May 14, 2020 -- Scientists tested a series of compounds for blocking the replication of SARS-CoV-2 using a novel proteomics approach. The method, which helped identify small-molecule drugs that may be effective at treating COVID-19 patients, was published in Nature on May 14.
Although there are many therapeutics under development, there are no established therapies for COVID-19, and treatment is currently based on supportive and symptomatic care. Therefore, the development of therapies specifically inhibiting SARS-CoV-2 infection or replication is urgently needed.
Molecular examination of infected cells by unbiased proteomics offers a strategy for revealing viral pathways and identifying potential drug targets. German researchers recently developed a method called multiplexed enhanced protein dynamics (mePROD) that allowed them to conduct this analysis of SARS-CoV-2 in cell culture.
First, the researchers established a permissive SARS-CoV-2 cell culture model in Caco-2 cells that would allow them to investigate different steps of the SARS-CoV-2 life cycle in cells. They subsequently quantified translatome (all mRNA fragments that are translated in a single cell) and proteome (all proteins expressed in a cell) changes by mePROD proteomics over time.
Overall, translation rates were not greatly impacted by SARS-CoV-2 infection. However, associating host and upregulated viral proteins with a role in viral amplification, the researchers found that there was an extensive increase in the host translation machinery. They tested two translation inhibitors -- cycloheximide (inhibitor of translation elongation) and emetine (inhibits 40S ribosomal protein S14) for their ability to inhibit SARS-CoV-2 replication. Both molecules were successful at blocking replication at nontoxic levels.
Alternatively, the proteome underwent extensive modulation 24 hours after infection. One group of proteins associated with cholesterol metabolism was reduced, while a second group of RNA modifiers (spliceosome; small nuclear RNAs) and proteins involved in carbon metabolism were increased during infection.
Pladienolide B, a spliceosome inhibitor targeting splicing factor SF3B1, effectively blocked SARS-CoV-2 replication. Therefore, the researchers suggested that splicing is an essential pathway for SARS-CoV-2 replication and a potential therapeutic target.
To test if blocking carbon metabolism (glycolysis) would inhibit SARS-CoV-2 replication, the researchers applied nontoxic levels of 2-deoxy-D-glucose (2-DG), an inhibitor of hexokinase, the rate-limiting enzyme in glycolysis. They found that 2-DG prevented SARS-CoV-2 replication in Caco-2 cells. A U.S. company, Moleculin Biotech, has a prodrug (WP1122) that is similar to 2-DG and it is planning a COVID-19-related clinical trial based on the results from this study.
Finally, the researchers detected a cluster of nucleic acid metabolism pathways that were also upregulated during infection. They showed that compounds that interfere with nucleic acid metabolism, such as ribavirin (produced by Bausch Health Americas), inhibited SARS-CoV-2 replication. Ribavirin is currently being tested in a COVID-19 clinical trial.
Collectively, the results suggest that the virus reshapes host cell translation, likely by increasing production of translation machinery components to compensate for host-cell translation inhibition. SARS-CoV-2 reshapes central cellular pathways and small molecule inhibitors, so targeting those pathways may prevent viral replication in cells.
"The successful use of substances that are components of already approved drugs to combat SARS-CoV-2 is a great opportunity in the fight against the virus," explained Jindrich Cinatl, PhD, from the Institute of Medical Virology at Goethe University, in a statement. "These substances are already well characterized, and we know how they are tolerated by patients. This is why there is currently a global search for these types of substances. In the race against time, our work can now make an important contribution as to which directions promise the fastest success."
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