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TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS
SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (T...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399917/ https://www.ncbi.nlm.nih.gov/pubmed/36921991 http://dx.doi.org/10.1093/jmcb/mjad017 |
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author | Cicka, Danielle Niu, Qiankun Qui, Min Qian, Kun Miller, Eric Fan, Dacheng Mo, Xiulei Ivanov, Andrey A Sarafianos, Stefan G Du, Yuhong Fu, Haian |
author_facet | Cicka, Danielle Niu, Qiankun Qui, Min Qian, Kun Miller, Eric Fan, Dacheng Mo, Xiulei Ivanov, Andrey A Sarafianos, Stefan G Du, Yuhong Fu, Haian |
author_sort | Cicka, Danielle |
collection | PubMed |
description | SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2–SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein–protein interaction. |
format | Online Article Text |
id | pubmed-10399917 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-103999172023-08-04 TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS Cicka, Danielle Niu, Qiankun Qui, Min Qian, Kun Miller, Eric Fan, Dacheng Mo, Xiulei Ivanov, Andrey A Sarafianos, Stefan G Du, Yuhong Fu, Haian J Mol Cell Biol Article SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2–SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein–protein interaction. Oxford University Press 2023-03-15 /pmc/articles/PMC10399917/ /pubmed/36921991 http://dx.doi.org/10.1093/jmcb/mjad017 Text en © The Author(s) (2023). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Article Cicka, Danielle Niu, Qiankun Qui, Min Qian, Kun Miller, Eric Fan, Dacheng Mo, Xiulei Ivanov, Andrey A Sarafianos, Stefan G Du, Yuhong Fu, Haian TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title | TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title_full | TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title_fullStr | TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title_full_unstemmed | TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title_short | TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS |
title_sort | tmprss2 and sars-cov-2 spike interaction assay for uhts |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10399917/ https://www.ncbi.nlm.nih.gov/pubmed/36921991 http://dx.doi.org/10.1093/jmcb/mjad017 |
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