According to Joyce Jose, assistant professor of biochemistry and molecular biology at Penn State, “The vaccines for SARS-CoV-2 are targeting the spike protein, but this protein is under strong selection pressure and, as we have seen with Omicron, can undergo significant mutations. Therapeutics that target parts of the virus other than the spike protein that are less likely to evolve are still urgently needed for SARS-CoV-2, says the study author.
The proteases Mpro and PLpro of SARS-CoV-2 are promising targets for antiviral drug development, according to previous research. For example, Paxlovid, a COVID-19 therapy from Pfizer, targets Mpro. Jose says that because these enzymes are relatively stable, drug-resistant mutations are unlikely to arise quickly.
Professor of biochemistry and molecular biology Katsuhiko Murakami noted that these virus proteases are necessary for SARS-CoV-2 replication in infected cells because of their ability to cleave or cut proteins.
When the SARS-CoV-2 virus enters a cell, it produces long proteins called polyproteins from its RNA genome, which must be cleaved into individual proteins in an ordered fashion by these proteases in order to start virus replication. To stop further spread of SARS-CoV-2, you could inhibit one of these proteases.
The findings were published in the journal Communications Biology on Tuesday, February 25th.
The researchers devised an assay for detecting inhibitors of the Mpro and PLpro proteases in human cells that was performed in vivo.
Although other assays exist, we designed our novel assay so that it could be conducted in live cells, allowing us to simultaneously measure the toxicity of inhibitors to human cells.”
An assay developed by the researchers tested the 64 compounds in a library for their ability to inhibit Mpro or PLpro by screening them for their ability to inhibit HIV and hepatitis C proteases as well as the cysteine-based proteases found in some protozoan parasites. Based on a 50% reduction in protease activity with 90% cell viability, the team identified eleven compounds that affected Mpro activity and five compounds that affected PLpro activity from the 64 compounds tested.
Compounds were monitored using confocal microscopy by an associate professor of biochemistry and molecular biology, Anoop Narayanan.
Our experiment was designed to show fluorescence in specific areas of the cell if we were able to inhibit proteases,” said Narayanan.
It was found that eight of 16 PLpro and Mpro inhibitors had dose-dependent antiviral activities against SARS-CoV-2 when tested on live human cells in a BSL-3 facility, the Eva J. Pell ABS-3 Laboratory for Advanced Biological Research at Penn State. Sitagliptin and Daclatasvir were found to inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate were found to inhibit Mpro, respectively. Additionally, MG-101 inhibited the spike protein’s protease processing, reducing the virus’s ability to infect cells.
Pretreatment with the selected inhibitors only MG-101 prevented the virus from entering cells, according to Narayanan.
A greater reduction in SARS-CoV-2 replication was achieved by using a combination of Mpro and PLpro inhibitors, as discovered by the researchers.
While PLpro and Mpro inhibitors are effective on their own, when combined, they have an even greater effect on the virus without increasing toxicity, Jose explained. A lot of power comes from the combination of these two inhibitors.”
An X-ray crystallographic structure of MG-101 in complex with the Mpro protease was obtained by the scientists, including postdoctoral scholar Manju Narwal, to investigate the mechanism by which the enzyme MG-101 inhibits.
Mpro’s active site was clearly visible to Narwal’s team, who were able to observe how MG-101 interacted with it. Polyprotein-binding proteases are essential for virus replication, and this inhibitor mimicks them and blocks the protease from binding to and cutting the polyprotein, which is an essential step in the virus’s replication. “
It’s possible to design new compounds that may be even more effective by learning how MG-101 binds to the active site,” Murakami said.
It is true that the team is currently designing new compounds based on the X-ray crystallographic structures that they have discovered. These new drugs will be put to the test in combination with those that have already shown promise in preclinical studies in mice.
However, even though they focused on the Delta variant of SARS-CoV-2, the scientists predicted that their drugs would be effective against Omicron and future SARS-CoV-2 variants.
“The development of broad-spectrum antiviral drugs against a wide range of coronaviruses is the ultimate treatment strategy for circulating and emerging coronavirus infections,” said Jose. To combat SARS-CoV-2, repurposing FDA-approved drugs that have proven effective in blocking the activities of SARS-CoV-2 Mpro and PLpro may be an effective strategy.”
