Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures
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Abstract
The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5’-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5’-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5’-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5’-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.
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SciScore for 10.1101/2020.12.05.409821: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Analysis of sequence conservation: Representative betacoronavirus sequences were selected according to official taxonomy as represented by the International Committee for the Taxonomy of Viruses.39 Multiple sequence alignments of coronavirus sequences were constructed using BLAST40] and MAAFT41 as implemented in GLUE. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 32 In brief, Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.1 i.u./cell in 96-well plates, virus inoculum was … SciScore for 10.1101/2020.12.05.409821: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Analysis of sequence conservation: Representative betacoronavirus sequences were selected according to official taxonomy as represented by the International Committee for the Taxonomy of Viruses.39 Multiple sequence alignments of coronavirus sequences were constructed using BLAST40] and MAAFT41 as implemented in GLUE. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources 32 In brief, Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.1 i.u./cell in 96-well plates, virus inoculum was removed 1 hour post-adsorption and replaced with media containing serial dilutions of the DMA compounds. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Analysis of sequence conservation: Representative betacoronavirus sequences were selected according to official taxonomy as represented by the International Committee for the Taxonomy of Viruses.39 Multiple sequence alignments of coronavirus sequences were constructed using BLAST40] and MAAFT41 as implemented in GLUE. BLAST40suggested: NoneGLUEsuggested: (GLUE, RRID:SCR_009211)CoV-2-5’UTR-FLuc-3’UTR, Cov-2-5’UTR-FLuc, and RLuc RNAs were in vitro synthesized from these plasmid templates using the MEGAscript T7 Transcription Kit (ThermoFisher Scientifc). ThermoFisher Scientifcsuggested: NoneResults 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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
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