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

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.

Keywords

heparin - coronavirus - SARS-CoV-2 - COVID-19 - spike - S1 - RBD - circular dichroism - surface plasmon resonance - molecular modelling

^∗ These authors are joint first authors.

^∗∗ Senior authors.

Publikationsverlauf

Eingereicht: 06. Juni 2020

Angenommen: 13. Oktober 2020

Artikel online veröffentlicht:
23. Dezember 2020

• References

• 1 Woznica A, Gerdt JP, Hulett RE, Clardy J, King N. Mating in the closest living relatives of animals is induced by a bacterial chondroitinase. Cell 2017; 170 (06) 1175.e11-1183.e11
• 2 Ori A, Wilkinson MC, Fernig DG. A systems biology approach for the investigation of the heparin/heparan sulfate interactome. J Biol Chem 2011; 286 (22) 19892-19904
• 3 Ireland EV, Woznica A, King N. Synergistic cues from diverse bacteria enhance multicellular development in a choanoflagellate. Appl Environ Microbiol 2020; 86 (11) e02920-19
• 4 Ori A, Wilkinson MC, Fernig DG. The heparanome and regulation of cell function: structures, functions and challenges. Front Biosci 2008; 13: 4309-4338
• 5 Nunes QM, Su D, Brownridge PJ. et al. The heparin-binding proteome in normal pancreas and murine experimental acute pancreatitis. PLoS One 2019; 14 (06) e0217633
• 6 Rudd TR, Preston MD, Yates EA. 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
• 7 Meneghetti MCZ, Hughes AJ, Rudd TR. et al. Heparan sulfate and heparin interactions with proteins. J R Soc Interface 2015; 12 (110) 0589
• 8 Cagno V, Tseligka ED, Jones ST, Tapparel C. Heparan sulfate proteoglycans and viral attachment: true receptors or adaptation bias?. Viruses 2019; 11 (07) 596
• 9 Chandra N, Liu Y, Liu JX. et al. Sulfated glycosaminoglycans as viral decoy receptors for human adenovirus type 37. Viruses 2019; 11 (03) 247
• 10 Xu D, Esko JD. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 2014; 83: 129-157
• 11 Vicenzi E, Canducci F, Pinna D. et al. Coronaviridae and SARS-associated coronavirus strain HSR1. Emerg Infect Dis 2004; 10 (03) 413-418
• 12 Vicenzi E, Pagani I, Ghezzi S. et al. Subverting the mechanisms of cell death: flavivirus manipulation of host cell responses to infection. Biochem Soc Trans 2018; 46 (03) 609-617
• 13 Ghezzi S, Cooper L, Rubio A. et al. Heparin prevents Zika virus induced-cytopathic effects in human neural progenitor cells. Antiviral Res 2017; 140: 13-17
• 14 WuDunn D, Spear PG. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J Virol 1989; 63 (01) 52-58
• 15 Skidmore MA, Kajaste-Rudnitski A, Wells NM. et al. Inhibition of influenza H5N1 invasion by modified heparin derivatives. MedChemComm 2015; 6: 640-646
• 16 Rusnati M, Coltrini D, Oreste P. et al. Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size. J Biol Chem 1997; 272 (17) 11313-11320
• 17 Harrop HA, Rider CC. 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
• 18 Clementi N, Criscuolo E, Diotti RA. et al. Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro. bioRxiv 2020; . Accessed November 1, 2020 at: https://www.biorxiv.org/content/10.1101/2020.03.29.014407v1
• 19 Yates EA, 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
• 20 Micsonai A, Wien F, Kernya L. et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci U S A 2015; 112 (24) E3095-E3103
• 21 Rudd TR, Skidmore MA, Guimond SE. et al. 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
• 22 Rudd TR, Guimond SE, Skidmore MA. et al. Influence of substitution pattern and cation binding on conformation and activity in heparin derivatives. Glycobiology 2007; 17 (09) 983-993
• 23 Duchesne L, Gentili D, Comes-Franchini M, Fernig DG. Robust ligand shells for biological applications of gold nanoparticles. Langmuir 2008; 24 (23) 13572-13580
• 24 Thakar D, Migliorini E, Coche-Guerente L. et al. A quartz crystal microbalance method to study the terminal functionalization of glycosaminoglycans. Chem Commun (Camb) 2014; 50 (96) 15148-15151
• 25 Migliorini E, Thakar D, Sadir R. et al. Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels. Biomaterials 2014; 35 (32) 8903-8915
