Hello and happy new year! I am back from my winter break and starting to get back to research. If you have followed my previous posts, you know that I have posted the druggability and genetic variability analysis of several protein targets of SARS-CoV-2. There are a few targets that I will be posting in the new year. For this post, I focus on nsp14.
Coronaviruses are RNA viruses. Viral RNA molecules are 5’capped to mimic host RNA and evade the immune system. Non-structural protein 14 (nsp14) is a bifunctional enzyme that comprises an RNA cap guanine N7-methyltransferase (N7-MTase) and a 3′-5′ exoribonuclease (ExoN) activity. Nsp14 binds to nsp10 to form the nsp14-nsp10 complex. The presence of nsp10 is important for nsp14 to have its ExoN activity. (Bouvet et al., 2012). The nsp14-nsp10 complex also catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to guanosine-P3-adenosine-5′,5′-triphosphate (GpppA or G3A). The replication fidelity of SARS-CoV-1 has been linked with nsp14 ExoN activity. The capping function of nsp14 is important for viral mRNA translation and evading host defense. Therefore, nsp14-nsp10 could be a potential target for anti-SARS-CoV-1, anti-SARS-Cov-2, and, more broadly, anti-coronavirus drugs.
In the absence of a SARS-CoV-2 nsp14 structure, we used the SARS-CoV-1 structure to map and analyze binding pockets. The crystal structure of the SARS-CoV-1 nsp14-nsp10 dimer was solved (Ma et al., 2015). It can be used as a model of the SARS-CoV-2 protein, as both proteins share 95% sequence identity and are 100% identical at the methyltransferase catalytic site.
We first used IcmPocketFinder (Molsoft, San Diego) on the crystal structure of the nsp14-nsp10 dimer bound to SAH (S-adenosyl-homocysteine, the de-methylated version of SAM) and GpppA (PDB: 5C8S). Two well-defined pockets are found at the surface of nsp14: the methyltransferase (MTase) active site (blue) and an allosteric site (red), as shown in figure 1 zenodo report. Using SiteMap (Halgren 2019), both pockets are predicted to be druggable, with Dscores of 0.99 and 1.05, respectively.
We then determined the surrounding residues for both pockets; the catalytic site of nsp14 is lined by 40 amino acid residues (Figure 2, zenodo report), and the allosteric site is surrounded by 28 residues (figure 3, zenodo report).
We conducted a broad survey of viral proteins from Alpha- and Betacoronaviruses to identify the most conserved druggable sites. By integrating variability and druggability data, we can infer the most promising strategies for developing broad-spectrum antiviral small-molecule inhibitors. This analysis can be valuable in the context of the emergence of future coronaviruses from circulating viral strains in bat reservoir species.
As part of our systematic survey, we also assessed the variability of amino acid lining the nsp14 catalytic MTase site and its allosteric site by performing multiple sequence alignment. 16 out of 40 residues lining the MTase active site are not conserved, and 24 residues were shown to be identical, which translates to 60% sequence identity at this site.
16 out of 28 (57%) residues lining the allosteric site of nap14 are not conserved, and 12 residues were shown to be identical, which translates to 43% sequence identity at this site.
Figures 6 and 7 of the zenodo report are the color-coded representations of nsp14 to highlight the identical and non-identical residues among the Alpha- and Betacoronavirus genera entries at both nsp14 sites.
In our next step, we wanted to look at the genetic diversity of NSP14 among SARS-CoV-2 samples from COVID-19 patients. Nicola De Maio, our collaborator from European Bioinformatics Institute (EBI), looked at more than 15000 SARS-CoV-2 samples to find the variants at its catalytic and allosteric sites.
The nine non-synonymous variants at the MTase site and eight at the allosteric site of nsp14 that Nicola observed among the SARS-CoV-2 samples are shown in table 1 of the zenodo report.
Based on our analysis, the MTase site of nsp14 is more druggable and more conserved across coronaviruses than its allosteric site. Hence our findings encourage the design of a broad-spectrum inhibitor against the catalytic site of nsp14.
Please contact me via the “Leave a comment” link at the top of this post. Stay Tuned for more updates on this project.