SARS-CoV-2 enzyme inhibitors among drugs screened for repurposing

In the context of the coronavirus disease 2019 (COVID-19) pandemic, with over 3.5 million deaths and counting as of June 9, 2021, the development of effective antivirals remains essential. This persistent need is largely due to the fact that most of the currently available vaccines target the spike protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which appears to constantly undergo rapid and critical mutations.

A new bioRxiv* preprint describes the identification of some promising pharmaceutical compounds that could be developed as antiviral drugs against COVID-19. These drugs have been obtained by screening already approved or late-stage experimental drugs.

Study: Drug Repurposing for the SARS-CoV-2 Papain-Like Protease. Image Credit: Blue Planet Studio /


The SARS-CoV-2 genome encodes ten open reading frames (ORFs), the largest of which is ORF1ab. ORF1ab comprises two different ORFs, both of which are translated as a single large polypeptide. This large polypeptide is then cleaved by two cysteine proteases that are encoded within the ORFs themselves which are known as non-structural protein 3 (NSP3) and NSP5.

These NSPs, which are also referred to as papain-like proteases (PLpro) and main proteases (Mpro), respectively, cleave all ORF polypeptides. This cleavage produces 15 NSPs that are essential for viral replication and infection.

PLpro acts to cleave peptide bonds when it recognizes the four-residue peptide LXGG. This protease also has deubiquitination activity, as well as the ability to proteolyze ubiquitin linkages and remove the interferon-stimulated gene 15 product (ISG15). These actions are important in regulating antiviral host innate immune responses and promoting successful infection by SARS-CoV-2.

PLpro is 83% conserved between SARS-CoV-2 and SARS-CoV, as well as across other coronaviruses. The conservation of this enzyme, therefore, offers the inhibition of PLpro as a possible broad-spectrum treatment and/or prevention strategy.

Drug repurposing has many advantages in the current pandemic as it bypasses most of the drug development process, thus saving time and money. The repurposing of already approved drugs also eliminates the need for toxicity studies, as these drugs have already been shown to be safe in humans.

Molecules with promise

The researchers looked among 37 cysteine protease inhibitors and 33 deubiquitinase inhibitors to find potential PLpro inhibitors. This screen yielded seven candidate drugs capable of inhibiting the enzyme at low concentrations, with 50% inhibition occurring at less than 60 μM (50% inhibitory concentration, IC50 of <60 μM).

The range of IC50 values extended from 0.56 μM for SJB2-043 to 35.0 μM for S130. Five of these drug candidates had IC50 values below 10 μM.

Among the four compounds that completely inhibited the enzymatic activity of PLpro at the highest concentration of 200 μM, one is known to inhibit SARS-CoV PLpro non-covalently, but with no effect on the activity of host proteases. Despite the fact that this compound, which was denoted to as GRL0617, is small, it retains high potency, which was demonstrated by its IC50 of 1.4 μM.

Allosteric inhibition proves important

Among these compounds, SBJ2-043, though one of three partial inhibitors of PLpro enzymatic activity, had an IC50 value of only 0.56 μM. This indicates a very high binding affinity, which may point to its potential use in building a chimeric proteasome targeting molecule.

It is also probable that SBJ2-043, like its structural analog SJB3-019a, produces allosteric inhibition of the enzyme, rather than directly competing with the substrate for the catalytic site. It appears that this binding affects the structure of the binding site by allosteric hindrance.

Further study of these molecules as they interact with PLpro may reveal novel targeting sites for PLpro inhibitors.

Molecular docking studies on the top hits helped clarify how these compounds bound to the PLpro active site by different modes.

What are the implications?

These findings indicate potential opportunities for repurposing these drugs to help in the ongoing fight to treat patients with severe COVID-19. Direct competitive inhibition is not the only criterion to identify drugs of value that can be adapted for this purpose, as allosteric inhibitors also exhibited impressively low IC50 values against PLpro activity.

Understanding how the structure of each of these molecules underlies its efficacy in SARS-CoV-2 inhibition may help develop even more potent PLpro inhibitors.

The next step will be to investigate the mechanism of action of these compounds, as this will help to increase their efficacy in inhibiting PLpro for better antiviral activity.

As a pilot study, the current work indicates that COVID-19 drug repurposing by targeting PLpro holds promises but in-depth analysis of repurposed drugs is necessary to avoid omitting allosteric inhibitors.”

*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.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.


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