• 26 Wang Q, Zhang Y, Wu L. et al. Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell 2020; 181 (04) 894.e9-904.e9
• 27 Lan J, Ge J, Yu J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 2020; 581 (7807): 215-220
• 28 Boittier E, Burns JM, Gandhi NS. et al. GlycoTorch vina: improved docking of sulfated sugars using QM-derived scoring functions. 2020 . Accessed November 1, 2020 at: https://chemrxiv.org/articles/preprint/GlycoTorch_Vina_Improved_Docking_of_Sulfated_Sugars_Using_QM-derived_Scoring_Functions/12279251
• 29 Best RB, Hummer G. Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. J Phys Chem B 2009; 113 (26) 9004-9015
• 30 Kirschner KN, Yongye AB, Tschampel SM. et al. GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J Comput Chem 2008; 29 (04) 622-655
• 31 Jorgensen WL, Chandrasekhar J, Madura JD. et al. Comparison of simple potential functions for simulating liquid water. J Chem Phys 1983; 79: 926-935
• 32 Case DA, Cerutti DS, Cheatham TEI. et al. Amber 2017 Reference Manual. Univ. California; San Francisco: 2017. . Accessed November 1, 2020 at: http://ambermd.org/doc12/Amber17.pdf
• 33 Pettersen EF, Goddard TD, Huang CC. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 2004; 25 (13) 1605-1612
• 34 Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res 2004; 32 (Web Server issue): W665-7
• 35 Onufriev A, Bashford D, Case DA. Exploring protein native states and large-scale conformational changes with a modified generalized born model. Proteins 2004; 55 (02) 383-394
• 36 Weiser J, Shenkin PS, Still WC. Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO). J Comput Chem 1999; 20: 217-230
• 37 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
• 38 Dixon B, Smith R, Artigas A. et al. Can nebulised heparin reduce time to extubation in SARS CoV 2 the CHARTER study protocol. medRxiv 2020; . Accessed November 1, 2020 at: http://medrxiv.org/lookup/doi/10.1101/2020.04.28.20082552
• 39 Schmith VD, Zhou JJ, Lohmer LRL. 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
• 40 Duchesne L, Octeau V, Bearon RN. et al. 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
• 41 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
• 42 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
• 43 Yates EA, Santini F, De Cristofano B. et al. 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
• 44 Rudd TR, Yates EA. Conformational degeneracy restricts the effective information content of heparan sulfate. Mol Biosyst 2010; 6 (05) 902-908
• 45 Lima MA, Hughes AJ, Veraldi N. et al. Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity. MedChemComm 2013; 4: 870-873
• 46 Chang YC, Wang Z, Flax LA. et al. Glycosaminoglycan binding facilitates entry of a bacterial pathogen into central nervous systems. PLoS Pathog 2011; 7 (06) e1002082
• 47 García B, Merayo-Lloves J, Martin C, Alcalde I, Quirós LM, Vazquez F. Surface proteoglycans as mediators in bacterial pathogens infections. Front Microbiol 2016; 7: 220
• 48 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
• 49 Guerrini M, Elli S, Mourier P. et al. 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
• 50 Mycroft-West CJ, Su D, Elli S. et al. The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 receptor binding domain undergoes conformational change upon heparin binding. bioRxiv 2020. Accessed November 1, 2020 at: https://doi.org/10.1101/2020.02.29.971093
• 51 Liu L, Chopra P, Li X. et al. SARS-CoV-2 spike protein binds heparan sulfate in a length- and sequence-dependent manner. bioRxiv 2020; . Accessed November 1, 2020 at: https://doi.org/10.1101/2020.05.10.087288
• 52 Kim SY, Jin W, Sood A. et al. Glycosaminoglycan binding motif at S1/S2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus (SARS-CoV-2) host cell entry. bioRxiv 2020;
• 53 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
• 54 Veraldi N, Hughes AJ, Rudd TR. et al. Heparin derivatives for the targeting of multiple activities in the inflammatory response. Carbohydr Polym 2015; 117: 400-407
• 55 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
• 56 Thachil J. The versatile heparin in COVID-19. J Thromb Haemost 2020; 18 (05) 1020-1022