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mSphere ; 6(6): e0071121, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1546463


The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N6-(Δ2-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 µM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.

Antiviral Agents/pharmacology , Bacteria/metabolism , Culture Media/chemistry , Metabolic Networks and Pathways , Microbiota/physiology , SARS-CoV-2/drug effects , Symbiosis/physiology , Bacteria/chemistry , Bacteria/classification , Bacteria/growth & development , Biological Assay , Cell Line, Tumor , Culture Media/pharmacology , Humans , Molecular Docking Simulation , Protease Inhibitors/pharmacology , Protein Binding
Proc Natl Acad Sci U S A ; 118(19)2021 05 11.
Article in English | MEDLINE | ID: covidwho-1254144


Backtracking, the reverse motion of the transcriptase enzyme on the nucleic acid template, is a universal regulatory feature of transcription in cellular organisms but its role in viruses is not established. Here we present evidence that backtracking extends into the viral realm, where backtracking by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) may aid viral transcription and replication. Structures of SARS-CoV-2 RdRp bound to the essential nsp13 helicase and RNA suggested the helicase facilitates backtracking. We use cryo-electron microscopy, RNA-protein cross-linking, and unbiased molecular dynamics simulations to characterize SARS-CoV-2 RdRp backtracking. The results establish that the single-stranded 3' segment of the product RNA generated by backtracking extrudes through the RdRp nucleoside triphosphate (NTP) entry tunnel, that a mismatched nucleotide at the product RNA 3' end frays and enters the NTP entry tunnel to initiate backtracking, and that nsp13 stimulates RdRp backtracking. Backtracking may aid proofreading, a crucial process for SARS-CoV-2 resistance against antivirals.

COVID-19/virology , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine Monophosphate/pharmacology , Antiviral Agents/pharmacology , COVID-19/genetics , COVID-19/metabolism , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Cryoelectron Microscopy/methods , DNA Helicases/metabolism , Genome, Viral , Humans , Molecular Dynamics Simulation , RNA Helicases/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , RNA-Dependent RNA Polymerase/metabolism , RNA-Dependent RNA Polymerase/physiology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Nonstructural Proteins/genetics