Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease

Self Cite

The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication–transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.

Section: Chemical Library Screen Design and Resultsmentioning

confidence: 99%

Section: Chemical Library Screen Design and Resultsmentioning

confidence: 99%

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

Bertolin

Weissmann

Zeng

et al. 2021

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“…To test the role of these 4 compounds on nsp16 activity, we purified nsp16 fused to its cofactor nsp10. Fusion of nsp14 to its cofactor nsp10 has been shown as an effective strategy to obtain active recombinant nsp14 exonuclease (Canal et al [30]). Following this strategy, we decided to fuse the methyltransferase nsp16 with its cofactor nsp10 that, similarly to nsp14, would ensure stoichiometric expression of both subunits as well as their association.…”

Section: Primary Identification Of Nsp14 Inhibitorsmentioning

confidence: 99%

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp14 RNA cap methyltransferase

Basu

Mak

Ulferts

et al. 2021

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The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause a fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. To identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play key roles in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2′-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified four compounds as potential inhibitors of nsp14, all of which also showed antiviral capacity in a cell-based model of SARS-CoV-2 infection. Three of the four compounds also exhibited synergistic effects on viral replication with remdesivir.

“…Compounds that inhibited viral replication without deleterious effects on uninfected cells were selected as candidate antivirals. The seven papers describing these results are published in this addition of the Biochemical Journal [1][2][3][4][5][6][7].…”

mentioning

confidence: 99%

“…Although both inhibited viral replication in cell culture experiments it remains to be established if inhibition is via Nsp13. The Nsp15 uridine specific endoribonuclease was shown to be inhibited by NSC95397 in vitro but it did not appear to exert any inhibitory effect in vivo [5]. Protease inhibitors are effective antivirals and have been licenced for the treatment of human immunodeficiency virus (HIV) and hepatitis C virus [10].…”

mentioning

confidence: 99%

An all-out assault on SARS-CoV-2 replication

Hay

2021

The coronavirus pandemic has had a huge impact on public health with over 165 million people infected, 3.4 million deaths and a hugely deleterious effect on most economies. While vaccination effectively protects against the disease it is likely that viruses will evolve that can replicate in hosts immunised with the present vaccines. Thus, there is a great unmet need for effective antivirals that can block the development of serious disease in infected patients. The seven papers published in this issue of the Biochemical Journal address this need by expressing and purifying components required for viral replication, developing biochemical assays for these components and using the assays to screen a library of pre-existing pharmaceuticals for drugs that inhibited the target in vitro and inhibited viral replication in cell culture. The candidate drugs obtained are potential antivirals that may protect against SARS-CoV-2 infection. While not all the antiviral candidates will make it through to the clinic, they will be useful tool compounds and can act as the starting point for further drug discovery programmes.