Inhibition of the Cell Uptake of Delta and Omicron SARS-CoV-2 Pseudoviruses by N-Acetylcysteine Irrespective of the Oxidoreductive Environment
Sebastiano La Maestra, Silvano Garibaldi, Roumen Balansky, Francesco D’agostini, Rosanna T Micale, Silvio De Flora
Cells, doi:10.3390/cells11203313
The binding of SARS-CoV-2 spikes to the cell receptor angiotensin-converting enzyme 2 (ACE2) is a crucial target both in the prevention and in the therapy of COVID-19. We explored the involvement of oxidoreductive mechanisms by investigating the effects of oxidants and antioxidants on virus uptake by ACE2-expressing cells of human origin (ACE2-HEK293). The cell uptake of pseudoviruses carrying the envelope of either Delta or Omicron variants of SARS-CoV-2 was evaluated by means of a cytofluorimetric approach. The thiol N-acetyl-L-cysteine (NAC) inhibited the uptake of both variants in a reproducible and dose-dependent fashion. Ascorbic acid showed modest effects. In contrast, neither hydrogen peroxide (H 2 O 2 ) nor a system-generating reactive oxygen species (ROS), which play an important role in the intracellular alterations produced by SARS-CoV-2, were able to affect the ability of either Delta or Omicron SARS-CoV-2 pseudoviruses to be internalized into ACE2-expressing cells. In addition, neither H 2 O 2 nor the ROS generating system interfered with the ability of NAC to inhibit that mechanism. Moreover, based on previous studies, a preventive pharmacological approach with NAC would have the advantage of decreasing the risk of developing COVID-19, irrespective of its variants, and at the same time other respiratory viral infections and associated comorbidities.
Author Contributions: Planning, methodology, formal analysis, and editing, S.L.M.; methodology and formal analysis, S.G.; methodology, R.B., F.D. and R.T.M.; supervision, conceptualization, and writing, S.D.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding excepting the supply of the materials specified under Acknowledgements.
Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable.
References
Akhter, Quéromès, Pillai, Kepenekian, Badar et al., The Combination of bromelain and acetylcysteine (BromAc) synergistically inactivates SARS-CoV-2, Viruses,
doi:10.3390/v13030425Angeli, Reboldi, Trapasso, Zappa, Spanevello et al., COVID-19, vaccines and deficiency of ACE2 and other angiotensinases. Closing the loop on the "Spike effect, Eur. J. Intern. Med,
doi:10.1016/j.ejim.2022.06.015Aruoma, Halliwell, Hoey, Butler, The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid, Free Radic. Biol. Med,
doi:10.1016/0891-5849(89)90066-XBartolini, Stabile, Bastianelli, Giustarini, Pierucci et al., SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione, Redox Biol,
doi:10.1016/j.redox.2021.102041Basi, Turkoglu, In vitro effect of oxidized and reduced glutathione peptides on angiotensin converting enzyme purified from human plasma, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci,
doi:10.1016/j.jchromb.2018.11.023Beyerstedt, Casaro, Rangel, COVID-19: Angiotensin-converting enzyme 2 (ACE2) expression and tissue susceptibility to SARS-CoV-2 infection, Eur. J. Clin. Microbiol. Infect. Dis,
doi:10.1007/s10096-020-04138-6Capettini, Montecucco, Mach, Stergiopulos, Santos et al., Role of renin-angiotensin system in inflammation, immunity and aging, Curr. Pharm. Des,
doi:10.2174/138161212799436593De Flora, Balansky, La Maestra, Rationale for the use of N-acetylcysteine in both prevention and adjuvant therapy of COVID-19, FASEB J,
doi:10.1096/fj.202001807De Flora, Grassi, Carati, Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment, Eur. Respir. J,
doi:10.1183/09031936.97.10071535De Flora, Izzotti, D'agostini, Balansky, Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points, Carcinogenesis,
doi:10.1093/carcin/22.7.999Debnath, Mitra, Dewaker, Prabhakar, Tadala et al., N-acetyl cysteine: A tool to perturb SARS-CoV-2 spike protein conformation, ChemRxiv,
doi:10.26434/chemrxiv.12687923.v2Delgado-Roche, Mesta, Oxidative stress as key player in severe acute respiratory syndrome coronavirus (SARS-CoV) infection, Arch. Med. Res,
doi:10.1016/j.arcmed.2020.04.019Duarte Lana, Lana, Rodrigues, Santos, Navani et al., Nebulization of glutathione and N-Acetylcysteine as an adjuvant therapy for COVID-19 onset, Adv. Redox Res,
doi:10.1016/j.arres.2021.100015Galli, Marcantonini, Giustarini, Albertini, Migni et al., How aging and oxidative stress influence the cytopathic inflammatory effects of SARS-CoV-2 infection: The role of cellular glutathione and cysteine metabolism, Antioxidants,
doi:10.3390/antiox11071366García-Sánchez, Miranda-Díaz, Cardona-Muñoz, The role of oxidative stress in physiopathology and pharmacological treatment with pro-and antioxidant properties in chronic diseases, Oxid. Med. Cell Longev
Grishin, Dolgova, Harms, Pickering, George et al., Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen, J. Mol. Biol,
doi:10.1016/j.jmb.2021.167357Hati, Bhattacharyya, Impact of thiol-disulfide balance on the binding of COVID-19 spike protein with angiotensin-converting enzyme 2 receptor, ACS Omega,
doi:10.1021/acsomega.0c02125Ivanov, Goc, Ivanova, Niedzwiecki, Rath, Inhibition of ACE2 Expression by ascorbic acid alone and its combinations with other natural compounds, Infect. Dis,
doi:10.1177/1178633721994605Jorge-Aarón, Rosa-Ester, N-acetylcysteine as a potential treatment for COVID-19, Future Microbiol
Khanna, Raymond, Jin, Charbit, Gitlin et al., Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro, bioRxiv
Laforge, Elbim, Frère, Hémadi, Massaad et al., Tissue damage from neutrophilinduced oxidative stress in COVID-19, Nat. Rev. Immunol,
doi:10.1038/s41577-020-0407-1Manček-Keber, Hafner-Bratkovič, Lainšček, Benčina, Govednik et al., Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs, FASEB J,
doi:10.1096/fj.202100560RMurae, Shimizu, Yamamoto, Kobayashi, Houri et al., The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione, Biochem. Biophys. Res. Commun,
doi:10.1016/j.bbrc.2022.01.106Shi, Zeida, Edwards, Mallory, Sastre et al., Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein, Proc. Natl. Acad. Sci,
doi:10.1073/pnas.2120419119Strålin, Karlsson, Johansson, Marklund, The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase, Arterioscler. Thromb. Vasc. Biol,
doi:10.1161/01.ATV.15.11.2032Van Den Brand, Haagmans, Van Riel, Osterhaus, Kuiken, The pathology and pathogenesis of experimental severe acute respiratory syndrome and influenza in animal models, J. Comp. Pathol,
doi:10.1016/j.jcpa.2014.01.004Vitiello, Ferrara, Auti, Di Domenico, Boccellino, Advances in the Omicron variant development, J. Intern. Med,
doi:10.1111/joim.13478Walls, Park, Tortorici, Wall, Mcguire et al., Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein, Cell,
doi:10.1016/j.cell.2020.02.058Çakırca, Damar Çakırca, Üstünel, Torun, Koyuncu et al., Thiol level and total oxidant/antioxidant status in patients with COVID-19 infection, Ir. J. Med. Sci,
doi:10.1007/s11845-021-02743-8
