Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity

  1. Luisa Diomede
  2. Sara Baroni
  3. Ada De Luigi
  4. Arianna Piotti
  5. Jacopo Lucchetti
  6. Claudia Fracasso
  7. Luca Russo
  8. Valerio Bonaldo
  9. Nicolò Panini
  10. Federica Filippini
  11. Fabio Fiordaliso
  12. Alessandro Corbelli
  13. Marten Beeg
  14. Massimo Pizzato
  15. Francesca Caccuri
  16. Marco Gobbi
  17. Emiliano Biasini
  18. Arnaldo Caruso
  19. Mario Salmona

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Abstract

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

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  1. Version published to 10.3390/v13091745
  2. ScreenIT

    SciScore for 10.1101/2021.07.30.454436: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membranes were incubated overnight at 4°C with anti-ACE2 mouse monoclonal antibody AC18Z (1:2000, Santa Cruz) or anti-β-actin mouse monoclonal antibody (1:5000, Sigma Aldrich)
    anti-β-actin
    suggested: None
    Anti-mouse IgG peroxidase conjugated (1:5000, Sigma Aldrich) was used as secondary antibody.
    Anti-mouse IgG
    suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
    FLAG-tagged ACE2 (AdipoGen) was captured on the chip by a previously immobilized anti-FLAG antibody (Merck Life Science S.r.l).
    ACE2
    suggested: None
    anti-FLAG
    suggested: None
    S protein (Euprotein), its S1 domain and its RBD (SinoBiological), all Fc-tagged, were captured on the same chip by a previously immobilized anti-Fc antibody (Merck Life Science S.r.l).
    anti-Fc
    suggested: None
    FlagACE-2, FcS, FcRBD or FcS1 were then flowed on the corresponding anti-tag antibodies at 30 µg/mL in 10 mM phosphate buffer containing 150 mM NaCl and 0.005% Tween 20 (PBST, pH 7.4), also used as running buffer.
    anti-tag
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, HEK293-T cells were seeded into 10 cm plates with DMEM containing 0.5 mg/mL geneticin G418 (Thermofisher).
    HEK293-T
    suggested: None
    Cell viability: Cells were seeded on 96-well plates (7.5 × 103 Vero cells/well and 2 × 104 HEK293-ACE2 cells/well) in complete DMEM medium with 10% FBS.
    Vero
    suggested: RRID:CVCL_ZW93)
    HEK293-ACE2
    suggested: None
    Virus: We successfully isolated SARS-CoV-2 in Vero E6 cells from the nasopharyngeal swab of a COVID-19 patient (31).
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    : 5′-GCT GGA TCC CCT AAT ATT ACA AAC TTG TGCC-3′; RBD-R: 5′-TGC CTC GAG CTC AAG TGT CTG TGGATC AC-3′) into pGEM T-easy vector (Promega, Madison, WI, USA).
    pGEM T-easy
    suggested: None
    Software and Algorithms
    SentencesResources
    The ZOE™ images were analyzed with Fiji software (see S1 Appendix).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Surface plasmon resonance: All analyses were done with a ProteOn XPR36 Protein Interaction Array system (Bio-Rad Laboratories, Hercules, CA) surface plasmon resonance (SPR) apparatus with six parallel flow channels that can immobilize up to six ligands on the same sensor chip.
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Statistical analysis: For statistical analyses were used with software version 7.03/8.0 (GraphPad) including all the data points, with the exception of experiments in which negative and/or positive controls did not give the expected outcome.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.07.30.454436v1 on bioRxiv