A genetic trap in yeast for inhibitors of SARS-CoV-2 main protease

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the E. coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor down to 10 μM by a robust growth increase. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates, and is rapidly adaptable for new variants of the protease with deviant site specificities.