Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors [post]

Hubert Sijben, Wieke van Oostveen, Peter Hartog, Laura Stucchi, Giovanna Maresca, Lia Scarabottolo, Adriaan IJzerman, Laura Heitman
2021 unpublished
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
more » ... 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.
doi:10.21203/ fatcat:nfpw4ymx3vhsnj7ourb4rbj5fa