Publications by authors named "Marion Holy"

39 Publications

Effects of Hydroxylated Mephedrone Metabolites on Monoamine Transporter Activity .

Front Pharmacol 2021 9;12:654061. Epub 2021 Apr 9.

Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria.

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.
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http://dx.doi.org/10.3389/fphar.2021.654061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063026PMC
April 2021

α-PPP and its derivatives are selective partial releasers at the human norepinephrine transporter: A pharmacological characterization of interactions between pyrrolidinopropiophenones and uptake1 and uptake2 monoamine transporters.

Neuropharmacology 2021 Apr 20;190:108570. Epub 2021 Apr 20.

Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Währingerstraße 13A, 1090, Vienna, Austria; AddRess Centre for Addiction Research and Science, Medical University of Vienna, Währingerstraße 13A, 1090, Vienna, Austria. Electronic address:

While classical cathinones, such as methcathinone, have been shown to be monoamine releasing agents at human monoamine transporters, the subgroup of α-pyrrolidinophenones has thus far solely been characterized as monoamine transporter reuptake inhibitors. Herein, we report data from previously undescribed α-pyrrolidinopropiophenone (α-PPP) derivatives and compare them with the pharmacologically well-researched α-PVP (α-pyrrolidinovalerophenone). Radiotracer-based in vitro uptake inhibition assays in HEK293 cells show that the investigated α-PPP derivatives inhibit the human high-affinity transporters of dopamine (hDAT) and norepinephrine (hNET) in the low micromolar range, with α-PVP being ten times more potent. Similar to α-PVP, no relevant pharmacological activity was found at the human serotonin transporter (hSERT). Unexpectedly, radiotracer-based in vitro release assays reveal α-PPP, MDPPP and 3Br-PPP, but not α-PVP, to be partial releasing agents at hNET (EC values in the low micromolar range). Furthermore, uptake inhibition assays at low-affinity monoamine transporters, i.e., the human organic cation transporters (hOCT) 1-3 and human plasma membrane monoamine transporter (hPMAT), bring to light that all compounds inhibit hOCT1 and 2 (IC values in the low micromolar range) while less potently interacting with hPMAT and hOCT3. In conclusion, this study describes (i) three new hybrid compounds that efficaciously block hDAT while being partial releasers at hNET, and (ii) highlights the interactions of α-PPP-derivatives with low-affinity monoamine transporters, giving impetus to further studies investigating the interaction of drugs of abuse with OCT1-3 and PMAT.
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http://dx.doi.org/10.1016/j.neuropharm.2021.108570DOI Listing
April 2021

Ga-Labelled Tropane Analogues for the Visualization of the Dopaminergic System.

ChemMedChem 2021 Mar 10;16(5):804-808. Epub 2020 Dec 10.

Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz, Fritz-Strassmann-Weg 2, 55128, Mainz, Germany.

The development of radiometal-labelled pharmaceuticals for neuroimaging could offer great potential due to easier handling during labelling and availability through radionuclide generator systems. Nonetheless, to date, no such tracers are available for positron emission tomography, primarily owing to the challenge of crossing the blood-brain barrier (BBB) and loss of affinity through chelator attachment. We have prepared a variety of Ga-labelled phenyltropanes showing that, through a simple hydrocarbon-linker, it is possible to introduce a chelator onto the lead structure while maintaining its high affinity for hDAT (human dopamine transporter) and simultaneously achieving adequate lipophilicity. One of the candidates, [ Ga]Ga-HBED-hexadiyne-tropane, showed an IC value of 66 nM, together with a log D of 0.96. A μPET study in a hemi-parkinsonian rat model showed a fast wash-out of the tracer, and no specific uptake in the brain, thus implying an inability to penetrate the BBB.
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http://dx.doi.org/10.1002/cmdc.202000820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984292PMC
March 2021

Cell-Based Radiotracer Binding and Uptake Inhibition Assays: A Comparison of Methods to Assess the Potency of Drugs That Target Monoamine Transporters.

