Application of a Combined Approach to Identify New Psychoactive Street Drugs and Decipher Their Mechanisms at Monoamine Transporters

Abstract

Psychoactive compounds can cause acute and long-term health problems and lead to addiction. In addition to well-studied and legally controlled compounds like cocaine, new psychoactive substances (NPS) are appearing in street drug markets as replacement strategies and legal alternatives. NPS are effectively marketed as "designer drugs" or "research chemicals" without any knowledge of their underlying pharmacological mode of action and their potential toxicological effects and obviously devoid of any registration process. As of 2016, the knowledge of structure-activity relationships for most NPS is scarce, and predicting detailed pharmacological activity of newly emerging drugs is a challenging task. Therefore, it is important to combine different approaches and employ biological test systems that are superior to mere chemical analysis in recognizing novel and potentially harmful street drugs. In this chapter, we provide a detailed description of techniques to decipher the molecular mechanism of action of NPS that target the high-affinity transporters for dopamine, norepinephrine, and serotonin. In addition, this chapter provides insights into a combined approach to identify and characterize new psychoactive street drugs of unknown content in a collaboration with the Austrian prevention project "checkit!."

An unsuspected role for organic cation transporter 3 in the actions of amphetamine

“…Radiotracer flux assays using [ 3 H]MPP + were carried out in rat cultured superior cervical ganglion cells (SCGs). The fluorescent substrate 4-(4-dimethylamino-styryl)- N -methylpyridinium (ASP + ) or [ 3 H]MPP + were used to measure substrate uptake and efflux in human embryonic kidney (HEK293) cells expressing either human OCT3 tagged with yellow fluorescent protein (YFPhOCT3), hVMAT2, or hNET, according to established methods [ 24 , 25 ]. Detailed descriptions of all methods can be found in the Supplementary information .…”

Stereochemistry of phase-1 metabolites of mephedrone determines their effectiveness as releasers at the serotonin transporter

“…Human embryonic 293 cells (HEK293 cells) were maintained at a subconfluent state in humidified atmosphere (37°C, 5% CO2) in Dulbecco's Modified Eagle Medium (DMEM), containing 10 % heat-inactivated fetal calf serum (FCS), streptomycin (100 µg x 100 mL -1 ) and penicillin (100 U x 100 mL -1 ). Geneticin (50 µg x mL -1 ) was added to select HEK293 cells stably expressing the human isoforms of DAT, NET and SERT as described in (Mayer et al, 2017). For uptake inhibition experiments cells expressing the desired transporter were seeded (40,000 cells per well in a final volume of 200 µL) onto poly-Dlysine (PDL) coated 96-well plates 24 hours prior to the experiment.…”

“…For uptake inhibition experiments cells expressing the desired transporter were seeded (40,000 cells per well in a final volume of 200 µL) onto poly-Dlysine (PDL) coated 96-well plates 24 hours prior to the experiment. The same procedure was applied for release experiments, with the only modification that PDLcoated glass-coverslips (diameter 5 mm) were placed into the 96-well plates (Mayer et al, 2017).…”

“…Uptake in the absence of the substance of interest was defined as 100% uptake. The IC50 values were determined by non-linear regression fits (GraphPad Prism version 5.0; log(inhibitor) vs. response -variable slope four parameters (Y= Bottom + (Top-Bottom)/(1+10^((LogIC50-X)*HillSlope)); fitting method: Least squares (ordinary) fit without weighting to minimize the absolute distances squared) as described in (Mayer et al, 2017).…”

“…Dynamic superfusion experiments were performed as previously described (Mayer et al, 2017;Sitte et al, 2000). Briefly, SERT expressing HEK293 cells were grown on PDLcoated glass coverslips and pre-loaded with [ 3 H]5-HT by exposing the cells to 0.4 µM [ 3 H]5-HT (in KHB) for 20 minutes at 37°C.…”