On March 11, 2020, the World Health Organization (WHO) announced the coronavirus disease 2019 (COVID-19) to be a pandemic. COVID-19 is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen, which is deemed to be highly contagious and is transmitted via human contact. To date, it has claimed over 2.9 million lives worldwide. Although the process of vaccination has commenced in many countries, the emergence of novel SARS-CoV-2 variants that have the potential to evade the vaccine-induced immune response has created the need for further research in developing more drugs and designing vaccines that could remain effective against such variants.

Study: Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease. Image Credit: Design_Cells / Shutterstock

SARS-CoV-2 belongs to the coronavirus family, subgenus Sarvecovirus, and genus Betacoronavirus. A phylogenetic study has revealed that this virus is closely related to SARS and more distantly related to Middle East respiratory syndrome coronavirus (MERS-CoV). In addition to vaccines, only a few antiviral drugs, such as dexamethasone and remdesivir, have been approved by the Food and Drug Administration (FDA) to be used to treat COVID-19. Further, Casirivimab and Imdevimab are also permitted under Emergency Use Authorization (EUA) by the FDA to treat COVID-19 patients. However, with the emergence of new variants and the uncertainty over the effectiveness of the approved vaccines against these variants, there is a need for more drugs to contain the pandemic.

During the process of infection, once the virus penetrates the host cell, two open reading frames, ORF1a and ORF1ab, are translated. ORF1ab is responsible for the production of 16 nonstructural proteins (nsp). Several viral proteases such as 3CLpro and PLpro (papain-like protease) produce 1-16 nsps. A new study has been published in bioRxiv* preprint server, which deals with identifying drugs that are effective as PLpro inhibitors.

PLpro is associated with the nsp3 protein, which produces viral non-structural proteins from a polyprotein precursor. Its main role is to cleave ubiquitin and ISG protein conjugates. Nsp3 is the largest non-structural protein that contains multiple domains. These are ubiquitin-like (Ubl-1), acidic-domain (AC domain), ADP-ribose-1”-phosphatase (ADRP) /macro/x-domain, SARS unique Domain (SUD), Ubl-2, PLpro domain, nucleic acid-binding domain (NAB), marker domain (G2M), double-pass transmembrane domains (TM 1-2 and TM 3-4), and the Y domain (subdomains Y1-3). Primarily, the current research deals with the expression and purification of PLpro. Researchers used both bacteria and insect cell systems to obtain purified protein that could preserve maximum enzymatic activity. In bacterial expression, PLpro was tagged with either His-Sumo or His-TEV at its N-terminus, whereas, for expression in insect cells, the protein was tagged with Flag-His at the N-terminus. For bacterial expression, the tags were removed by Ulp1 or TEV proteases, but for the insect cells system, the tags remained on the final protein.

In the present research, scientists used the quenched Förster (fluorescence) resonance energy transfer (FRET) technique to examine the protease activity. They found that the bacterial His-TEV-PLpro, after the removal of tags, was more active when compared to the insect cell’s Flag-His-PLpro, at the same enzyme concentration. However, similar activity was spotted at two bacterial expression proteins, i.e., His-TEV and His-Sumo-PLpro. As His-Sumo PLpro provided a higher yield after purification, it was selected for other experiments involved in this study.

Researchers performed high-throughput screening for over 5000 chemicals, which were obtained from the High-Throughput Screening (HTS) facility in Francis Crick Institute. HTS helped to identify seven drugs having inhibitory effects against PLpro.

Previous studies had shown compounds such as tanshinone derivatives (Tanshinone IIA and Cryptotanshinone) and dihydrotanshinone I can inhibit the PLpro from SARS-CoV-1. Another study had reported that GRL-0617 was effective against SARS-CoV-1/ -2 PLpro. To further examine these compounds, a gel-based PLpro protease assay was performed, where two of the seven drugs, Ursodiol and Pyrocatechuic acid, did not show the inhibitory effect on PLpro cleavage and was regarded as false positives. Further, even though GRL-0617, Tanshinone IIA and Cryptotanshinone could inhibit PLpro cleavage, they could only do so at a higher concentration. Dihydrotanshinone I was found to be the most effective inhibitor.

Researchers have also conducted orthogonal assays to study the effect of the drugs obtained in the HTS study to inhibit isopeptidase activity of PLpro. Dihydrotanshinone I was found to be the most potent for the inhibition of both K48-linked Ub3 and pro-ISG15 cleavage. Further, the ability of these compounds to inhibit viral growth in a cell culture-based assay was carried out. Researchers have used VERO E6 cells, which were infected with SARS-CoV-2, in this experiment. Several compounds exhibited various effects on the cells, such as cytotoxicity. However, dihydrotanshinone I was found to be the most potent compound that could efficiently inhibit the SARS-CoV-2 proliferation at an EC50 of 8 µM. Additionally, even at an increased concentration, this compound did not exhibit any cytotoxicity.  

The present research has revealed that dihydrotanshinone I can be used as an effective compound for the treatment of COVID-19 disease as it acts as an nsp3 PLpro inhibitor. This is a natural compound that is extracted from a lipophilic fraction of Salvia miltiorrhiza.

*Important Notice

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.

Written by

Dr. Priyom Bose

Priyom holds a Ph.D. in Plant Biology and Biotechnology from the University of Madras, India. She is an active researcher and an experienced science writer. Priyom has also co-authored several original research articles that have been published in reputed peer-reviewed journals. She is also an avid reader and an amateur photographer.