'Second-generation' mephedrone analogs, 4-MEC and 4-MePPP, differentially affect monoamine transporter function

Abstract

The nonmedical use of synthetic cathinones is increasing on a global scale. 4-Methyl-N-methylcathinone (mephedrone) is a popular synthetic cathinone that is now illegal in the United States and other countries. Since the legislative ban on mephedrone, a number of 'second-generation' analogs have appeared in the street drug marketplace, including 4-methyl-N-ethylcathinone (4-MEC) and 4'-methyl-α-pyrrolidinopropiophenone (4-MePPP). Here we characterized the interactions of 4-MEC and 4-MePPP with transporters for 5-HT (SERT) and dopamine (DAT) using molecular, cellular, and whole-animal methods. In vitro transporter assays revealed that 4-MEC displays unusual 'hybrid' activity as a SERT substrate (ie, 5-HT releaser) and DAT blocker, whereas 4-MePPP is a blocker at both transporters but more potent at DAT. In vivo microdialysis experiments in rat brain demonstrated that 4-MEC (1-3 mg/kg, i.v.) produced large increases in extracellular 5-HT, small increases in dopamine, and minimal motor stimulation. In contrast, 4-MePPP (1-3 mg/kg, i.v.) produced selective increases in dopamine and robust motor stimulation. Consistent with its activity as a SERT substrate, 4-MEC evoked inward current in SERT-expressing Xenopus oocytes, whereas 4-MePPP was inactive in this regard. To examine drug-transporter interactions at the molecular level, we modeled the fit of 4-MEC and 4-MePPP into the binding pockets for DAT and SERT. Subtle distinctions in ligand-transporter binding were found that account for the differential effects of 4-MEC and 4-MePPP at SERT. Collectively, our results provide key information about the pharmacology of newly emerging mephedrone analogs, and give clues to structural requirements that govern drug selectivity at DAT vs SERT.

Figure 1

Chemical structure of 4-methyl-N-ethylcathinone (4-MEC) and 4′-methyl-α-pyrrolidinopropiophenone (4-MePPP), as compared with N-methylcathinone (methcathinone) and 4-methyl-N-methylcathinone (mephedrone).

Figure 2

Effects of 4-MEC and 4-MePPP on transporter-mediated uptake and release in rat brain synaptosomes. Inhibition of [³H]5-HT uptake (a) and [³H]DA uptake (b) by 4-MEC, 4-MePPP, and mephedrone. Release of preloaded [³H]5-HT from SERT (c) and [³H]MPP+ from DAT (d) evoked by 4-MEC, 4-MePPP, and mephedrone. Data are mean±SD for N=3 separate experiments performed in triplicate.

Figure 3

Effects of i.v. administration of saline, 4-MEC, and 4-MePPP on neurochemistry and behavior in rats undergoing microdialysis in nucleus accumbens. Effects of saline, 4-MEC, and 4-MePPP on dialysate 5-HT (a) and dialysate dopamine (DA) (b). Effects of saline, 4-MEC, and 4-MePPP on forward locomotion (Motor) (c) and stereotypic movements (Stereo) (d). Data are mean±SEM expressed as % baseline for N=6–7 rats/group. Arrows indicate time of injections and numbers indicate i.v. mg/kg doses. *P<0.05 compared with saline-injected control at specific time points.

Figure 4

Inhibition of uptake and stimulation of efflux produced by 4-MEC and 4-MePPP in HEK293 cells stably expressing hSERT and hDAT. Inhibition of [³H]5-HT uptake by hSERT (a) and [³H]DA uptake by hDAT (b) in cells. Efflux of [³H]5-HT (c) and [³H]MPP+ (d) via hSERT and hDAT, respectively. Effects of 10 μM 4-MEC and 10 μM 4-MePPP on efflux were carried out in the presence of Krebs HEPES buffer (KHP) or 10 μM monensin. Arrows show the administration of 4-MEC/4-MePPP. Data are mean±SD for n=3 separate experiments.

Figure 5

Dose–response effects of 4-MEC and 4-MePPP on SERT-generated currents in Xenopus oocytes. Representative electrophysiological traces for 4-MEC (a) and 4-MePPP (b). Concentration–response curves, pooled from different oocytes for 4-MEC and 4-MePPP (c). Data in (c) are mean±SEM for n=8 oocytes from two independent preparations. Maximal current for 4-MEC was measured at 30 μM and no current was observed for 4-MePPP.

Figure 6

Central binding site of (a) DAT with docking poses of 4-MEC (cyan) and 4-MePPP (orange), (b) SERT with the Thr439 hydroxyl pointing toward the S1 site, and (c) SERT with the Thr439 methyl pointing toward the S1 site. The poses of both ligands in DAT are very consistent whereby the 4-methyl group points into subsite B. The same consistency is found in SERT for 4-MEC, but not for 4-MePPP, presumably because of an unfavorable fit into this subpocket forced by the larger substituent on the nitrogen atom.