Development of a fluorescence based, high-throughput SARS-CoV-2 3CL^proreporter assay

In late 2019 a human coronavirus, now known as SARS-CoV-2, emerged, likely from a zoonotic reservoir. This virus causes COVID-19 disease, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CL^pro. 3CL^prois an attractive target for antiviral therapeutics as it is essential for processing newly translated viral proteins, and the viral lifecycle cannot be completed without protease activity. In this work, we present a new assay to identify inhibitors of the SARS-CoV-2 main protease, 3CL^pro. Our reporter is based on a GFP-derived protein that only fluoresces after cleavage by 3CL^pro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level-2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread.