Effects of Hydroxylated Mephedrone Metabolites on Monoamine Transporter Activity in vitro

Abstract

Mephedrone is a largely abused psychostimulant. It elicits the release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Stereoselective metabolic reactions are involved in the inactivation and the elimination of its chemical structure. However, during these processes, several structures are generated and some of them have been reported to be still pharmacologically active. In this study 1) we have newly synthetized several putative mephedrone metabolites, 2) compared their activity at monoamine transporters, 3) generated quantitative structure activity relationships, and 4) exploited the chemical structure of the putative metabolites to screen a urine sample from a drug user and dissect mephedrone metabolism. We have found that most of the tested metabolites are weak inhibitors of monoamine transporters and that all of them are more potent at DAT and NET in comparison to SERT. The only exception is represented by the COOH-metabolite which shows no pharmacological activity at all three monoamine transporters. The enantioselectivity of mephedrone and its metabolites is present mainly at SERT, with only minor effects at DAT and NET being introduced when the β-keto group is reduced to an OH-group. Importantly, while at DAT the putative metabolites did not show changes in inhibitory potencies, but rather changes in their substrate/blocker profile, at SERT they showed mainly changes in inhibitory potencies. Molecular modeling suggests that the hydrophobic nature of a specific SERT subpocket may be involved in such loss of affinity. Finally, the assessment of the putative metabolites in one urine sample of mephedrone user displayed two previously uncharacterized metabolites, 4-COOH-nor-mephedrone (4-COOH-MC) and dihydro-4- nor-mephedrone (dihydro-4-MC). These results confirm and expand previous studies highlighting the importance of the stereochemistry in the pharmacodynamics of phase-1 metabolites of mephedrone, established their structure-activity relationships at DAT, NET and SERT and pave the way for a systematic dissection of mephedrone metabolic routes. Given the number of structures found having residual and modified pharmacological profiles, these findings may help in understanding the complex subjective effects of administered mephedrone. Moreover, the dissection of mephedrone metabolic routes may help in developing new therapies for treating psychostimulants acute intoxications.

Serotonin-releasing agents with reduced off-target effects

“…In the case of S-4-MMC and S-4-MC, the addition of a methyl-group to the 4-position increased the potency at SERT and rendered the substances nonselective with respect to DAT and SERT (DAT/SERT ratio =1.06 and 1.9, respectively; Table 1). In agreement with our previous studies on mephedrone metabolites 19,20 , we detected a leftward-shift for all the S-enantiomers when compared to the corresponding R-enantiomers (Fig. 1 j-m; Table 1) at SERT, which further improved the relative potency at SERT versus DAT (all DAT/SERT ratios are summarized in Table 1).…”

“…In our efforts to identify SERT-preferring releasing agents, we chose β-keto-amphetamine-derived drugs (i.e., cathinone-derived drugs) as a starting scaffold based on the following rationale: i) the extant literature has identified basic structure-activity-relationships (SAR) for cathinone compounds 16 ; ii) stereochemistry dictates the pharmacological profile of monoamine release induced by cathinones at SERT, but not DAT [17][18][19][20] , and iii) pre-clinical data indicate a reduced neurotoxic potential for cathinonederived drugs when compared to their amphetamine counterparts e.g. MDMA 21,22 .…”

A Novel and Selective Dopamine Transporter Inhibitor, (S)-MK-26, Promotes Hippocampal Synaptic Plasticity and Restores Effort-Related Motivational Dysfunctions

“…Human embryonic kidney 293 cells (HEK293 cells) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM), containing 10% heat-inactivated fetal calf serum (FCS; 100 mL/L), streptomycin (100 μg/100 mL) and penicillin (100 U × HEK293). Geneticin (50 μg/mL) was added to select cells stably expressing the human isoforms of dopamine, norepinephrine and serotonin transporter (hDAT, hNET, and hSERT, respectively) as described earlier [ 181 , 182 ]. Cell lines were maintained in culture in humidified atmosphere (37 °C, 5% CO 2 ), and passaged when reached 80–90% confluency.…”

Overlap and Specificity in the Substrate Spectra of Human Monoamine Transporters and Organic Cation Transporters 1, 2, and 3

“…While inhibitors of MATs—as well as OCTs—have been studied intensively [ 23 , 24 , 25 , 26 , 27 ] and ligand- as well as structure-based determinants of inhibition have been identified [ 28 , 29 , 30 , 31 ], less is known about the substrate spectra of MATs and OCTs. Especially with MATs, relatively little data is available beyond the narrow substrate group of the neurotransmitters themselves.…”