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High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses

Bara Cihlova, Andrea Huskova, Jiri Böserle, Radim Nencka, View ORCID ProfileEvzen Boura, View ORCID ProfileJan Silhan
doi: https://doi.org/10.1101/2020.10.27.357418
Bara Cihlova
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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Andrea Huskova
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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Jiri Böserle
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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Radim Nencka
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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Evzen Boura
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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  • ORCID record for Evzen Boura
  • For correspondence: jan.silhan@uochb.cas.cz evzen.boura@uochb.cas.cz
Jan Silhan
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
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  • ORCID record for Jan Silhan
  • For correspondence: jan.silhan@uochb.cas.cz evzen.boura@uochb.cas.cz
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Abstract

Spanish flu and other influenza outbreaks, the recent Zika epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases that are caused by RNA viruses. Perhaps less well known yet dangerous RNA viruses cause deadly diseases such as polio, Ebola, measles, rubella, yellow fever, dengue fever and many others. To combat a particular viral disease by diminishing its spread and number of fatal cases, effective vaccines and antivirals are indispensable. Therefore, quick access to the means of discovery of new treatments for any epidemic outbreak is of great interest and in vitro biochemical assays are the basis of drug discovery. The recent outbreak of the coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands an affordable and reliable assay for testing antivirals. Here, we developed a quick and inexpensive high-throughput fluorescent assay to test inhibitors of viral proteases. Accordingly, we employed this assay to sample inhibitors for papain-like protease from SARS-CoV-2. In addition, we validated this assay for screening inhibitors of flaviviral protease from the tick-borne encephalitis virus to emphasize a broad range of applications of our approach. This fluorescent high-throughput assay is based on fluorescent energy transfer (FRET) between two distinct fluorescent proteins (eGFP and mCherry) connected via a substrate polypeptide. When the substrate is cleaved, FRET is abolished and the change in fluorescence corresponds to reaction progress. Our data show that this assay can be used for testing the inhibitors in the 96 or 384 well plates format with robust and reproducible outcomes.

Competing Interest Statement

The authors have declared no competing interest.

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  • ↵1 Co-first author

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted February 03, 2021.
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High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses
Bara Cihlova, Andrea Huskova, Jiri Böserle, Radim Nencka, Evzen Boura, Jan Silhan
bioRxiv 2020.10.27.357418; doi: https://doi.org/10.1101/2020.10.27.357418
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High-throughput fluorescent assay for inhibitor screening of proteases from RNA viruses
Bara Cihlova, Andrea Huskova, Jiri Böserle, Radim Nencka, Evzen Boura, Jan Silhan
bioRxiv 2020.10.27.357418; doi: https://doi.org/10.1101/2020.10.27.357418

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