Cathinones

The dopamine and serotonin transporter proteins (DAT, SERT) play a vital role in behavior and mental illness. Although their substrate transport has been studied extensively, the molecular basis of their selectivity is not completely understood yet. In this study, we exploit molecular dynamics simulations combined with mutagenesis studies to shed light on the driving factors for DAT-over-SERT selectivity of a set of cathinones. Results indicate that these compounds can adopt two binding modes of which one is more favorable. In addition, free energy calculations indicated the substrate binding site (S1) as the primary recognition site for these ligands. By simulating DAT with SERT-like mutations, we hypothesize unsubstituted cathinones to bind more favorably to DAT, due to a Val152 offering more space, as compared  to the bulkier Ile172 in SERT. This was supported by uptake inhibition measurements, which showed an increase in activity in SERT-I172V.

• Para-CF3-substitution of MCAT allows to selectively target SERT over DAT.
• MCAT and para-CF3-MCAT are partial releasers of SERT.
• The partial release evoked by para-CF3-MCAT involves the allosteric site of SERT.
• The substrate permeation pathway of SERT affords different binding modes.

The transporters for dopamine (DAT) and serotonin (SERT) are important targets in the treatment of psychiatric disorders including major depression, anxiety and attention-deficit hyperactivity disorder. Drugs acting at these transporters can act as inhibitors or as releasers. In addition, it has been recently appreciated that some compounds are less efficacious releasers than amphetamine. Thus, they are classified as partial releasers. Compounds can act on both SERT and DAT or display exquisite selectivity for either SERT or DAT, but the structural basis for selectivity is poorly understood. The trifluoromethyl-substitution of methcathinone in the para-position has been shown to dramatically shift the selectivity of methcathinone (MCAT) towards SERT. Here, we examined MCAT, para-trifluoromethyl-methcathinone (p-CF3-MCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF 3-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the p-CF3-group, but not of other substituents with larger van der Waal's volume, lipophilicity or polarity, converted the DAT-selective MCAT into a SERT-selective partial releaser. Electrophysiological and superfusion experiments, together with kinetic modelling, showed that p-CF3-MCAT, but not MCAT, trapped a fraction of SERTs in an inactive state by occupying the S2-site. These findings define a new mechanism of action for partial releasers, which is distinct from the other two known binding modes underlying partial release. Our observations highlight the fact that the substrate permeation pathway of monoamine transporters supports multiple binding modes, which can be exploited for drug design.

A new LCMS approach is tested for the analysis of controlled substances using bath salts (cathinones) as a model system. Since the structure of cathinones have a polar functionality (the amine group) as well as an aromatic component, separation on the LC column could be achieved in the reversed-phase(RP) or aque- ous normal phase (ANP) modes. Since silica hydride columns can function in both modes they have con- siderable potential for the analysis of substances that have both polar and nonpolar functionalities. Four different silica hydride-based columns (C18, phenyl, cholesterol and diol) are tested in either the RP or ANP and RP modes using representative compounds to determine their applicability to bath salt analysis. A protocol for the analysis of a related illicit drug, benzylpiperazine, is developed on another silica hydride column (Diamond Hydride) in the ANP mode. Further testing is done on physiological samples, saliva (with 78–98% recovery) and hair, in order to determine that the approach outlined in this study could be utilized for these and other controlled substances in biological matrices.

The nonmedical use of 'designer' cathinone analogs, such as 4-methylmethcathinone (mephedrone) and 3,4-methylenedioxymethcathinone (methylone), is increasing worldwide, yet little information is available regarding the mechanism of action for these drugs. Here, we employed in vitro and in vivo methods to compare neurobiological effects of mephedrone and methylone with those produced by the structurally related compounds, 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine. In vitro release assays using rat brain synaptosomes revealed that mephedrone and methylone are nonselective substrates for plasma membrane monoamine transporters, similar to MDMA in potency and selectivity. In vivo microdialysis in rat nucleus accumbens showed that i.v. administration of 0.3 and 1.0 mg/kg of mephedrone or methylone produces dose-related increases in extracellular dopamine and serotonin (5-HT), with the magnitude of effect on 5-HT being greater. Both methcathinone analogs were w...