Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay.
Anne Zwartsen, Anouk H A Verboven, Regina G D M Van Kleef, Fiona M J Wijnolts, Remco H S Westerink, Laura Hondebrink
Toxicology in vitro : an international journal published in association with BIBRA December 1, 2017 DOI: 10.1016/j.tiv.2017.05.010 via PubMed
Summary
AI-generated from the abstractA new high-throughput fluorescent assay can detect how illicit drugs and new psychoactive substances (NPS) inhibit monoamine reuptake transporters (DAT, NET, SERT). The assay uses a fluorescent monoamine-mimicking substrate in human embryonic kidney cells expressing these transporters. It successfully discriminated between common drugs (cocaine, amphetamine, MDMA), several NPS (e.g., α-PVP, 2C-B, 25B-NBOMe), and the antidepressant fluoxetine. Most IC50 values matched those from traditional radiometric assays and estimated human brain concentrations, though phenethylamines showed higher IC50 values on SERT, possibly due to experimental differences. The fluorescent assay is simpler, works under physiological conditions, requires no special facilities, and allows kinetic measurements, making it a good alternative to radiometric methods.
Study at a glance
| Characteristics | Experimental laboratory study Peer reviewed |
|---|---|
| Population | Human embryonic kidney (HEK293) cells expressing DAT, NET, or SERT |
| Interventions | Cocaine dl-amphetamine MDMA 4-fluoroamphetamine PMMA α-PVP 5-APB 2C-B 25B-NBOMe 25I-NBOMe methoxetamine fluoxetine |
| Keywords | Designer drugs Dopamine transporter Drug screening Fluorescent monoamine substrate Norepinephrine transporter |
| Citations | 59 |
| Key finding | A fluorescent microplate reader-based assay reliably detects inhibition of monoamine transporters by various drugs and NPS, with most IC50 values consistent with radiometric assays and estimated human brain concentrations. |
Abstract
The prevalence and use of new psychoactive substances (NPS) is increasing and currently over 600 NPS exist. Many illicit drugs and NPS increase brain monoamine levels by inhibition and/or reversal of monoamine reuptake transporters (DAT, NET and SERT). This is often investigated using labor-intensive, radiometric endpoint measurements. We investigated the applicability of a novel and innovative assay that is based on a fluorescent monoamine mimicking substrate. DAT, NET or SERT-expressing human embryonic kidney (HEK293) cells were exposed to common drugs (cocaine, dl-amphetamine or MDMA), NPS (4-fluoroamphetamine, PMMA, α-PVP, 5-APB, 2C-B, 25B-NBOMe, 25I-NBOMe or methoxetamine) or the antidepressant fluoxetine. We demonstrate that this fluorescent microplate reader-based assay detects inhibition of different transporters by various drugs and discriminates between drugs. Most IC50 values were in line with previous results from radiometric assays and within estimated human brain concentrations. However, phenethylamines showed higher IC50 values on hSERT, possibly due to experimental differences. Compared to radiometric assays, this high-throughput fluorescent assay is uncomplicated, can measure at physiological conditions, requires no specific facilities and allows for kinetic measurements, enabling detection of transient effects. This assay is therefore a good alternative for radiometric assays to investigate effects of illicit drugs and NPS on monoamine reuptake transporters.