Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2

  1. Youngchang Kim
  2. Jacek Wower
  3. Natalia Maltseva
  4. Changsoo Chang
  5. Robert Jedrzejczak
  6. Mateusz Wilamowski
  7. Soowon Kang
  8. Vlad Nicolaescu
  9. Glenn Randall
  10. Karolina Michalska
  11. Andrzej Joachimiak

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Abstract

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme’s active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

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  1. Version published to 10.1038/s42003-021-01735-9
  2. ScreenIT

    SciScore for 10.1101/2020.06.26.173872: (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
    Primary antibody was washed with PBS and PBS-T and then cells were incubated in secondary antibody (ImmPRESS Horse Anti-Mouse IgG Polymer Reagent, Peroxidase; Vector Laboratories) for 60 min at room temperature.
    Anti-Mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and Virus: A549 cells expressing human ACE2 (kind gift of Dr. Benjamin Tenoever, Mt. Sinai School of Medicine) were infected under biosafety level 3 conditions with SARS-CoV-2 (nCoV/Washington/1/2020, kindly provided by the National Biocontainment Laboratory, Galveston, TX).
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    Intensities were converted to structure factor amplitudes in the Ctruncate program 32,33 from the CCP4 package 34 and using the apo-form SARS-CoV-2 Nsp15 structure (PDB id: 6VWW) as a search model, the structures were determined using molrep 35, all implemented in the HKL3000 software package.
    CCP4
    suggested: (CCP4, RRID:SCR_007255)
    The initial solutions were refined, both rigid-body refinement and regular restrained refinement by REFMAC 34,36 as a part of HKL3000.
    REFMAC
    suggested: (Refmac, RRID:SCR_014225)
    The models including the ligands were manually adjusted using COOT 37 and then iteratively refined using COOT and PHENIX 38.
    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 did not find any issues relating to the usage of bar graphs.

    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.

    About SciScore

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  3. Version published to 10.1101/2020.06.26.173872v2 on bioRxiv
  4. Version published to 10.1101/2020.06.26.173872v1 on bioRxiv