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 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.

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition

“…Recently label-free techniques, as a new technology, were introduced to study transporter activity [16,19,31,32]. We previously introduced the so-called TRACT assay requiring a GPCR partner to detect SLC-transport activity via a mutual substrate/agonist [17][18][19]. Endogenously, SLCs and GPCRs are not always coexpressed, and thus would require transient expression of either the SLC or GPCR.…”

“…Label-free cellular impedance measurements were performed using an xCELLigence Real-Time Cell Analyzer (RTCA) SP system (ACEA biosciences, San Diego, CA, USA) as described previously [17,18]. In short, the xCELLigence biosensor detects changes in cell morphology, adhesion, and proliferation through impedance of the electron flow.…”

“…Therefore, label-free methodologies to study transporters are currently emerging as an alternative to traditional uptake assays [16]. Recently, our group introduced the xCELLigencebiosensor as a successful tool to evaluate transport activity of the equilibrative nucleoside transporter 1 (ENT1), dopamine (DAT) and norepinephrine (NET) transporters indirectly via attenuated G protein-coupled receptor (GPCR) signaling [17][18][19]. This impedance-based assay detects GPCR-induced changes in cell morphology, adhesion, and proliferation on intact cells [20] and provides a more physiologically relevant condition to assess transport activity than the conventional uptake assays without the need to label substrates.…”

Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

“…JumpIn cells with doxycycline (dox)-inducible transgene overexpression were generated for either one of the five human EAAT subtypes by the RESOLUTE consortium. JumpIn cells were cultured and transfected as described previously ( Sijben et al, 2021a ; 2021b ). In short, cells were cultured in culture medium supplemented with 200 μg/ml hygromycin B and 5 μg/ml blasticidin.…”

“…Alternatively, indirect assays based on fluorescent probes and reporters such as membrane potential dyes ( Jensen and Bräuner-Osborne, 2004 ), glutamate sensors ( Armbruster et al, 2020 ), and intracellular anion sensors ( Zielewicz and Grewer, 2019 ) have proven successful to infer glutamate transport activity, although they require the introduction of non-physiological chemical labels. Recently, we reported on a label-free impedance-based method to assess activity and inhibition of nucleoside ( Vlachodimou et al, 2019 ), dopamine ( Sijben et al, 2021a ), and norepinephrine transporters ( Sijben et al, 2021b ) via activation of congruent G protein-coupled receptors (GPCRs) by their endogenous substrate in live cells, termed the TRACT assay. Importantly, impedance-based biosensors have the advantage to record any changes in cellular morphology upon cell perturbation, such as receptor activation and acute cytotoxicity ( Lundstrom, 2017 ), which opens a broader application of label-free assays to study transport proteins.…”