2021
DOI: 10.1128/msystems.01087-21
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A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease

Abstract: 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.

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Cited by 17 publications
(16 citation statements)
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“…In yeast and other eukaryotes, Ub fusion proteins are cleaved by Ub-specific proteases directly C-terminal to the Ub, revealing the N-terminal residue of the fused protein, regardless of sequence ( Bachmair et al, 1986 ). Expression of functionally active M pro is toxic to yeast cells ( Alalam et al, 2021 ). To control the expression level of M pro while limiting its toxic side effects, we placed Ub-M pro under control of the inducible and engineered LexA-ER-AD TF ( Ottoz et al, 2014 ).…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…In yeast and other eukaryotes, Ub fusion proteins are cleaved by Ub-specific proteases directly C-terminal to the Ub, revealing the N-terminal residue of the fused protein, regardless of sequence ( Bachmair et al, 1986 ). Expression of functionally active M pro is toxic to yeast cells ( Alalam et al, 2021 ). To control the expression level of M pro while limiting its toxic side effects, we placed Ub-M pro under control of the inducible and engineered LexA-ER-AD TF ( Ottoz et al, 2014 ).…”
Section: Resultsmentioning
confidence: 99%
“…Expression of Ub-M pro in cells engineered with the split TF exhibited a β-estradiol-dependent decrease in GFP reporter activity that required the presence of catalytically functional M pro protein ( Figure 1—figure supplement 1d ). The final screen leverages the toxicity of M pro expression in yeast, which likely results from cleavage of essential yeast proteins by the protease ( Alalam et al, 2021 ; Figure 1c ). Increasing concentrations of β-estradiol correlates with a decrease in yeast growth rate that is dependent on the presence of catalytically functional M pro ( Figure 1—figure supplement 1b ).…”
Section: Resultsmentioning
confidence: 99%
“…Taken together, these results demonstrate that a non-pathogenic, rapid, inexpensive and highly accessible yeast-based method can be used to characterize mutants for both their effects on M pro activity and their responses to inhibitor compounds. There are reports using yeast as a tool to screen for M pro inhibitors or perform mutational analysis 33,34 . These systems incorporate M pro reporters and modification of M pro to carry a N-terminal serine.…”
Section: Discussionmentioning
confidence: 99%
“…Several methods have been developed or adapted for quantifying the potency of the SARS-CoV-2 M-pro inhibitors [ 73 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 ]. These methods demonstrate the mechanism of action of antiviral drugs and do not require cells infected with SARS-CoV-2 or a laboratory with biosafety level 3 containment facilities.…”
Section: Sars-cov-2 M-pro Inhibitorsmentioning
confidence: 99%