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NSP16 Shows Promise for Anti-Viral Therapeutics

by Bellbrook Labs / Tuesday, 20 September 2022 / Published in Emerging Targets, HTS Assays, Innate Immunity
NSP16 Target for COVID-19 Therapeutics

SARS-CoV-2 virus (Covid-19) possesses one of the largest genomes of any RNA virus. While it naturally encodes structural proteins among its 29 genes, it also produces non-structural proteins that are necessary to perpetuate infection. Non-structural protein 16 (NSP16) is one such protein.1

NSP16 Camouflages Viral mRNA

NSP16 is an m7GpppA-specific, S-adenosyl-L-methionine-dependent 2’-O-methyltransferase (2’-O-MTase) that “caps” virally transcribed mRNA to protect it from host innate immune defenses. This capping involves the transfer of a methyl group from NSP16’s S-adenosyl-L-methionine (SAM) cofactor to the 2’ hydroxyl ribose sugar at the 5’ end of the viral mRNA. This type 1 cap consists of a N-methylated guanosine triphosphate and C-2’-O-methyl-ribosyladenine and is fabricated in the cytoplasm. In human cells, this cap is installed in the nucleus by host proteins, such as CMTr1. For both host and pathogen, this cap serves to mark their mRNA as non-threatening, add stability, and increase translation efficiency. NSP16 induced mRNA capping effectively camouflages viral mRNA from host innate immune surveillance mechanisms, such as IFIT and RIG-I. As a result, the type 1 interferon defense remains effectively disengaged.2

However, unlike all other known 2’-O-MTases, NSP16 absolutely requires the assistance of another protein, SARS-CoV-2’s NSP10, to bind SAM or viral mRNA. In detail, NSP10 activates NSP16 by stabilizing its SAM and RNA binding pockets, permitting a stable catalytic complex to form. While it may initially seem like a good idea to interfere with SAM binding to impede the capping process, SAM is required by several host enzymes.3

Cryptic Pockets as Hopeful Ways to Inhibit NSP16 Activity

Recent work has focused upon what are known as “cryptic pockets” that occur as part of regular protein conformational shifts. These conformational shift sites, if stabilized or “jammed open” may offer new ways to inhibit enzymatic activity. NSP16 has just such a pocket in the form of the space between its Beta 3 and Beta 4 sheets. When Beta 4 moves away from Beta 3, it not only closes the SAM binding site, but also disrupts the NSP10 binding region. Any inhibitor that could occupy and stabilize this conformation, should effectively inactivate NSP16. To date, this pocket has been found to be highly conserved in the MERS and SARS virus families, but not at all among host 2’-O-MTases. This is a very promising avenue for future therapeutic endeavors.4

BellBrook’s own AptaFluor SAH Methyltransferase Assay may prove indispensable in finding selective inhibitors that act on select cryptic pockets. Essentially, when SAM is used in methylation by methyltransferases, S-adenosylhomocysteine (SAH) is produced as a by-product. This assay utilizes an aptamer to selectively bind to any generated SAH. Time-resolved fluorescence resonance energy transfer detection further enhances the exquisite sensitivity of this proprietary assay, detecting SAH at concentrations as low as 0.6 nM.

Check Out The Aptafluor SAH Assay

References

1. Chang, L-J and Chen, T-H. (2021) NSP16 2′-O-MTase in Coronavirus Pathogenesis: Possible Prevention and Treatments Strategies. Viruses, 13(4):538. Review. https://doi.org/10.3390/v13040538

2. Krafcikova, P. et al. (2020) Structural analysis of the SARS-CoV-2 methyltransferase complex involved in RNA cap creation bound to sinefungin. Nature Communications, 11:3717. https://doi.org/10.1038/s41467-020-17495-9

3. Sk, M.F. et al. (2020) Computational Investigation of Structural Dynamics of SARS-CoV-2 Methyltransferase-Stimulatory Factor Heterodimer nsp16/nsp10 Bound to the Cofactor SAM. Frontiers in Molecular Biosciences, 7:590165. https://doi.org/10.3389/fmolb.2020.590165

4.  Vithani, N. et al. (2021) SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential. Biophysical Journal, 120(14), 2880-2889. https://doi.org/10.1016/j.bpj.2021.03.024

Tagged under: AptaFluor SAH Methyltransferase Assay, COVID-19, Innate Immunity, NSP16

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