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1.  Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters☆ 
Neurochemistry International  2014;73(100):32-41.
Highlights
•We quantified adulterants in street drugs sold as cocaine.•We analyzed effects of the most common adulterant levamisole, on neurotransmitter transporters.•Differences in the selectivity of levamisole can be explained by homology modelling and docking.•Aminorex, a metabolite of levamisole, modulates neurotransmitter transporters directly.•Depending on the transporter, aminorex acts as a blocker or as a releaser.
Psychostimulants such as amphetamine and cocaine are illicitly used drugs that act on neurotransmitter transporters for dopamine, serotonin or norepinephrine. These drugs can by themselves already cause severe neurotoxicity. However, an additional health threat arises from adulterant substances which are added to the illicit compound without declaration. One of the most frequently added adulterants in street drugs sold as cocaine is the anthelmintic drug levamisole. We tested the effects of levamisole on neurotransmitter transporters heterologously expressed in HEK293 cells. Levamisole was 100 and 300-fold less potent than cocaine in blocking norepinephrine and dopamine uptake, and had only very low affinity for the serotonin transporter. In addition, levamisole did not trigger any appreciable substrate efflux. Because levamisole and cocaine are frequently co-administered, we searched for possible allosteric effects; at 30 μM, a concentration at which levamisole displayed already mild effects on norepinephrine transport it did not enhance the inhibitory action of cocaine. Levamisole is metabolized to aminorex, a formerly marketed anorectic drug, which is classified as an amphetamine-like substance. We examined the uptake-inhibitory and efflux-eliciting properties of aminorex and found it to exert strong effects on all three neurotransmitter transporters in a manner similar to amphetamine. We therefore conclude that while the adulterant levamisole itself has only moderate effects on neurotransmitter transporters, its metabolite aminorex may exert distinct psychostimulant effects by itself. Given that the half-time of levamisole and aminorex exceeds that of cocaine, it may be safe to conclude that after the cocaine effect “fades out” the levamisole/aminorex effect “kicks in”.
doi:10.1016/j.neuint.2013.11.010
PMCID: PMC4077236  PMID: 24296074
SERT, serotonin transporter; NET, norepinephrine transporter; DAT, dopamine transporter; 5-HT, serotonin; DA, dopamine; KHB, Krebs–Ringer–HEPES buffer; HPLC, high performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; Levamisole; Aminorex; Neurotransmitter transporter; Cocaine; Adulterant
2.  Missense dopamine transporter mutations associate with adult parkinsonism and ADHD 
The Journal of Clinical Investigation  2014;124(7):3107-3120.
Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.
doi:10.1172/JCI73778
PMCID: PMC4071392  PMID: 24911152
3.  Powerful Cocaine-Like Actions of 3,4-Methylenedioxypyrovalerone (MDPV), a Principal Constituent of Psychoactive ‘Bath Salts' Products 
Neuropsychopharmacology  2012;38(4):552-562.
The abuse of psychoactive ‘bath salts' containing cathinones such as 3,4-methylenedioxypyrovalerone (MDPV) is a growing public health concern, yet little is known about their pharmacology. Here, we evaluated the effects of MDPV and related drugs using molecular, cellular, and whole-animal methods. In vitro transporter assays were performed in rat brain synaptosomes and in cells expressing human transporters, while clearance of endogenous dopamine was measured by fast-scan cyclic voltammetry in mouse striatal slices. Assessments of in vivo neurochemistry, locomotor activity, and cardiovascular parameters were carried out in rats. We found that MDPV blocks uptake of [3H]dopamine (IC50=4.1 nℳ) and [3H]norepinephrine (IC50=26 nℳ) with high potency but has weak effects on uptake of [3H]serotonin (IC50=3349 nℳ). In contrast to other psychoactive cathinones (eg, mephedrone), MDPV is not a transporter substrate. The clearance of endogenous dopamine is inhibited by MDPV and cocaine in a similar manner, but MDPV displays greater potency and efficacy. Consistent with in vitro findings, MDPV (0.1–0.3 mg/kg, intravenous) increases extracellular concentrations of dopamine in the nucleus accumbens. Additionally, MDPV (0.1–3.0 mg/kg, subcutaneous) is at least 10 times more potent than cocaine at producing locomotor activation, tachycardia, and hypertension in rats. Our data show that MDPV is a monoamine transporter blocker with increased potency and selectivity for catecholamines when compared with cocaine. The robust stimulation of dopamine transmission by MDPV predicts serious potential for abuse and may provide a mechanism to explain the adverse effects observed in humans taking high doses of ‘bath salts' preparations.
doi:10.1038/npp.2012.204
PMCID: PMC3572453  PMID: 23072836
addiction & Substance Abuse; blocker; catecholamines; designer drugs; neuropharmacology; Psychostimulants; releaser; transporter; designer drug; dopamine; cathinone; monoamine transporter; uptake blocker
4.  A Combined Approach Using Transporter-Flux Assays and Mass Spectrometry to Examine Psychostimulant Street Drugs of Unknown Content 
ACS Chemical Neuroscience  2012;4(1):182-190.
