6′,6′-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication
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Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6′,6′-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5’ end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a ∼100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication.
Importance
Currently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.
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SciScore for 10.1101/2021.05.20.445077: (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 Calu-3 cells (ATCC HTB-55) were cultured in EMEM medium complemented with 10% FCS, penicillin/streptomycin, 2 mM L-glutamine, non-essential amino acids and sodium pyruvate (Life technologies). Calu-3suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)Cytopathic effect (CPE) reduction assay: Cells were seeded in 96-well flat bottom plates in 100 μl at a density of 10000 cells/well of Huh7, 15000 cells/well of MRC-5 or 20000 cells/well of Vero cells. Verosuggested: CLS Cat# …SciScore for 10.1101/2021.05.20.445077: (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 Calu-3 cells (ATCC HTB-55) were cultured in EMEM medium complemented with 10% FCS, penicillin/streptomycin, 2 mM L-glutamine, non-essential amino acids and sodium pyruvate (Life technologies). Calu-3suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)Cytopathic effect (CPE) reduction assay: Cells were seeded in 96-well flat bottom plates in 100 μl at a density of 10000 cells/well of Huh7, 15000 cells/well of MRC-5 or 20000 cells/well of Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Dose response assay: To evaluate the effect of compound treatment on viral progeny titers, confluent monolayers of Vero, Huh7 or MRC-5 were seeded in 24-well plates. Huh7suggested: NoneMRC-5suggested: NoneExperimental Models: Organisms/Strains Sentences Resources MERS-CoV (strain EMC/2012; (3, 4)), SARS-CoV (Frankfurt-1 strain,(88)), SARS-CoV-2/Leiden-0002 (GenBank accession nr. MERS-CoVsuggested: NoneRecombinant DNA Sentences Resources (DFA) and its adenine phosphoramidate pro-drug (pDFA) were designed and synthesized, designated as 2c and 3a, respectively, as described in a previous report (41). pDFAsuggested: NoneResults from OddPub: Thank you for sharing your data.
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:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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