Two notorious SARS-CoV-2 proteases—Mpro and PLpro—were inhibited by drugs that have already been approved for indications other than COVID-19. The identification of potentially useful SARS-CoV-2 antiviral drugs is obviously welcome, but in this case, the findings are especially encouraging. Why? Because the discovery of the inhibitors was accomplished with a novel screening strategy, one that could be used in additional screening studies.

Details about the protease inhibitors—and the screening strategy—appeared in Communications Biology, in an article titled, “Identification of SARS-CoV-2 inhibitors targeting Mpro and PLpro using in-cell protease assay.” The in-cell protease assay (ICP) indicated in the article’s title is just one part of the screening strategy. Other parts include antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity.

The assay strategy was developed by scientists at Pennsylvania State University and their colleagues at Cardiff University and the University of California, San Diego. These scientists noted that proteases are attractive targets because they are well conserved and crucial for virus replication.

“The SARS-CoV-2 vaccines target the spike protein, but this protein is under strong selection pressure and, as we have seen with Omicron, can undergo significant mutations,” said Penn State’s Joyce Jose, PhD, the senior author of the current study. “There remains an urgent need for SARS-CoV-2 therapeutic agents that target parts of the virus other than the spike protein that are not as likely to evolve.”

One of the study’s co-authors, Penn State’s Katsuhiko Murakami, PhD, noted that Mpro and PLpro are essential for SARS-CoV-2 replication in infected cells because of their protein-cleaving capabilities.

“SARS-CoV-2 produces long proteins, called polyproteins, from its RNA genome that must be cleaved into individual proteins by these proteases in an ordered fashion leading to the formation of functional virus enzymes and proteins to start virus replication once it enters a cell,” he explained. “If you inhibit one of these proteases, further spread of SARS-CoV-2 in the infected person could be stopped.”

To rapidly identify inhibitors of the Mpro and PLpro proteases, the researchers led by Jose designed a novel assay. “Other assays are available,” Jose said, “[but] we designed our novel assay so it could be conducted in live cells, which enabled us to simultaneously measure the toxicity of the inhibitors to human cells.”

The researchers used their assay to test a library of 64 compounds—including inhibitors of HIV and hepatitis C proteases; cysteine proteases, which occur in certain protozoan parasites; and dipeptidyl peptidase, a human enzyme involved in type 2 diabetes—for their ability to inhibit Mpro or PLpro.

“After prioritizing compounds based on inhibitory activity against the target protease combined with low cellular toxicity, six inhibitors for Mpro (MG-101, Lycorine HCl, BMS-707035, Atazanavir, Lomibuvir, and Nelfinavir mesylate) and two inhibitors for PLpro (Sitagliptin and Daclastavir) were selected,” the article’s authors wrote. “Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the Delta variant.”

The researchers also reported that MG-101 hindered the virus’s ability to infect cells by inhibiting protease processing of the spike protein: “The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site.”

“By understanding how the MG-101 compound binds to the active site, we can design new compounds that may be even more effective,” Murakami asserted. Indeed, the team is in the process of designing new compounds based on the structures they determined by X-ray crystallography. They also plan to test the combination drugs that they already demonstrated to be effective in vitro in mice.

Although the scientists studied the Delta variant of SARS-CoV-2, they said the drugs will likely be effective against Omicron and future variants because they target parts of the virus that are unlikely to mutate significantly.

“The development of broad-spectrum antiviral drugs against a wide range of coronaviruses is the ultimate treatment strategy for circulating and emerging coronavirus infections,” Jose declared. “Our research shows that repurposing certain FDA-approved drugs that demonstrate effectiveness at inhibiting the activities of Mpro and PLpro may be a useful strategy in the fight against SARS-CoV-2.”

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