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Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament
The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune respo...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Shared Science Publishers OG
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551747/ https://www.ncbi.nlm.nih.gov/pubmed/31225471 http://dx.doi.org/10.15698/cst2018.04.131 |
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author | Sborgi, Lorenzo Ude, Johanna Dick, Mathias S. Vesin, Jonathan Chambon, Marc Turcatti, Gerardo Broz, Petr Hiller, Sebastian |
author_facet | Sborgi, Lorenzo Ude, Johanna Dick, Mathias S. Vesin, Jonathan Chambon, Marc Turcatti, Gerardo Broz, Petr Hiller, Sebastian |
author_sort | Sborgi, Lorenzo |
collection | PubMed |
description | The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z’-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens. |
format | Online Article Text |
id | pubmed-6551747 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Shared Science Publishers OG |
record_format | MEDLINE/PubMed |
spelling | pubmed-65517472019-06-20 Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament Sborgi, Lorenzo Ude, Johanna Dick, Mathias S. Vesin, Jonathan Chambon, Marc Turcatti, Gerardo Broz, Petr Hiller, Sebastian Cell Stress Research Article The protein ASC is a central component of most inflammasome complexes, forming functional oligomeric filaments that activate large amounts of pro-caspase-1 for further IL-1β processing and the induction of Gasdermin D-dependent cell death. The central role of inflammasomes in the innate immune response pose them as new molecular targets for therapy of diverse acute, chronic and inherited autoinflammatory pathologies. In recent years, an increasing number of molecules were proposed to modulate inflammasome signalling by interacting with different components of inflammasome complexes. However, the difficult in vitro reconstitution of the inflammasome has limited the development of specific on-target biochemical assays for compound activity confirmation and for drug discovery in high throughput screening setups. Here we describe a homogeneous, pH-based ASC oligomerization assay that employs fluorescence anisotropy (FA) to monitor the in vitro filament formation of the PYD domain of human ASC. The absence of additional solubility tags as well as of proteolytic enzymes to initiate the filament reaction makes this assay suitable for testing the direct effect of small molecules on filament formation in high throughput format. The ability of the assay to detect modulators of filament formation was confirmed by using a non-filament forming PYD mutant. The high and reproducible Z’-factor of 0.7 allowed to screen 10,100 compounds by high-throughput screening (HTS) aiming to identify inhibitors of ASC filament. While none of these molecules was able to inhibit ASC filament formation in vitro, the assay is directly amenable to screen other compound classes or validate candidate molecules from other screens. Shared Science Publishers OG 2018-03-21 /pmc/articles/PMC6551747/ /pubmed/31225471 http://dx.doi.org/10.15698/cst2018.04.131 Text en Copyright: © 2018 Sborgi et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged. |
spellingShingle | Research Article Sborgi, Lorenzo Ude, Johanna Dick, Mathias S. Vesin, Jonathan Chambon, Marc Turcatti, Gerardo Broz, Petr Hiller, Sebastian Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title | Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title_full | Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title_fullStr | Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title_full_unstemmed | Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title_short | Assay for high-throughput screening of inhibitors of the ASC-PYD inflammasome core filament |
title_sort | assay for high-throughput screening of inhibitors of the asc-pyd inflammasome core filament |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551747/ https://www.ncbi.nlm.nih.gov/pubmed/31225471 http://dx.doi.org/10.15698/cst2018.04.131 |
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