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Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases
Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier B.V.
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113757/ https://www.ncbi.nlm.nih.gov/pubmed/24393668 http://dx.doi.org/10.1016/j.antiviral.2013.12.012 |
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author | van der Linden, Lonneke Ulferts, Rachel Nabuurs, Sander B. Kusov, Yuri Liu, Hong George, Shyla Lacroix, Céline Goris, Nesya Lefebvre, David Lanke, Kjerstin H.W. De Clercq, Kris Hilgenfeld, Rolf Neyts, Johan van Kuppeveld, Frank J.M. |
author_facet | van der Linden, Lonneke Ulferts, Rachel Nabuurs, Sander B. Kusov, Yuri Liu, Hong George, Shyla Lacroix, Céline Goris, Nesya Lefebvre, David Lanke, Kjerstin H.W. De Clercq, Kris Hilgenfeld, Rolf Neyts, Johan van Kuppeveld, Frank J.M. |
author_sort | van der Linden, Lonneke |
collection | PubMed |
description | Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation. |
format | Online Article Text |
id | pubmed-7113757 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Elsevier B.V. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71137572020-04-02 Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases van der Linden, Lonneke Ulferts, Rachel Nabuurs, Sander B. Kusov, Yuri Liu, Hong George, Shyla Lacroix, Céline Goris, Nesya Lefebvre, David Lanke, Kjerstin H.W. De Clercq, Kris Hilgenfeld, Rolf Neyts, Johan van Kuppeveld, Frank J.M. Antiviral Res Article Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation. Elsevier B.V. 2014-03 2014-01-04 /pmc/articles/PMC7113757/ /pubmed/24393668 http://dx.doi.org/10.1016/j.antiviral.2013.12.012 Text en Copyright © 2013 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article van der Linden, Lonneke Ulferts, Rachel Nabuurs, Sander B. Kusov, Yuri Liu, Hong George, Shyla Lacroix, Céline Goris, Nesya Lefebvre, David Lanke, Kjerstin H.W. De Clercq, Kris Hilgenfeld, Rolf Neyts, Johan van Kuppeveld, Frank J.M. Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title | Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title_full | Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title_fullStr | Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title_full_unstemmed | Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title_short | Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases |
title_sort | application of a cell-based protease assay for testing inhibitors of picornavirus 3c proteases |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113757/ https://www.ncbi.nlm.nih.gov/pubmed/24393668 http://dx.doi.org/10.1016/j.antiviral.2013.12.012 |
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