Front Pharmacol 2020 19;11:673. Epub 2020 May 19.

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria.

High-affinity monoamine transporters are targets for prescribed medications and stimulant drugs of abuse. Therefore, assessing monoamine transporter activity for candidate medications and newly-emerging drugs of abuse provides essential information for industry, academia, and public health. Radiotracer binding and uptake inhibition are the gold standard assays for determining drug-transporter interaction profiles. The combined results from such assays yield a unique biochemical fingerprint for each compound. Over time, different assay methods have been developed to assess transporter activity, and the comparability of data across various assay platforms remains largely unclear. Here, we compare the effects of six well-established stimulants in two different cell-based uptake inhibition assays, one method using adherent cells and the other using suspended cells. Furthermore, we compare the data from transfected cell lines derived from different laboratories and data reported from rat synaptosomes. For transporter inhibitors, IC values obtained by the two experimental methods were comparable, but using different transfected cell lines yielded disparate results. For transporter substrates, differences between the two cell lines were less pronounced but the drugs displayed different inhibition potencies when evaluated by the two methods. Our study illustrates the inherent limitations when comparing transporter inhibition data from different laboratories and stresses the importance of including appropriate control experiments with reference compounds when investigating new drugs of interest.
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http://dx.doi.org/10.3389/fphar.2020.00673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248194PMC
May 2020

para-Trifluoromethyl-methcathinone is an allosteric modulator of the serotonin transporter.

Neuropharmacology 2019 12 24;161:107615. Epub 2019 Apr 24.

Institute of Pharmacology, Medical University, Vienna, Austria. Electronic address:

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 (pCFMCAT) and other analogues to understand (i) the determinants of selectivity and (ii) the effects of the para-CF-substitution of MCAT on the transport cycle. We systematically tested different para-substituted MCATs by biochemical, computational and electrophysiological approaches: addition of the pCFgroup, 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 pCFMCAT, 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. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
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http://dx.doi.org/10.1016/j.neuropharm.2019.04.021DOI Listing
December 2019

α-Fluorination of carbonyls with nucleophilic fluorine.

Nat Chem 2019 04 4;11(4):329-334. Epub 2019 Mar 4.

Institute of Organic Chemistry, University of Vienna, Vienna, Austria.

Given the unique properties of fluorine, and the ability of fluorination to change the properties of organic molecules, there is significant interest from medicinal chemists in innovative methodologies that enable the synthesis of new fluorinated motifs. State-of-the-art syntheses of α-fluorinated carbonyl compounds invariably rely on electrophilic fluorinating agents, which can be strongly oxidizing and difficult to handle. Here we show that reversing the polarity of the enolate partner to that of an enolonium enables nucleophilic fluorinating agents to be used for direct chemoselective α-C-H-fluorination of amides. Reduction of these products enables facile access to β-fluorinated amines and the value of this methodology is shown by the easy preparation of a number of fluorinated analogues of drugs and agrochemicals. A fluorinated analogue of citalopram, a marketed antidepressant drug, is presented as an example of the preserved biological activity after fluorination.
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http://dx.doi.org/10.1038/s41557-019-0215-zDOI Listing
April 2019

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

Neuropharmacology 2019 04 2;148:199-209. Epub 2019 Jan 2.

Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Vienna, Austria; Center for Addiction Research and Science - AddRess, Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria. Electronic address:

