Targeting Enzymes of SARS-CoV-2 : RNA cap Methyltransferases.

Curing infections triggered by potentially lethal pathogens such as the novel SARS-CoV-2 necessitates a comprehensive understanding of the virus. Knowledge of the genome, the proteins it encodes, and the vital role of these proteins in the virus’s lifecycle are essential factors in developing successful therapeutic interventions.

One scientific approach involves inhibiting or modifying enzymes utilized by the virus for interaction and replication; for example, RNA cap MTases that defend virions from the host’s cellular innate immunity.

Eukaryotic mRNA has a distinct 5’ cap structure which typically consists of an N7-methylated GTP molecule connected via a 5′–5′ triphosphate bond to the first transcribed nucleotide methylated at the ribose-2’-O position.

To commandeer host translation machinery for propagation, several RNA viruses - including SARS-CoV-2 - have successfully evolved their own mRNA cap MTases to obtain similar cap structures. NSP14 and NSP16 (which are N7- and 2’-O- MTase respectively) cap viral RNAs so that they are able to elude host immune recognition.

Several recombinant versions of essential SARS-CoV-2 active protein targets exist for potential COVID-19 therapeutics.

  • NSP10/NSP16 Methyltransferase, Active (C19NS-E301H) - Recombinant 2019-nCoV NSP10/NSP16 methyltransferase includes full length NSP10 (A4254-Q4392) and NSP16 (S6799-N7096) expressed in E. coli using C-terminal His tag.
  • NSP14 Methyltransferase, Active (C19NS-E311H) - Recombinant SARS-CoV-2 (COVID-19) NSP14 Methyltransferase (5925-6452) was expressed in E. coli using a C-terminal His-tag.

NSP14 of SARS-CoV-2 includes two domains with distinct functions: The N-terminal domain (ExoN) is provided with exoribonuclease activity, which is able to synergize with the RNA proofreading activity of other enzymes. Similarly, ExoN knockout mutants of SARS-CoV and murine hepatitis virus were found to accumulate a considerable number of mutations.

The carboxy-terminal segment of NSP14 contains (guanine N7, or m7G)-MTase activity, and this participates in the viral mRNA cap synthesis. Curiously, the association of NSP10 substantially improves the ExoN activity of NSP14 while having no effect on m7G MTase activity.

It was also recently shown that NSP14 is among three other SARS-CoV-2 proteins with the potential to effectively suppress primary interferon production and interferon signaling during the course of viral infection.

The second viral RNA cap MTase NSP16 – can form an obligatory complex with NSP10, efficiently converting mRNA target from the Cap-0 (m7G0pppA1) to the Cap-1 form (m7G0pppA1m). It does this by methylating the ribose 2’-O of the first transcribed nucleotide (general adenosine in CoVs) of the nascent mRNA by utilizing S-adenosylmethionine as the methyl donor. This last Cap-1 structure is crucial for the virus to evade the host immune response.

Because of this, the ablation of NSP16 activity should prompt an immune response to CoV infection, limiting pathogenesis. Through vaccination with NSP16-defective SARS-CoV, or through the use of antibodies to disrupt the NSP16-NSP10 complex formation, mice were able to survive what would have usually been a lethal dose.

Recently, a high-resolution structure of NSP16/NSP10 co-crystallized with adenosine - the nucleoside drug - was found to display an allosteric ligand binding site that is unique to the NSP16/NSP10 complex of SARS-CoV-2. This provides a further potential target in the development of new antiviral therapies.

Activity of SARS-CoV-2 RNA cap methyltransferases NSP14 (SignalChem Catalog No. Nxxxxx, A), and NSP16 (SignalChem Catalog No. Nxxxxx, B), as detected in Methyltransferase-GloTM assays.Figure 5. Activity of SARS-CoV-2 RNA cap methyltransferases NSP14 (SignalChem Catalog No. Nxxxxx, A), and NSP16 (SignalChem Catalog No. Nxxxxx, B), as detected in Methyltransferase-GloTM assays. Image Credit: SignalChem Biotech Inc.

SignalChem is a world leader in the areas of cell signaling and protein engineering. Its scientists have been undertaking research around the new coronavirus ever since its initial outbreak, investing their resources and expertise in properly understanding the protein interaction map of the SARS-CoV-2. Their goal in this work is to successfully engineer active recombinant SARS-CoV-2 and human proteins.

SignalChem plans to provide other researchers across the globe with these elements of drug discovery research projects. With the assistance of advanced AI drug development software and engineering technology, SignalChem has already launched projects with a view to developing expression systems of key COVID-19 related proteins. For example, hot drug targets such as proteases and methyltransferases.

These catalytically active proteins are considered to be fully functional and ready for use in research projects aimed at the development of drugs directed against SARS-CoV-2, as well as other coronavirus infections.

About SignalChem Biotech Inc.

SignalChem is a biotech company focused on the research, development, and production of innovative and high-quality human recombinant cell signaling products.

Throughout the years, Signalchem has capitalized on its core expertise in cellular signaling, molecular biology, and protein biochemistry to generate more than 2,000 functional protein products and has established itself as the leader in the industry to produce highly active human recombinant signaling enzymes, especially protein kinases, disease-related mutant kinases, lipid kinases, epigenetic enzymes, ubiquitination-related enzymes, and neurodegenerative disease-related enzymes and proteins.

SignalChem strives to support scientists in academia, pharma and biotech companies around the world by creating effective research tools to advance the basic research in life sciences and to facilitate the efforts in drug discovery and development.


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Last updated: Oct 9, 2020 at 10:40 AM

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