Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

  1. Sneha Munshi
  2. Krishna Neupane
  3. Sandaru M. Ileperuma
  4. Matthew T.J. Halma
  5. Jamie A. Kelly
  6. Clarissa F. Halpern
  7. Jonathan D. Dinman
  8. Sarah Loerch
  9. Michael T. Woodside

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Abstract

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use −1 programmed ribosomal frameshifting (−1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates −1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs—the most likely source of future zoonoses—as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed −1 PRF significantly in several of them, while having limited to no effect on −1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit −1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

Article activity feed

  1. Rapid Reviews Infectious Diseases

    Pavel Baranov

    Review 3: "Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses"

    This preprint examines the therapeutic potential of small molecule ligands that inhibit ribosomal frameshifting.The authors find the tested ligands inhibit ribosomal frameshifting in vitro for a panel of coronaviruses and reviewers find the preprint's strength of evidence strong.

  2. Rapid Reviews Infectious Diseases

    Yitzhak Pilpel

    Review 2: "Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses"

    This preprint examines the therapeutic potential of small molecule ligands that inhibit ribosomal frameshifting.The authors find the tested ligands inhibit ribosomal frameshifting in vitro for a panel of coronaviruses and reviewers find the preprint's strength of evidence strong.

  3. Rapid Reviews Infectious Diseases

    Ewan Plant

    Review 1: "Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses"

    This preprint examines the therapeutic potential of small molecule ligands that inhibit ribosomal frameshifting.The authors find the tested ligands inhibit ribosomal frameshifting in vitro for a panel of coronaviruses and reviewers find the preprint's strength of evidence strong.

  5. ScreenIT

    SciScore for 10.1101/2021.08.06.455424: (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

    Experimental Models: Cell Lines
    SentencesResources
    Bi-fluorescence frameshift measurements: A549 (CRL-185) cells were purchased from the American Type Culture Collection (Manassas, VA).
    A549
    suggested: None
    Recombinant DNA
    SentencesResources
    For measuring −1 PRF efficiency in the panel of CoV frameshift signals used to test inhibitors, we used a dual-luciferase reporting system based on a plasmid containing the sequence for Renilla luciferase and the multiple cloning site (MCS) from the plasmid pMLuc-1 (Novagen) upstream of the firefly luciferase sequence in the plasmid pISO (addgene), as described previously (19,26).
    pMLuc-1
    suggested: None
    pISO
    suggested: None
    To remove rNTPs and polymerase, mRNA was purified using weak anion exchange as described (34). (3) Construction of bi-fluorescent reporter for cell-based assays: The reporter plasmid pJD2261 was constructed by cloning a gBlock (IDT) encoding AcGFP, the HIV-1 −1 PRF signal, mCherry, and insulator A2 described previously(35) into PstI-digested pUC19 (Genbank accession L09137).
    pJD2261
    suggested: None
    pUC19
    suggested: RRID:Addgene_50005)
    Next, the CMV promoter from pJD2044 was inserted into KpnI/PstI-digested fluorescent reporter.
    pJD2044
    suggested: None
    The SARS-CoV-2 frameshift reporter was made by digesting pJD2514 [19] using SalI and BamHI, gel-purifying the SARS-CoV-2 −1 PRF insert, and then ligating it into SalI/BamHI-digested pJD2261 vector.
    pJD2514
    suggested: None
    Software and Algorithms
    SentencesResources
    Secondary structural predictions were performed on the pKiss (31) and Hotknots (32,33) web servers using default settings, with the proposed structures (Fig.
    pKiss
    suggested: (pKiss, RRID:SCR_017256)
    All data analysis was carried out in Graphpad Prizm 8 and 9.
    Graphpad
    suggested: (GraphPad, RRID:SCR_000306)

    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.

    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.

  6. Version published to 10.1101/2021.08.06.455424v1 on bioRxiv