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

Abstract 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 informa...

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Autores principales:	Hubert J. Sijben, Wieke M. van Oostveen, Peter B. R. Hartog, Laura Stucchi, Andrea Rossignoli, Giovanna Maresca, Lia Scarabottolo, Adriaan P. IJzerman, Laura H. Heitman
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f31bd47fc7a84a55966bc22ad396c6be
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spelling	oai:doaj.org-article:f31bd47fc7a84a55966bc22ad396c6be2021-12-02T17:34:49ZLabel-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors10.1038/s41598-021-91700-72045-2322https://doaj.org/article/f31bd47fc7a84a55966bc22ad396c6be2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91700-7https://doaj.org/toc/2045-2322Abstract 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.Hubert J. SijbenWieke M. van OostveenPeter B. R. HartogLaura StucchiAndrea RossignoliGiovanna MarescaLia ScarabottoloAdriaan P. IJzermanLaura H. HeitmanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hubert J. Sijben Wieke M. van Oostveen Peter B. R. Hartog Laura Stucchi Andrea Rossignoli Giovanna Maresca Lia Scarabottolo Adriaan P. IJzerman Laura H. Heitman Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors
description	Abstract 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.
format	article
author	Hubert J. Sijben Wieke M. van Oostveen Peter B. R. Hartog Laura Stucchi Andrea Rossignoli Giovanna Maresca Lia Scarabottolo Adriaan P. IJzerman Laura H. Heitman
author_facet	Hubert J. Sijben Wieke M. van Oostveen Peter B. R. Hartog Laura Stucchi Andrea Rossignoli Giovanna Maresca Lia Scarabottolo Adriaan P. IJzerman Laura H. Heitman
author_sort	Hubert J. Sijben
title	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_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_sort	label-free high-throughput screening assay for the identification of norepinephrine transporter (net/slc6a2) inhibitors
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f31bd47fc7a84a55966bc22ad396c6be
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Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors

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