The illicit consumption of psychoactive compounds may cause short and long-term health problems and addiction. This is also true for amphetamines and cocaine, which target monoamine transporters. In the recent past, an increasing number of new compounds with amphetamine-like structure such as mephedrone or 3,4-methylenedioxypyrovalerone (MDPV) entered the market of illicit drugs. Subtle structural changes circumvent legal restrictions placed on the parent compound. These novel drugs are effectively marketed “designer drugs” (also called “research chemicals”) without any knowledge of the underlying pharmacology, the potential harm or a registration of the manufacturing process. Accordingly new entrants and their byproducts are identified postmarketing by chemical analysis and their pharmacological properties inferred by comparison to compounds of known structure. However, such a heuristic approach fails, if the structures diverge substantially from a known derivative. In addition, the understanding of structure–activity relations is too rudimentary to predict detailed pharmacological activity. Here, we tested a combined approach by examining the composition of street drugs using mass spectrometry and by assessing the functional activity of their constituents at the neuronal transporters for dopamine, serotonin, and norepinephrine. We show that this approach is superior to mere chemical analysis in recognizing novel and potentially harmful street drugs.
doi:10.1021/cn3001763
PMCID: PMC3547486  PMID: 23336057
Amphetamine; bath-salts; mass spectrometry; combo; psychostimulants; 2C-B; MDPV
7.  Dopamine Transporter Phosphorylation Site Threonine 53 Regulates Substrate Reuptake and Amphetamine-stimulated Efflux* 
The Journal of Biological Chemistry  2012;287(35):29702-29712.
Background: DAT activity is regulated by protein kinases.
Results: We identify Thr53 as a DAT phosphorylation site in rat striatum by mass spectrometry and a phospho-specific antibody; Thr53 mutation reduced dopamine influx and ablated transporter-mediated efflux.
Conclusion: Phosphorylation of DAT Thr53 is involved in transport activity.
Significance: These results identify Thr53 phosphorylation of DAT in vivo and elucidate associated functional properties.
In the central nervous system, levels of extraneuronal dopamine are controlled primarily by the action of the dopamine transporter (DAT). Multiple signaling pathways regulate transport activity, substrate efflux, and other DAT functions through currently unknown mechanisms. DAT is phosphorylated by protein kinase C within a serine cluster at the distal end of the cytoplasmic N terminus, whereas recent work in model cells revealed proline-directed phosphorylation of rat DAT at membrane-proximal residue Thr53. In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr53 in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr53 occurred with a stoichiometry of ∼50% and was strongly increased by phorbol esters and protein phosphatase inhibitors, demonstrating modulation of the site by signaling pathways that impact DAT activity. Mutations of Thr53 to prevent phosphorylation led to reduced dopamine transport Vmax and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.
doi:10.1074/jbc.M112.367706
PMCID: PMC3436161  PMID: 22722938
ERK; MAP Kinases (MAPKs); Mass Spectrometry (MS); Protein Kinase C (PKC); SH3 Domains; 1-Methyl-4-phenylpyridinium (MPP+); PP1/2A; cis-trans Isomerization; Phospho-specific Antibody; Proline-directed Phosphorylation
8.  Probing Binding Pocket of Serotonin Transporter by Single Molecular Force Spectroscopy on Living Cells* 
The Journal of Biological Chemistry  2011;287(1):105-113.
Background: The serotonin transporter is the site of action of antidepressants and amphetamines.
Results: Single molecular force spectroscopy allowed for mapping the energy landscape involved in MFZ2-12/SERT binding.
Conclusion: Our data indicate that the outer vestibule imposes a barrier on the entry of MFZ2-12 into the SERT substrate binding site.
Significance: Our results provide a useful framework for a further exploration of antidepressant binding.
