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Research Article
Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors
Laura Heitman
Leiden University
Corresponding Author
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The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological 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 inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in 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.
Keywords
Norepinephrine transporter (NET / SLC6A2), GPCR, impedance, label-free, HTS, solute carriers
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Reviewer #3 agreed
On 16 Jan, 2021
Reviewer #1 agreed
On 16 Jan, 2021
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On 16 Jan, 2021
Reviewer #6 agreed
On 16 Jan, 2021
Reviewer #4 agreed
On 16 Jan, 2021
Reviewer #2 agreed
On 16 Jan, 2021
Reviewer #7 agreed
On 16 Jan, 2021
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This preprint is under consideration at Scientific Reports. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research Article
Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors
Laura Heitman
Leiden University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Revision
Version 1
Posted 05 Jan, 2021
No community comments so far
Editorial decision: Major revision
On 07 Feb, 2021
Review #2 received
Received 07 Feb, 2021
Review #3 received
Received 07 Feb, 2021
Review #1 received
Received 07 Feb, 2021
Review #4 received
Received 07 Feb, 2021
Reviewer #3 agreed
On 16 Jan, 2021
Reviewer #1 agreed
On 16 Jan, 2021
Reviewer #5 agreed
On 16 Jan, 2021
Reviewer #6 agreed
On 16 Jan, 2021
Reviewer #4 agreed
On 16 Jan, 2021
Reviewer #2 agreed
On 16 Jan, 2021
Reviewer #7 agreed
On 16 Jan, 2021
Reviewers invited
Invitations sent on 15 Jan, 2021
Editor assigned
On 12 Jan, 2021
Editor invited
On 01 Jan, 2021
Submission checks complete
On 01 Jan, 2021
First submitted
On 23 Dec, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological 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 inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in 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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.