Measuring inhibition of monoamine reuptake transporters by new psychoactive substances (NPS) in real-time using a high-throughput, fluorescence-based assay

• Published in: Toxicology in vitro 2017-12, Vol.45 (Pt 1), p.60-71
• Main Authors: Zwartsen, Anne, Verboven, Anouk H.A., van Kleef, Regina G.D.M., Wijnolts, Fiona M.J., Westerink, Remco H.S., Hondebrink, Laura
• Format: Article
• Language: English
• Subjects: Amphetamines; Amphetamines - pharmacology; Assaying; Bioassays; Brain; Cocaine; Cocaine - pharmacology; Designer drugs; Dopamine; Dopamine transporter; Drug abuse; Drug screening; Drug testing; Drugs; Fluorescence; Fluorescent monoamine substrate; Fluoxetine; HEK293 Cells; Humans; Inhibition; Kidneys; MDMA; Mimicry; Molecular Structure; N-Methyl-3,4-methylenedioxyamphetamine - pharmacology; Narcotics; Neurotransmitter Transport Proteins - antagonists & inhibitors; Norepinephrine transporter; Polymethyl methacrylate; Polymethylmethacrylate; Psychotropic drugs; Psychotropic Drugs - chemistry; Psychotropic Drugs - pharmacology; Serotonin; Serotonin transporter; Substrates
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2017.05.010

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. •The fluorescence-based assay is uncomplicated, requires no specific facilities and allows for kinetic measurements at physiological conditions.•Commonly used illicit drugs and NPS concentration-dependently inhibit monoamine reuptake at expected brain concentrations.•The fluorescence-based assay shows comparable results on hDAT and hNET to radiometric assays.•The fluorescence-based assay shows different results for phenethylamines on hSERT compared to radiometric assays.•This high-throughput fluorescent assay is a good alternative for radiometric assays to investigate drug-induced inhibition of monoamine reuptake transporters.