Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics

Abstract

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

SciScore for 10.1101/2021.07.21.453274: (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	Plaque numbers were scored in at least 3 replicates per dilution by independent readers, who were blinded with respect to the source of the supernatant.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Cells and Virus: African green monkey kidney cells (Vero, subtype E6) and the human lung epithelial cell line (Calu-3 cells) were cultured in high glucose DMEM and low glucose DMEM medium, both complemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2.	Calu-3 suggeste…

SciScore for 10.1101/2021.07.21.453274: (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	Plaque numbers were scored in at least 3 replicates per dilution by independent readers, who were blinded with respect to the source of the supernatant.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Experimental Models: Cell Lines
Sentences	Resources
Cells and Virus: African green monkey kidney cells (Vero, subtype E6) and the human lung epithelial cell line (Calu-3 cells) were cultured in high glucose DMEM and low glucose DMEM medium, both complemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO2.	Calu-3 suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
48-72 h supernatants were collected and the harvested virus was quantified as PFU/mL by titering in Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
Recombinant DNA
Sentences	Resources
The pRSFDuet-1 plasmids (Novagen) encoding SARS-CoV-2 nsp14 or nsp10 engineered with an N-terminal His-SUMO tag were prepared as follows: SARS-CoV-2 RNA isolated from the supernatant of SARS-CoV-2-infected Vero E6 cells was provided by Benjamin R. tenOever48.	pRSFDuet-1 suggested: RRID:Addgene_165451)
PCR products were digested with BamHI and XhoI and subsequently ligated into BamHI and XhoI-digested pRSFDuet-His6-sumo vector and sequence verified.	pRSFDuet-His6-sumo suggested: None
Software and Algorithms
Sentences	Resources
The molecular docking calculations were performed using GOLD 2020.2 software (Cambridge Crystallographic Data Centre, Cambridge, UK)54	GOLD suggested: (GOLD, RRID:SCR_000188)
The figures for the docking poses of the largest docking score value was generated with PyMOL Delano Scientific LLC software (Schrödinger, New York, USA)55	PyMOL suggested: (PyMOL, RRID:SCR_000305)

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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Combination of Antiviral Drugs to Inhibit SARS-CoV-2 Polymerase and Exonuclease as Potential COVID-19 Therapeutics

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