Cell culture studies by researchers at the University of Liverpool and Institut Pasteur in Paris suggest COVID-19 can be targeted using existing drugs. The new findings, published in Nature Communications, examine proteolysis during viral infection, and may help pinpoint targeted treatments against the virus.
SARS-CoV-2 has been responsible for more than 227 million infections, and more than 4.6 million deaths worldwide during the pandemic. There is only one FDA approved treatment—the antiviral drug remdesivir (Veklury), which is used to treat COVID-19 in hospitalized adults and children who are age 12 or older. Currently more than 6,500 clinical trials of treatments or vaccines for COVID-19 are active, but the understanding of the basic biology of the disease is still ongoing.
Proteolytic cleavage plays a crucial role in the life cycle of SARS-CoV-2 and inhibitors targeting both viral and cellular proteases have been shown to inhibit SARS-CoV-2 replication in cell culture. In this study, the researchers write, they “present a first unbiased study of proteolysis during SARS-CoV-2 infection, and its implications for viral antigens, as well as cellular proteins that may represent options for antiviral intervention.”
Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication, and inhibitors targeting proteases have already shown success at inhibiting SARS-CoV-2 in cell culture models.
As the researchers write: “Proteolytic cleavage of the two coronavirus polyproteins generates the various viral proteins needed to form a replication complex, required for transcription and replication of the viral genome and subgenomic mRNAs.” The viral enzymes involved are papain-like (PLP, nsp3) and main proteases (Mpro, nsp5). Besides cleaving viral substrates, these enzymes also act on cellular proteins, modifying or neutralizing substrate activity.
These researchers used mass spectrometry to study proteolytic cleavage events during SARS-CoV-2 infection.
“Mass spectrometry-based approaches to identify protease substrates have existed for a number of years however, they have seen only limited application to the study of viral substrates, and had not been previously applied to the study of proteolysis during coronavirus infection,” explains lead author Emmott Edward, a Fellow at the University of Liverpool’s Institute of Systems, Molecular, and Integrative Biology.
The team found previously unknown cleavage sites in multiple viral proteins, including major antigenic proteins S and N, which are the main targets for vaccine and antibody testing efforts.
They discovered significant increases in cellular cleavage by SARS-CoV-2 main protease (Mpro) and identified 14 potential high-confidence substrates of the main and papain-like proteases, validating a subset with in vitro assays.
They went on to show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, showed a dose-dependent reduction in SARS CoV-2 titres.
Both Bafetinib (an experimental cancer drug) and Sorafenib (an approved drug used to treat kidney and liver cancer) showed SARS-CoV-2 inhibition at concentrations that did not result in cytotoxicity in a human cell line model of infection.
Emmott said: “An improved understanding of the exact ways in which proteolytic cleavage is regulated, modulates protein activity, and serves to benefit viral replication will be crucial for targeting cellular substrates of viral proteases as a therapeutic strategy.
“As further SARS-CoV-2 variants emerge, the incorporation of post translational modification data from studies such as this can also support efforts to predict phenotypes from genetic data on emerging variants.”
