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A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease
10.1128/mSystems.01087-21
Hanna Alalam
Sunniva Sigurdardóttir
Catarina Bourgard
Ievgeniia Tiukova
Ross D. King
Morten Grøtli
Per Sunnerhagen
Methods and Protocols
Methods and Protocols
COVID-19
small molecules
Saccharomyces cerevisiae
genetic engineering
MazF toxin
SARS-CoV-2
antiviral agents
genetic selection system
American Society for Microbiology
Alalam et al.
Copyright © 2021 Alalam et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
20211221
2021
ABSTRACT
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 the 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 Escherichia 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 by a robust growth increase, discernible down to 1 μM. 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.
IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARS-CoV-2 main protease or even for other proteases.
20210912
20211103
2379-5077
Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version)2022-01-11T04:04:50-08:00
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