Researchers unveil new SARS-CoV-2 inhibitor that targets viral immune evasion

In a recent quest for an effective antiviral compound, UK researchers have purified a specific enzyme of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) known as nsp15 and optimized fluorescent biochemical endoribonuclease assays to screen a custom chemical library with over 5,000 commercial compounds. The study is currently available on the bioRxiv* preprint server.

SARS-CoV-2 is a causative agent of coronavirus disease 2019 (COVID-19), a human disease that resulted in millions of deaths, burdened global health systems to near-breaking point, and imperiled economies of countries and families in an unprecedented fashion.

Although vaccination endeavors are well underway, not all countries can access them, and specific antiviral treatments to combat this disease are currently lacking. Remdesivir is the only antiviral drug approved for the treatment of COVID-19; however, its effectiveness remains questionable.

But where to look for effective new drugs? The answer may lie in the structure of the virus. It is known that SARS-CoV-2 encodes sixteen non-structural proteins (nsp) that have various enzymatic activities with pivotal roles in viral genome replication, transcription and host immune evasion.

Considering the latter, one crucial aspect of host immune evasion is exerted by the uridine-directed endoribonuclease activity of nsp15. Hence, nsp15 inhibitors might be a useful way to potentiate the host immune response directed against SARS-CoV-2.

In March 2020, a research group led by Dr. Berta Canal from the Francis Crick Institute in London initiated a sizeable project to pinpoint inhibitors of various SARS-CoV-2 enzymes from a custom chemical library containing over 5000 compounds.

Screening a vast library of compounds

In this study, the researchers have expressed and purified a series of N-terminally tagged versions of nsp15 from bacteria and insect cells. More specifically, nsp15 was eluted as a high molecular weight complex during size exclusion chromatography.

Then, they have performed the screen with nsp15 enzyme and the adequate amount of substrate, which produced reactions that were linear over the period of reaction monitoring that lasted 15 minutes – against a library of over five thousand commercial chemical compounds.

The quest then narrowed down to the NCS95397 compound, which demonstrated steadfast inhibition activity in different experiments. In order to study its specificity towards nsp15, the scientists tested whether RNase A and benzonase activities are also inhibited by the NSC95397.

Finally, this group wanted to explore whether the inhibition of the nsp15 endoribonuclease activity can also be synergized with remdesivir antiviral activity, thus they have performed specific infectivity assays in the presence of both NSC95397 and remdesivir.

A viable inhibitor of nsp15

In short, this study has found that NSC95397 is a viable inhibitor of nsp15, but not a general nuclease inhibitor in vitro. Nevertheless, albeit NSC95397 inhibited nsp15 activity in vitro, it did not impede the proliferation of SARS-CoV-2 in Vero E6 cells, which is a frequently used cell model for viral growth under laboratory conditions.

Furthermore, the results have shown that nsp15 activity is additionally stimulated at high substrate concentrations, implying that nsp15 can be observed as an allosteric enzyme, which confirms predictions based on structural cryogenic electron microscopy studies of the SARS-CoV-2 nsp15 hexamer.

Finally, no additive effect of NSC95397 and remdesivir has been observed on SARS-CoV-2 infectivity in Vero E6 cells. Still, the assays showed a rather high host cell toxicity in response to NSC95397, which will have to be addressed in further research endeavors.

Eliciting a robust immune response

“Because of its role in immune evasion, treatment with nsp15 inhibitors could be useful early in SARS-CoV-2 infection to help elicit a robust immune response to clear the virus”, say study authors in this bioRxiv paper. “As an alternative strategy, nsp15 inhibitors could be tested in combination with other treatments to screen for synthetic antiviral effects”, they add.

Nonetheless, since we deal with an endoribonuclease, unrestricted nsp15 activity might result in unwanted cleavage of the viral genome; hence, the regulation of its activity is likely necessary for balancing its immune evasion function with viral genome integrity.

In any case, further studies are necessary in order to appraise the exact effect of nsp15 inhibition on host immune evasion, and those studies should use a higher compound to enzyme or substrate ratios for much better detection of nsp15 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. Tomislav Meštrović

Written by

Dr. Tomislav Meštrović

Dr. Tomislav Meštrović is a medical doctor (MD) with a Ph.D. in biomedical and health sciences, specialist in the field of clinical microbiology, and an Assistant Professor at Croatia's youngest university - University North. In addition to his interest in clinical, research and lecturing activities, his immense passion for medical writing and scientific communication goes back to his student days. He enjoys contributing back to the community. In his spare time, Tomislav is a movie buff and an avid traveler.

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