Mephedrone (4-methyl-N-methylcathinone) is a psychostimulant that promotes release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Metabolic breakdown of mephedrone results in bioactive metabolites that act as substrate-type releasers at monoamine transporters and stereospecific metabolism of mephedrone has been reported. This study compared the effects of the enantiomers of the phase-1 metabolites nor-mephedrone, 4-hydroxytolyl-mephedrone (4-OH-mephedrone) and dihydro-mephedrone on (i) DAT, NET and SERT mediated substrate fluxes, (ii) determined their binding affinities towards a battery of monoamine receptors and (iii) examined the relative abundance of the enantiomers in human urine. Each of the enantiomers tested inhibited uptake mediated by DAT, NET and SERT. No marked differences were detected at DAT and NET. However, at SERT, the S-enantiomers of nor-mephedrone and 4-OH-mephedrone were several times more potent than the corresponding R-enantiomers. Moreover, the R-enantiomers were markedly less effective as releasers at SERT. S-nor-mephedrone displayed moderate affinities towards human alpha, human 5-HT and rat and mouse trace amine-associated receptor 1. These results demonstrate that stereochemistry dictates the pharmacodynamics of the phase-1 metabolites of mephedrone at SERT, but not at DAT and NET, which manifests in marked differences in their relative potencies, i.e. DAT/SERT ratios. Chiral analysis of urine samples demonstrated that nor-mephedrone predominantly exists as the S-enantiomer. Given the asymmetric abundance of the enantiomers in biological samples, these findings may add to our understanding of the subjective effects of administered mephedrone, which indicate pronounced effects on the serotonergic system.
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http://dx.doi.org/10.1016/j.neuropharm.2018.12.032DOI Listing
April 2019

Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity.

J Biol Chem 2019 03 26;294(10):3419-3431. Epub 2018 Dec 26.

the Department of Biomedical Sciences, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202-9037, and

Several protein kinases, including protein kinase C, Ca/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, we defined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation.
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http://dx.doi.org/10.1074/jbc.RA118.005251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416449PMC
March 2019

The synthetic cathinones, butylone and pentylone, are stimulants that act as dopamine transporter blockers but 5-HT transporter substrates.

Psychopharmacology (Berl) 2019 Mar 22;236(3):953-962. Epub 2018 Oct 22.

Designer Drug Research Unit (DDRU), Intramural Research Program (IRP), NIDA, NIH, DHHS, 333 Cassell Drive, Suite 4400, Baltimore, MD, 21224, USA.

Rationale: Synthetic cathinones continue to emerge in recreational drug markets worldwide. 1-(1,3-Benzodioxol-5-yl)-2-(methylamino)butan-1-one (butylone) and 1-(1,3-benzodioxol-5-yl)-2-(methylamino)pentan-1-one (pentylone) are derivatives of the cathinone compound, 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), that are being detected in drug products and human casework.

Objectives: The purpose of the present study was to examine the neuropharmacology of butylone and pentylone using in vitro and in vivo methods.

Methods: In vitro uptake and release assays were carried out in rat brain synaptosomes and in cells expressing human dopamine transporters (DAT) and 5-HT transporters (SERT). In vivo microdialysis was performed in the nucleus accumbens of conscious rats to assess drug-induced changes in neurochemistry.

Results: Butylone and pentylone were efficacious uptake blockers at DAT and SERT, though pentylone was more DAT-selective. Both drugs acted as transporter substrates that evoked release of [H]5-HT at SERT, while neither evoked release at DAT. Consistent with the release data, butylone and pentylone induced substrate-associated inward currents at SERT but not DAT. Administration of butylone or pentylone to rats (1 and 3 mg/kg, i.v.) increased extracellular monoamines and motor activity, but pentylone had weaker effects on 5-HT and stronger effects on motor stimulation.

Conclusions: Our data demonstrate that increasing the α-carbon chain length of methylone creates "hybrid" transporter compounds which act as DAT blockers but SERT substrates. Nevertheless, butylone and pentylone elevate extracellular dopamine and stimulate motor activity, suggesting both drugs possess significant risk for abuse.
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http://dx.doi.org/10.1007/s00213-018-5075-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476708PMC
March 2019

"Polytox" synthetic cathinone abuse: A potential role for organic cation transporter 3 in combined cathinone-induced efflux.

Neurochem Int 2019 02 21;123:7-12. Epub 2018 Sep 21.

Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Waehringer Straße 13A, 1090, Vienna, Austria; Center for Addiction Research and Science, Medical University Vienna, Waehringerstrasse 13 A, 1090, Vienna, Austria. Electronic address:

Synthetic cathinone derivatives are a new class of psychoactive substances (NPS), also known as "bath salts", designed to exert psychostimulant effects resembling those of well-known psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). As major constituents of bath salts, the cathinone derivatives 3,4-methylenedioxypyrovalerone (MDPV) and 4-methylmethcathinone (mephedrone), have received considerable media attention. MDPV and mephedrone interfere with the function of the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT), resulting in increased extracellular levels of these monoamines, though their mechanism of action differs. MDPV acts as a non-transported inhibitor of DAT, NET and SERT, whereas mephedrone promotes transporter-mediated release in an amphetamine-like fashion. MDPV and mephedrone are often taken together, creating a conundrum in as much as non-transported inhibitors, like MDPV, prevent mephedrone-induced reverse transport via DAT, NET and SERT. Here we provide evidence supporting a role for organic cation transporter 3 (OCT3) in the actions of mephedrone, which may account for its ability to enhance effects of MDPV. We show that mephedrone can induce substrate efflux via OCT3 in the presence of MDPV. Real-time recordings of the fluorescent OCT3 substrate (4-(4-dimethylamino)styryl)-N-methylpyridinium (ASP) and radiotracer-flux studies using [H]1-methyl-4-phenyl-pyridinium (MPP), demonstrated that OCT3 is MDPV-insensitive when expressed in human embryonic kidney (HEK293) cells. Ex vivo experiments performed in cultured superior cervical ganglia (SCG) cells, rich in NET and OCT3, revealed that mephedrone induces [H]MPP release in an OCT3-dependent manner when NET is fully occupied with MDPV. These results extend our recent findings that OCT3 is key in the mechanism of action of amphetamine-induced substrate release. OCT3 likewise appears to be a mechanism through which mephedrone can induce release of monoamines, thereby accounting for the paradoxically more potent psychostimulant effects of MDPV taken together with mephedrone, and greater risk for deleterious side effects.
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http://dx.doi.org/10.1016/j.neuint.2018.09.008DOI Listing
February 2019

The psychostimulant (±)-cis-4,4'-dimethylaminorex (4,4'-DMAR) interacts with human plasmalemmal and vesicular monoamine transporters.

Neuropharmacology 2018 08 23;138:282-291. Epub 2018 Jun 23.

Medical University of Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Währingerstraße 13A, 1090, Vienna, Austria; Center for Addiction Research and Science, Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria. Electronic address:

(±)-cis-4,4'-Dimethylaminorex (4,4'-DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4'-DMAR on human high-affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4'-DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4'-DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC values < 2 μM). Release assays identified 4,4'-DMAR as a substrate type releaser, capable of inducing transporter-mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4'-DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4-methylenedioxymethylamphetamine (MDMA). This study identified 4,4'-DMAR as a potent non-selective monoamine releasing agent. In contrast to the known effects of aminorex and 4-methylaminorex, 4,4'-DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4'-DMAR may result in long-term neurotoxicity.
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http://dx.doi.org/10.1016/j.neuropharm.2018.06.018DOI Listing
August 2018

Identification and characterization of the Fasciola hepatica sodium- and chloride-dependent taurine transporter.

PLoS Negl Trop Dis 2018 04 27;12(4):e0006428. Epub 2018 Apr 27.

Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria and Gaston H. Glock Research Laboratories for Exploratory Drug Development, Vienna, Austria.

