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A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the therma...

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Autores principales: Martinez, Natalia J., Asawa, Rosita R., Cyr, Matthew G., Zakharov, Alexey, Urban, Daniel J., Roth, Jacob S., Wallgren, Eric, Klumpp-Thomas, Carleen, Coussens, Nathan P., Rai, Ganesha, Yang, Shyh-Ming, Hall, Matthew D., Marugan, Juan J., Simeonov, Anton, Henderson, Mark J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013488/
https://www.ncbi.nlm.nih.gov/pubmed/29930256
http://dx.doi.org/10.1038/s41598-018-27834-y
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author Martinez, Natalia J.
Asawa, Rosita R.
Cyr, Matthew G.
Zakharov, Alexey
Urban, Daniel J.
Roth, Jacob S.
Wallgren, Eric
Klumpp-Thomas, Carleen
Coussens, Nathan P.
Rai, Ganesha
Yang, Shyh-Ming
Hall, Matthew D.
Marugan, Juan J.
Simeonov, Anton
Henderson, Mark J.
author_facet Martinez, Natalia J.
Asawa, Rosita R.
Cyr, Matthew G.
Zakharov, Alexey
Urban, Daniel J.
Roth, Jacob S.
Wallgren, Eric
Klumpp-Thomas, Carleen
Coussens, Nathan P.
Rai, Ganesha
Yang, Shyh-Ming
Hall, Matthew D.
Marugan, Juan J.
Simeonov, Anton
Henderson, Mark J.
author_sort Martinez, Natalia J.
collection PubMed
description Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.
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spelling pubmed-60134882018-06-27 A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase Martinez, Natalia J. Asawa, Rosita R. Cyr, Matthew G. Zakharov, Alexey Urban, Daniel J. Roth, Jacob S. Wallgren, Eric Klumpp-Thomas, Carleen Coussens, Nathan P. Rai, Ganesha Yang, Shyh-Ming Hall, Matthew D. Marugan, Juan J. Simeonov, Anton Henderson, Mark J. Sci Rep Article Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available. Nature Publishing Group UK 2018-06-21 /pmc/articles/PMC6013488/ /pubmed/29930256 http://dx.doi.org/10.1038/s41598-018-27834-y Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Martinez, Natalia J.
Asawa, Rosita R.
Cyr, Matthew G.
Zakharov, Alexey
Urban, Daniel J.
Roth, Jacob S.
Wallgren, Eric
Klumpp-Thomas, Carleen
Coussens, Nathan P.
Rai, Ganesha
Yang, Shyh-Ming
Hall, Matthew D.
Marugan, Juan J.
Simeonov, Anton
Henderson, Mark J.
A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title_full A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title_fullStr A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title_full_unstemmed A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title_short A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase
title_sort widely-applicable high-throughput cellular thermal shift assay (cetsa) using split nano luciferase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013488/
https://www.ncbi.nlm.nih.gov/pubmed/29930256
http://dx.doi.org/10.1038/s41598-018-27834-y
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