A dual mechanism of action of AT-527 against SARS-CoV-2 polymerase

  1. Ashleigh Shannon
  2. Véronique Fattorini
  3. Bhawna Sama
  4. Barbara Selisko
  5. Mikael Feracci
  6. Camille Falcou
  7. Pierre Gauffre
  8. Priscila El Kazzi
  9. Adrien Delpal
  10. Etienne Decroly
  11. Karine Alvarez
  12. Cécilia Eydoux
  13. Jean-Claude Guillemot
  14. Adel Moussa
  15. Steven S. Good
  16. Paolo La Colla
  17. Kai Lin
  18. Jean-Pierre Sommadossi
  19. Yingxiao Zhu
  20. Xiaodong Yan
  21. Hui Shi
  22. François Ferron
  23. Bruno Canard

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Abstract

The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp8 2 -RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3′ end of the RNA product strand. Its modified ribose group (2′-fluoro, 2′-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.

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  1. Version published to 10.1038/s41467-022-28113-1
  2. Version published to 10.21203/rs.3.rs-624370/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2021.03.23.436564: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Nsp8 was probed with mouse monoclonal anti-nsp8 (5A10) from GeneTex (GTX632696), and HRP-conjugated rabbit anti-mouse secondary (Agilent Dako), washing 3X in PBS.T between each antibody.
    anti-nsp8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV cofactor protein nsp10 was expressed under the control of a Tet-promoter in a pASK vector in E. coli NEB Express C2523 cells (New England Biolabs) carrying the pRare2LacI (Novagen) plasmid.
    C2523
    suggested: None
    SARS-CoV nsp14 was expressed from a pDEST14 vector in E. coli strain NEB Express C2566 cells (New England Biolabs) carrying the pRare2, in the presence of Ampicillin (100 μM/mL) and Chloramphenicol (17 μg/mL).
    C2566
    suggested: None
    Software and Algorithms
    SentencesResources
    Nsp8 was probed with mouse monoclonal anti-nsp8 (5A10) from GeneTex (GTX632696), and HRP-conjugated rabbit anti-mouse secondary (Agilent Dako), washing 3X in PBS.T between each antibody.
    Agilent Dako
    suggested: None
    All movies were automatically recorded using SerialEM (Mastronarde, 2005) at a magnification of 105K, with a physical pixel size of 0.83 Å.
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    CTF estimation, 2D classification, 3D classification and refinements were all performed in cryoSPARC.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    The model was manually built in Coot (Emsley et al., 2010) with the guidance of the Cryo-EM map, and in combination with real space refinement using Phenix (Afonine et al., 2018).
    Coot
    suggested: (Coot, RRID:SCR_014222)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 33. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

    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.

  4. Version published to 10.1101/2021.03.23.436564 on bioRxiv