Small-molecule ligands can inhibit −1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses
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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
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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.
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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.
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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.
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Strength of evidence
Reviewers: Ewan Plant (US FDA) | 📗📗📗📗◻️
Yitzhak Pilpel (Weizmann Institute) | 📘📘📘📘📘
Pavel Baranov (University of College Cork) | 📒📒📒◻️◻️ -
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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Bi-fluorescence frameshift measurements: A549 (CRL-185) cells were purchased from the American Type Culture Collection (Manassas, VA). A549suggested: NoneRecombinant DNA Sentences Resources 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) … 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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Bi-fluorescence frameshift measurements: A549 (CRL-185) cells were purchased from the American Type Culture Collection (Manassas, VA). A549suggested: NoneRecombinant DNA Sentences Resources 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-1suggested: NonepISOsuggested: NoneTo 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). pJD2261suggested: NonepUC19suggested: RRID:Addgene_50005)Next, the CMV promoter from pJD2044 was inserted into KpnI/PstI-digested fluorescent reporter. pJD2044suggested: NoneThe 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. pJD2514suggested: NoneSoftware and Algorithms Sentences Resources Secondary structural predictions were performed on the pKiss (31) and Hotknots (32,33) web servers using default settings, with the proposed structures (Fig. pKisssuggested: (pKiss, RRID:SCR_017256)All data analysis was carried out in Graphpad Prizm 8 and 9. Graphpadsuggested: (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.
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