As efforts to find effective antivirals to combat the SARS-CoV-2-caused COVID-19 pandemic continue, a new study explores the viral enzyme PLpro, encoded in the nonstructural protein nsp3, and its inhibitors. The study appeared as a preprint on the bioRxiv* server in December 2020.
Development of nanobodies that inhibit Nsp3. Image Credit: https://www.biorxiv.org/content/10.1101/2020.12.09.417741v1.full.pdf
The Nsp3 Protein
The viral genome encodes 16 non-structural proteins, of which nsp3 is the largest. This is a membrane protein with multiple domains and is essential within the replication-transcription complex formed on the host membranes. It is also a protease, encoding the papain-like protease PLpro that is required for the release of several other non-structural proteins from the polypeptide.
The proteolytic activity of PLpro is not confined to viral polypeptides. It also removes ubiquitin and ISG15 (a ubiquitin-like modifier, interferon-stimulated gene 15) from proteins, undoing important post-translational changes within innate immune pathways targeting viruses, and inflammatory signaling pathways. In effect, PLpro disrupts these pathways. These effects increase its value for viral replication, making it an excellent target for antiviral development.
While similar in many respects to the PLpro encoded by SARS-CoV, it has a structure that could be compared to that of an open hand, with subdomains corresponding to the thumb, palm, and fingers to engage its substrate, forming the S1 site. The current study explores the enzyme site of nsp3 as well as the protease activity of the full-length protein.
PLpro Less Active than Nsp3
The researchers found that nsp3 is a more active protease than PLpro, which was unable to cleave nsp1-2 to release nsp1, even at increasing PLpro concentrations.
However, an extended version of nsp3 was able to cleave all three substrates actively, while maintaining enzyme specificity and despite its not possessing any additional catalytic sites beyond PLpro. Moreover, nsp3 shows faster and more complete cleavage of the other two substrates compared to PLpro.
Further research is required to understand the structure of longer nsp3 variants, either by themselves or in complex with any of its substrates. The interactome of this protein is also poorly understood.
The scientists, therefore, used a truncated model to obtain a high enough level of expression within cells, since the full-length molecule was expressed at very low concentrations.
Nsp3 Activity Related to Innate Immune Function
Examining the interactions of both PLpro and nsp3 – the truncated version – they found that many of the interacting proteins showed a common tendency to be stimulated by interferons and by antiviral signaling proteins. ISG15 was also a significant interacting protein, along with IFIT1 and IFIT2, both interferon-induced proteins that sense single-stranded viral RNAs to prevent the expression of viral mRNAs. Several other antiviral and interferon-related proteins were also found to interact with both proteases.
Other interactors were associated with translation, mostly for both proteins but in one or two cases, with only one, like the RNA binding protein Fragile X mental retardation syndrome-related protein 1 (FXR1). Unique nsp3 interactors may have been missed because of the lack of part of the nsp3 in this truncated version, hindering its trafficking to the right subcellular compartments, or because it is a middleman, mediating the binding of other nsps to create replication complexes.
Poor Correlation Between In Vitro and Cell-Based Inhibitor Studies
The scientists used a high-throughput assay to screen ~2,000 FDA-approved compounds to find potential drugs that may inhibit this enzyme at low concentrations. They identified five compounds that could inhibit both PLpro and nsp3. This comprised thioguanine, nordihydroguaiaretic acid, disulfiram, auranofin, and tideglusib.
Despite their good in vitro performance, none of the screened molecules fulfilled their promise, failing to inhibit viral replication in a cell model. This not only highlights the limitations of repurposing drugs to find lead compounds, but also the need to use nsp3 for screening. This is because PLpro is not capable of efficient cleavage of viral polypeptides, while nsp3 can.
The use of Nsp3 will help to identify allosteric inhibitors that can prevent proteolysis of all three substrates by nsp3. Again, combining inhibitors of PLpro in combination with Mpro and other essential RNA-dependent RNA polymerases may help arrive at a more effective antiviral therapeutic strategy. Not only were the identified inhibitors inefficient in cell-based assays, but they showed off-target effects, on other ubiquitin-like molecules.
The investigators, therefore, built PLpro-binding nanobodies that bind to the S1 site with high affinity, inhibiting PLpro. These were found to be probably specific in their binding to this site. Further work is necessary to demonstrate their structure.
What are the Implications?
The researchers were able to uncover relevant differences in the protease activity of nsp3 and the catalytic PLpro region alone. Their work suggests the importance of measuring the inhibitory capacity of the potential drug lead compounds on full-length nsp3 as well as the ability to prevent the cleavage of viral polypeptides.
Moreover, the nanobodies developed here could help greatly to examine the cellular substrates of nsp3 and its mechanism of working at the three substrates.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.