Carbohydrate PL-M binds galectin-3 to inhibit SARS-CoV-2 viral entry into cells
Main Article Content
Abstract
Galectin-3 (Gal-3) binds to glycans on the spike protein S1 domain of the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) virus, thereby facilitating viral entry into cells. Because little is known about how to antagonize Gal-3 to block viral activity, we developed a pectin-derived polysaccharide of a(1-6)-D-mannopyranose (termed “ProLectin M” or PL-M) and studied its effect on Vero cells being infected with SARS-CoV-2 virus. Using this novel glycovirology approach, we demonstrated a significant reduction in viral load with no demonstrable cytotoxicity. We also evaluated the efficacy of PL-M in a randomized, double-blinded, placebo-controlled clinical study in 34 patients with mild to moderately severe COVID-19. Overall, treatment with PL-M significantly (p = 0.001) increased RT-PCR cycle counts for N and ORF genes on days 3 (Ct values 32.09 ± 2.39 and 30.69 ± 3.38, respectively) and 7 (Ct values 34.91 ± 0.39 and 34.85 ± 0.61, respectively) compared to placebo. From day 3 post-treatment, all subjects were RT-PCR negative for both genes in the PL-M treatment group, whereas placebo subjects remained positive. On the molecular level, our NMR studies show that PL-M binds relatively strongly to Gal-3, supporting the idea of an antagonist effect on the lectin. Gal-3 also binds strongly to sugar binding sites on the SARS-CoV2 virus spike protein S1 domain as evidenced by competitive lactose binding. In this regard, PL-M competes with the spike protein for binding to Gal-3 and thereby compromises viral entry into susceptible target cells, thus helping to explain our positive clinical effect from PL-M on the course of viral infection. This report provides a brief review of what is already known about PL-M and its effects on SARS-CoV2 virus, as well as new results on an expanded clinical trial with PL-M and NMR studies on the molecular mechanism of action of PL-M, Gal-3, and the SARS-CoV2 viral spike protein S1 from the Omicron variant.
Keywords:
Galectin-3, ProLectin-M, SARS-CoV-2, Clinical Trial, NMR, Spike protein
Article Details
How to Cite
MILLER, Michelle C. et al.
Carbohydrate PL-M binds galectin-3 to inhibit SARS-CoV-2 viral entry into cells.
Medical Research Archives, [S.l.], v. 12, n. 8, aug. 2024.
ISSN 2375-1924.
Available at: <https://esmed.org/MRA/mra/article/view/5616>. Date accessed: 06 sep. 2024.
doi: https://doi.org/10.18103/mra.v12i8.5616.
Section
Research Articles
The Medical Research Archives grants authors the right to publish and reproduce the unrevised contribution in whole or in part at any time and in any form for any scholarly non-commercial purpose with the condition that all publications of the contribution include a full citation to the journal as published by the Medical Research Archives.
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3. Harvey WT. SARS-CoV-2 variants, spike mutations and immune escape. Nature Rev Microbiol. 2021;19:409-424.
4. Bano I, Sharif M, Alam S. Genetic drift in the genome of SARS CoV-2 and its global health concern. J Med Virol. 2022;94:88-98.
5. Khandia, R. Emergence of SARS-CoV-2 Omicron (B.1.1.529) variant, salient features, high global health concerns and strategies to counter it amid ongoing COVID-19 pandemic. Environ Res. 2022;209:112816, doi:10.1016/j.envres.2022.112816.
6. Chi X, Yan R, Zhang J, Zhang G, Zhang Y, Hao M, Zhang Z, Fan P, Dong Y, Yang Y. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2. Science 2020;369:650–655.
7. Behloul N, Baha S, Shi R, Meng J. Role of the GTNGTKR motif in the N-terminal receptor-binding domain of the SARS-CoV-2 spike protein. Virus Res. 2020;286:198058, doi:10.1016/j.virusres.2020.198058.
8. Wang J, Jiang M, Chen X, Montaner LJ. Cytokine storm and leukocyte changes in mild versus severe SARS-CoV-2 infection: Review of 3939 COVID-19 patients in China and emerging pathogenesis and therapy concepts. J Leukoc Biol. 2020;108:17-41.
9. Aleem A, Samad A, Vaqar S. Emerging Variants of SARS-CoV-2 and Novel Therapeutics Against Coronavirus (COVID-19). Stat Pearls. Treasure Island (FL) 2024; PMID: 34033342.
10. Haslwanter D, Dieterle ME, Wec AZ, O'Brien CM. A combination of receptor-binding domain and N-terminal domain neutralizing antibodies limits the generation of SARS-CoV-2 spike neutralization-escape mutants. Mbio 2021;12:doi.org/10.1128/ mbio.02473.
