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1.
Front Immunol ; 13: 844749, 2022.
Article in English | MEDLINE | ID: covidwho-1809396

ABSTRACT

SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA-dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Escherichia coli/genetics , Humans , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics
2.
Engineering (Beijing) ; 2021 Jun 26.
Article in English | MEDLINE | ID: covidwho-1284079

ABSTRACT

Many microorganisms have mechanisms that protect cells against attack from viruses. The fermentation components of Streptomyces sp. 1647 exhibit potent anti-influenza A virus (IAV) activity. This strain was isolated from soil in southern China in the 1970s, but the chemical nature of its antiviral substance(s) has remained unknown until now. We used an integrated multi-omics strategy to identify the antiviral agents from this streptomycete. The antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) analysis of its genome sequence revealed 38 biosynthetic gene clusters (BGCs) for secondary metabolites, and the target BGCs possibly responsible for the production of antiviral components were narrowed down to three BGCs by bioactivity-guided comparative transcriptomics analysis. Through bioinformatics analysis and genetic manipulation of the regulators and a biosynthetic gene, cluster 36 was identified as the BGC responsible for the biosynthesis of the antiviral compounds. Bioactivity-based molecular networking analysis of mass spectrometric data from different recombinant strains illustrated that the antiviral compounds were a class of structural analogues. Finally, 18 pseudo-tetrapeptides with an internal ureido linkage, omicsynins A1-A6, B1-B6, and C1-C6, were identified and/or isolated from fermentation broth. Among them, 11 compounds (omicsynins A1, A2, A6, B1-B3, B5, B6, C1, C2, and C6) are new compounds. Omicsynins B1-B4 exhibited potent antiviral activity against IAV with the 50% inhibitory concentration (IC50) of approximately 1 µmol∙L-1 and a selectivity index (SI) ranging from 100 to 300. Omicsynins B1-B4 also showed significant antiviral activity against human coronavirus HCoV-229E. By integrating multi-omics data, we discovered a number of novel antiviral pseudo-tetrapeptides produced by Streptomyces sp. 1647, indicating that the secondary metabolites of microorganisms are a valuable source of novel antivirals.

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