The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). The interaction energies involved in binding of such compounds to the transporter are unknown. Here, we used atomic force microscopy (AFM) to probe single molecular interactions between the serotonin transporter and MFZ2-12 (a potent cocaine analog) in living CHOK1 cells. For the AFM measurements, MFZ2-12 was immobilized on AFM tips by using a heterobifunctional cross-linker. By varying the pulling velocity in force distance cycles drug-transporter complexes were ruptured at different force loadings allowing for mapping of the interaction energy landscape. We derived chemical rate constants from these recordings and compared them with those inferred from inhibition of transport and ligand binding: koff values were in good agreement with those derived from uptake experiments; in contrast, the kon values were scaled down when determined by AFM. Our observations generated new insights into the energy landscape of the interaction between SERT and inhibitors. They thus provide a useful framework for molecular dynamics simulations by exploring the range of forces and energies that operate during the binding reaction.
doi:10.1074/jbc.M111.304873
PMCID: PMC3249061  PMID: 22033932
Atomic Force Microscopy; Molecular Docking; Molecular Imaging; Serotonin Transporters; Spectroscopy; Binding Reaction; Force Spectroscopy; MFZ2-12
10.  The N Terminus of Monoamine Transporters Is a Lever Required for the Action of Amphetamines* 
The Journal of Biological Chemistry  2010;285(14):10924-10938.
The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N terminus in mediating the action of amphetamines by focusing initially on the highly conserved threonine residue at position 81, a candidate site for phosphorylation by protein kinase C. Molecular dynamics simulations of the wild type SERT, compared with its mutations SERTT81A and SERTT81D, suggested structural changes in the inner vestibule indicative of an opening of the inner vestibule. Predictions from this model (e.g. the preferential accumulation of SERTT81A in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERTT81A (and the homologous mutations in noradrenaline and dopamine) failed to support amphetamine-induced efflux, and this was not remedied by aspartate at this position. Amphetamine-induced currents through SERTT81A were comparable with those through the wild type transporter. Both abundant Na+ entry and accumulation of SERTT81A in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating the first 64 amino acids and by tethering the N terminus to an additional transmembrane helix. Either modification abolished amphetamine-induced efflux. We therefore conclude that the N terminus of monoamine transporters acts as a lever that sustains reverse transport.
doi:10.1074/jbc.M109.083154
PMCID: PMC2856298  PMID: 20118234
Phosphorylation; Transport/Neurotransmitter; Oocyte; Transport Drugs; Zinc; Amphetamine; Carrier-mediated Efflux; Fluorescence Resonance Energy Transfer Microscopy; Serotonin
12.  A Combined Approach Using Transporter-Flux Assays and Mass Spectrometry to Examine Psychostimulant Street Drugs of Unknown Content 
ACS Chemical Neuroscience  2012;4(1):182-190.
The illicit consumption of psychoactive compounds may cause short and long-term health problems and addiction. This is also true for amphetamines and cocaine, which target monoamine transporters. In the recent past, an increasing number of new compounds with amphetamine-like structure such as mephedrone or 3,4-methylenedioxypyrovalerone (MDPV) entered the market of illicit drugs. Subtle structural changes circumvent legal restrictions placed on the parent compound. These novel drugs are effectively marketed “designer drugs” (also called “research chemicals”) without any knowledge of the underlying pharmacology, the potential harm or a registration of the manufacturing process. Accordingly new entrants and their byproducts are identified postmarketing by chemical analysis and their pharmacological properties inferred by comparison to compounds of known structure. However, such a heuristic approach fails, if the structures diverge substantially from a known derivative. In addition, the understanding of structure–activity relations is too rudimentary to predict detailed pharmacological activity. Here, we tested a combined approach by examining the composition of street drugs using mass spectrometry and by assessing the functional activity of their constituents at the neuronal transporters for dopamine, serotonin, and norepinephrine. We show that this approach is superior to mere chemical analysis in recognizing novel and potentially harmful street drugs.
doi:10.1021/cn3001763
PMCID: PMC3547486  PMID: 23336057
Amphetamine; bath-salts; mass spectrometry; combo; psychostimulants; 2C-B; MDPV
13.  Probing the Selectivity of Monoamine Transporter Substrates by Means of Molecular Modeling 
Molecular Informatics  2013;32(5-6):409-413.
doi:10.1002/minf.201300013
PMCID: PMC3743209  PMID: 23956802
Serotonin transporter (SERT); Dopamine transporter (DAT); Substrate selectivity; Fenfluramine; Docking; Common scaffold clustering

Results 1-13 (13)