11. Wang C, Wang Z, Wang G, Johnson Y-NL, Zhang K, Li W. COVID-19 in early 2021: current status and looking forward. Signal Transduc Targeted Ther. 2021;6:1-14.
12. Singanayagam A, Hakki S, Dunning J, Madon KJ, Crone MA, Koycheva A, Derqui-Fernandez N, Barnett JL, Whitfield MG, Varro R. Community transmission and viral load kinetics of the SARS-CoV-2 delta (B.1.617.2) variant in vaccinated and unvaccinated individuals in the UK: a prospective longitudinal cohort study. Lancet Infect Dis. 2022;22:183-195.
13. Eyre DW, Taylor D, Purver M, Chapman D, Fowler T, Pouwels KB, Walker AS, Peto TEA. Effect of Covid-19 vaccination on transmission of alpha and delta variants. New Engl J Med. 2022;386:744-756.
14. de Gier B, Andeweg S, Backer JA, Hahné SJ, van den Hof S, de Melker HE, Knol MJ. Vaccine effectiveness against SARS-CoV-2 transmission to household contacts during dominance of Delta variant (B. 1.617. 2). Eurosurveillance 2021;26:2100977.
15. Li F. Receptor recognition mechanisms of coronaviruses: A decade of structural studies. J. Virol. 2015;89:1954–1964.
16. Cui J, Li F, Shi Z-L. Origin and evolution of pathogenic coronaviruses. Nature Rev. Microbiol. 2019;17:181-192.
17. Tortorici MA. Structural basis for human coronavirus attachment to sialic acid receptors. Nat Struct Mol Biol. 2019;26:481-489.
18. Gong Y, Qin S, Dai L, Tian Z. The glycosylation in SARS-CoV-2 and its receptor ACE2. Signal Transduc Targeted Ther. 2021;6:396.
19. Mousavizadeh L, Ghasemi S. Genotype and phenotype of COVID-19: Their roles in pathogenesis. J Microbiol Immunol Infect. 2021;54:159-163.
20. Sivaraman H, Er SY, Choong YK, Gavor E, Sivaraman J. Structural basis of SARS-CoV-2–and SARS-CoV–receptor binding and small-molecule blockers as potential therapeutics. Ann Rev Pharmacol Toxicol. 2021;61:465-493.
21. Xia S, Liu M, Wang C, Xu W. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 2020;30:343–355.
22. Díaz-Alvarez L, Ortega E. The Many Roles of Galectin-3, a Multifaceted Molecule, in Innate Immune Responses against Pathogens. Mediators Inflamm. 2017;9247574, doi:10.1155/2017/9247574.
23. Machala EA, McSharry BP, Rouse BT, Abendroth A, Slobedman B. Gal power: The diverse roles of galectins in regulating viral infections. J. Gen. Virol. 2019;100:333-349.
24. Wang WH, Lin CY, Chang MR, Urbina AN, Assavalapsakul W, Thitithanyanont A, Chen YH, Liu FT, Wang SF. The role of galectins in virus infection - A systemic literature review. J Microbiol Immunol Infect. 2020;53:925-935.
25. Caniglia JL, Asuthkar S, Tsung AJ, Guda MR, Velpula KK. Immunopathology of galectin-3: an increasingly promising target in COVID-19. F1000Res 2020;9:1078, doi:10.12688/f1000research.25979.2.
26. Lee Y-K. Carbohydrate Ligands for COVID-19 Spike Proteins. Viruses 2022;14: doi:10.3390/v14020330.
27. Sato S, St-Pierre C, Bhaumik P, Nieminen J. Galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (DAMPs) and as receptors for pathogen-associated molecular patterns (PAMPs). Immunol Rev. 2009;230:172-187.
28. Del Valle DM, Kim-Schulze S, Huang H-H, Beckmann ND, Nirenberg S, Wang B, Lavin Y. An inflammatory cytokine signature predicts COVID-19 severity and survival. Nature Med. 2020;26:1636-1643.
29. Zhang C, Wu Z, Li J, Zhao H, Wang GQ. Cytokine release syndrome in severe COVID-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality. Int J Antimicrob Agents 2020;55: doi:10.1016/j.ijantimicag.2020.105954.
30. Cervantes-Alvarez E, la Rosa NL, la Mora MS, Valdez-Sandoval P, Palacios-Jimenez M, Rodriguez-Alvarez F, Vera-Maldonado BI, Aguirre-Aguilar E, Escobar-Valderrama JM, Alanis-Mendizabal J. Galectin-3 as a potential prognostic biomarker of severe COVID-19 in SARS-CoV-2 infected patients. Sci Rep. 2022;12:1856.
