z, Flax L A. Glycosaminoglycan binding facilitates entry of a bacterial pathogen into central nervous systems. PLoS Pathog. 2011;7(06):e1002082.
- García B, Merayo-Lloves J, Martin C, Alcalde I, Quirós L M, Vazquez F. Surface proteoglycans as mediators in bacterial pathogens infections. Front Microbiol. 2016;7:220.
- Milewska A, Zarebski M, Nowak P, Stozek K, Potempa J, Pyrc K. Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells. J Virol. 2014;88(22):13221–13230.
- Guerrini M, Elli S, Mourier P. An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence. Biochem J. 2013;449(02):343–351.
- Mycroft-West C J, Su D, Elli S.The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 receptor binding domain undergoes conformational change upon heparin bindingbioRxiv 2020. Accessed November 1, 2020 at:
- Liu L, Chopra P, Li X.SARS-CoV-2 spike protein binds heparan sulfate in a length- and sequence-dependent manner bioRxiv 202010.1101/2020.05.10.087288. Accessed November 1, 2020 at:
- Kim S Y, Jin W, Sood A. Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry. bioRxiv. 2020 doi: 10.1101/2020.04.14.041459.
- Tang N, Bai H, Chen X, Gong J, Li D, Sun Z. Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J Thromb Haemost. 2020;18(05):1094–1099.
- Veraldi N, Hughes A J, Rudd T R. Heparin derivatives for the targeting of multiple activities in the inflammatory response. Carbohydr Polym. 2015;117:400–407.
- Mousavi S, Moradi M, Khorshidahmad T, Motamedi M. Anti-inflammatory effects of heparin and its derivatives: a systematic review. Adv Pharmacol Sci. 2015;2015:507151.
- Thachil J. The versatile heparin in COVID-19. J Thromb Haemost. 2020;18(05):1020–1022.
Source: PubMed