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Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors

The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but...

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Autores principales: Sijben, Hubert J., van Oostveen, Wieke M., Hartog, Peter B. R., Stucchi, Laura, Rossignoli, Andrea, Maresca, Giovanna, Scarabottolo, Lia, IJzerman, Adriaan P., Heitman, Laura H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192900/
https://www.ncbi.nlm.nih.gov/pubmed/34112854
http://dx.doi.org/10.1038/s41598-021-91700-7
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author Sijben, Hubert J.
van Oostveen, Wieke M.
Hartog, Peter B. R.
Stucchi, Laura
Rossignoli, Andrea
Maresca, Giovanna
Scarabottolo, Lia
IJzerman, Adriaan P.
Heitman, Laura H.
author_facet Sijben, Hubert J.
van Oostveen, Wieke M.
Hartog, Peter B. R.
Stucchi, Laura
Rossignoli, Andrea
Maresca, Giovanna
Scarabottolo, Lia
IJzerman, Adriaan P.
Heitman, Laura H.
author_sort Sijben, Hubert J.
collection PubMed
description The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.
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spelling pubmed-81929002021-06-14 Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors Sijben, Hubert J. van Oostveen, Wieke M. Hartog, Peter B. R. Stucchi, Laura Rossignoli, Andrea Maresca, Giovanna Scarabottolo, Lia IJzerman, Adriaan P. Heitman, Laura H. Sci Rep Article The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors. Nature Publishing Group UK 2021-06-10 /pmc/articles/PMC8192900/ /pubmed/34112854 http://dx.doi.org/10.1038/s41598-021-91700-7 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Sijben, Hubert J.
van Oostveen, Wieke M.
Hartog, Peter B. R.
Stucchi, Laura
Rossignoli, Andrea
Maresca, Giovanna
Scarabottolo, Lia
IJzerman, Adriaan P.
Heitman, Laura H.
Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title_full Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title_fullStr Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title_full_unstemmed Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title_short Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
title_sort label-free high-throughput screening assay for the identification of norepinephrine transporter (net/slc6a2) inhibitors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192900/
https://www.ncbi.nlm.nih.gov/pubmed/34112854
http://dx.doi.org/10.1038/s41598-021-91700-7
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