A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease

Alalam, Hanna; Sigurdardóttir, Sunniva; Bourgard, Catarina; Tiukova, Ievgeniia A.; King, Ross D.; Grötli, Morten; Sunnerhagen, Per

doi:10.1128/msystems.01087-21

Cited by 15 publications

(16 citation statements)

References 31 publications

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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%

“…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%

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Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

Flynn

Samant

Schneider-Nachum

et al. 2022

eLife

120

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With the continual evolution of new strains of SARS-CoV-2 that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high-throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

Flynn

Samant

Schneider-Nachum

et al. 2022

eLife

120

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“…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%

A yeast-based system to study SARS-CoV-2 M^pro structure and to identify nirmatrelvir resistant mutations

Lewandowski

et al. 2022

Preprint

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The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

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“…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%

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

Macip

Garcia-Segura

Mestres-Truyol

et al. 2021

IJMS

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In this review, we collected 1765 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M-pro inhibitors from the bibliography and other sources, such as the COVID Moonshot project and the ChEMBL database. This set of inhibitors includes only those compounds whose inhibitory capacity, mainly expressed as the half-maximal inhibitory concentration (IC50) value, against M-pro from SARS-CoV-2 has been determined. Several covalent warheads are used to treat covalent and non-covalent inhibitors separately. Chemical space, the variation of the IC50 inhibitory activity when measured by different methods or laboratories, and the influence of 1,4-dithiothreitol (DTT) are discussed. When available, we have collected the values of inhibition of viral replication measured with a cellular antiviral assay and expressed as half maximal effective concentration (EC50) values, and their possible relationship to inhibitory potency against M-pro is analyzed. Finally, the most potent covalent and non-covalent inhibitors that simultaneously inhibit the SARS-CoV-2 M-pro and the virus replication in vitro are discussed.

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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.

Cited by 15 publications

References 31 publications

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

A yeast-based system to study SARS-CoV-2 M^pro structure and to identify nirmatrelvir resistant mutations

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

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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.

Cited by 15 publications

References 31 publications

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms

A yeast-based system to study SARS-CoV-2 Mpro structure and to identify nirmatrelvir resistant mutations

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

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A yeast-based system to study SARS-CoV-2 M^pro structure and to identify nirmatrelvir resistant mutations