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

doi:10.1016/j.tiv.2017.05.010

Anne Zwartsen, Anouk H.A Verboven + Show 4 more

Open Access

https://doi.org/10.1016/j.tiv.2017.05.010

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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.

Highlights

The use of illicit drugs is high and 5% of the population worldwide used an illicit drug in the last year
To determine the effects of drugs on fluorescent substrate uptake, cells were pre-incubated with fluorescent substrate solution for 12 min, during which FU increased in control wells for all three transporters (Fig. 3)
Since MDMA is known to reverse hSERT (Verrico et al, 2007; Rudnick and Wall, 1992; Mlinar and Corradetti, 2003), we investigated the effect of MDMA on single cells expressing hSERT

Summary

Introduction

The use of illicit drugs is high and 5% of the population worldwide used an illicit drug in the last year. Used drugs include cocaine, amphetamine and 3,4-methylenedioxy-N-methylamphetamine (MDMA). While the prevalence of use of these common drugs is decreasing, the use of new psychoactive substances (NPS) is steadily increasing (UNODC, 2016). A European survey conducted amongst young European adults (15–24 year old) reported a lifetime prevalence for NPS use of 8% (Flash Eurobarometer 401, 2014). In the Netherlands, the Drugs Information and Monitoring System (DIMS) offers a drug testing service to drug users. NPS are sold as common illicit drugs, the use of NPS as a drug of choice is increasing. The most frequently detected NPS in drug

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