31. Kulkarni R, Prasad A. Exosomes derived from HIV-1 infected DCs mediate viral trans-infection via fibronectin and galectin-3. Sci. Rep. 2017;7:1-14.
32. Okamoto M, Hidaka A, Toyama M, Baba M. Galectin-3 is involved in HIV-1 expression through NF-κB activation and associated with Tat in latently infected cells. Virus Res. 2019;260:86-93.
33. Dumic J, Dabelic S, Flögel M. Galectin-3: An open-ended story; Review. Biochim Biophys Acta 2006;1760:616 – 635.
34. Zhao Z, Xu X, Cheng H, Miller MC, He Z, Gu H, Zhang Z, Raz A, Tai G, Mayo KH, Zhou Y. Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation. Proc Natl Acad Sci USA. 2021;118:doi: 10.1073/pnas.2021074118).
35. Nesmelova IV, Dings RPM, Mayo KH. Understanding galectin structure-function relationships to design effective antagonists, Chapter 2 in “Galectins” (ed., Klyosov, A.) 2008 Oxford University Press, New York.
36. Barondes SH, Castronovo V, Cooper DNW, Cummings RD. Galectins: A Family of Animal b-Galactoside Binding Lectins. Cell 1994;76:597-596.
37. Mayo KH. Galectins: From theory to therapy. IDrugs 2008;11:97-100.
38. Miller MC, Ribeiro JP, Roldós V, Martín-Santamaría S, Cañada FJ, Nesmelova IA, André S, Pang M, Klyosov AA, Baum LG, Jiménez-Barbero J, Gabius H-J, Mayo KH. Structural aspects of binding of a-linked digalactosides to human galectin-1. Glycobiol. 2011;21:1627-1641.
39. Miller M, Nesmelova IV, Klyosov A, Platt D, Mayo KH. The carbohydrate binding domain on galectin-1 is more extensive for a complex glycan than for simple saccharides: implications for galectin-glycan interactions at the cell surface. Biochem J. 2009a;421:211-221.
40. Ippel H, Miller MC, Vértesy S, Zheng Y, Cañada FJ, Suylen D, Umemoto K, Romano C, Hackeng T, Tai G, Leffler H, Kopitz J, André S, Kübler D, Jiménez-Barbero J, Oscarson S, Gabius H-J, Mayo KH. Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR. Glycobiol. 2016;26:888-903.
41. Miller MC, Klyosov A, Mayo KH. The a-galactomannan Davanat binds galectin-1 at a site different from the conventional galectin carbohydrate binding site. Glycobiol. 2009b;19:1034-1045.
42. Miller MC, Klyosov A, Mayo KH. Structural Features for a-galactomannan binding to galectin-1. Glycobiol. 2012;22:543-551.
43. Miller MC, Ippel H, Suylen D, Klyosov AA, Traber PG, Hackeng T, Mayo KH. Binding of Polysaccharides to Human Galectin-3 at a Non-Canonical Site in its Carbohydrate Recognition Domain. Glycobiol. 2016;26:88-99.
44. Sigamani A. Galectin antagonist use in mild cases of SARS-CoV-2; pilot feasibility randomised, open label, controlled trial. medRxiv. 2020;doi:10.1101/2020.12.03. 20238840.
45. Sigamani A, Mayo KH, Miller MC, Chen-Walden H, Reddy S, Platt D. An Oral Galectin Inhibitor in COVID-19-A Phase II Randomized Controlled Trial. Vaccines 2023;11:731-739.
46. Sigamani A, Chen-Walden H, Pahan J, Miller MC, Mayo KH, Platt D. Carbohydrate prolectin-M, a galectin-3 antagonist, blocks SARS-CoV-2 activity. Intern J Health Sci. 2022;6:6671–6683.
47. Portacci A, Diaferia F, Santomasi C, Dragonieri S, Boniello E, Di Serio F, Carpagnano GE. Galectin-3 as prognostic biomarker in patients with COVID-19 acute respiratory failure. Respir Med. 2021;187:106556.
48. Milanetti E. In-Silico Evidence for a Two Receptor Based Strategy of SARS-CoV-2. Front Mol Biosci. 2021;8:690655, doi:10.3389/fmolb.2021.690655.
49. Delaglio F. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 1995;6:277-293.
50. Johnson BA, Blevins RA. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 1994;4:603-614.
51. Ippel H, Miller MC, Berbis MA, Suylen D, Andre S, Hackeng TM, Canada FJ, Weber C, Gabius H-J, Jimenez-Barbero J, Mayo KH. 1H, 13C, and 15N backbone and side-chain chemical shift assignments for the 36 proline-containing, full length 29 kDa human chimera-type galectin-3. Biomol NMR Assign. 2015;9:59-63.
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