The parasitic liver fluke Fasciola hepatica infests mainly ruminants, but it can also cause fasciolosis in people, who ingest the metacercariae encysted on plants. The drug of choice to treat fasciolosis is triclabendazole (TBZ), which has been on the market for several decades. This is also true for the other available drugs. Accordingly, drug-resistant flukes have been emerging at an increasing rate making it desirable to identify alternative drug targets. Here, we focused on the fact that adult F. hepatica persists in the hostile environment of the bile ducts of infected organisms. A common way to render bile acids less toxic is to conjugate them to taurine (2-aminoethanesulfonic acid). We cloned a transporter from the solute carrier-6 (SLC6) family, which was most closely related to the GABA-transporter-2 of other organisms. When heterologously expressed, this F. hepatica transporter supported the high-affinity cellular uptake of taurine (KM = 12.0 ± 0.5 μM) but not of GABA. Substrate uptake was dependent on Na+- and Cl- (calculated stoichiometry 2:1). Consistent with the low chloride concentration in mammalian bile, the F. hepatica transporter had a higher apparent affinity for Cl- (EC50 = 14±3 mM) than the human taurine transporter (EC50 = 55±7 mM). We incubated flukes with unconjugated bile acids in the presence and absence of taurine: taurine promoted survival of flukes; the taurine transporter inhibitor guanidinoethansulfonic acid abolished this protective effect of taurine. Based on these observations, we conclude that the taurine transporter is critical for the survival of liver flukes in the bile. Thus, the taurine transporter represents a candidate drug target.
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http://dx.doi.org/10.1371/journal.pntd.0006428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942844PMC
April 2018

Cocaine adulteration.

J Chem Neuroanat 2017 Oct 12;83-84:75-81. Epub 2017 Jun 12.

Medical University Vienna, Center for Physiology and Pharmacology, Institute of Pharmacology, Waehringerstrasse 13a, 1090 Vienna, Austria; Center for Addiction Research and Science - Medical University Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria. Electronic address:

Cocaine is a naturally occurring and illicitly used psychostimulant drug. Cocaine acts at monoaminergic neurotransmitter transporters to block uptake of the monoamines, dopamine, serotonin and norepinephrine. The resulting increase of monoamines in the extracellular space underlies the positively reinforcing effects that cocaine users seek. In turn, this increase in monoamines underlies the development of addiction, and can also result in a number of severe side effects. Currently, cocaine is one of the most common illicit drugs available on the European market. However, cocaine is increasingly sold in impure forms. This trend is driven by cocaine dealers seeking to increase their profit margin by mixing ("cutting") cocaine with numerous other compounds ("adulterants"). Importantly, these undeclared compounds put cocaine consumers at risk, because consumers are not aware of the additional potential threats to their health. This review describes adulterants that have been identified in cocaine sold on the street market. Their typical pharmacological profile and possible reasons why these compounds can be used as cutting agents will be discussed. Since a subset of these adulterants has been found to exert effects similar to cocaine itself, we will discuss levamisole, the most frequently used cocaine cutting agent today, and its metabolite aminorex.
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http://dx.doi.org/10.1016/j.jchemneu.2017.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610562PMC
October 2017

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

Curr Top Behav Neurosci 2017;32:333-350

Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University Vienna, Waehringerstrasse 13a, A-1090, Vienna, Austria.

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!."
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http://dx.doi.org/10.1007/7854_2016_63DOI Listing
July 2017

Combined Simulation and Mutation Studies to Elucidate Selectivity of Unsubstituted Amphetamine-like Cathinones at the Dopamine Transporter.

Mol Inform 2017 05 8;36(5-6). Epub 2016 Nov 8.

University of Vienna, Department of Pharmaceutical Chemistry, Althanstrasse 14, 1090, Vienna, Austria.

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.
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http://dx.doi.org/10.1002/minf.201600094DOI Listing
May 2017

A Novel Heterocyclic Compound CE-104 Enhances Spatial Working Memory in the Radial Arm Maze in Rats and Modulates the Dopaminergic System.

Front Behav Neurosci 2016 22;10:20. Epub 2016 Feb 22.

Department of Pharmaceutical Chemistry, University of Vienna Vienna, Austria.

Various psychostimulants targeting monoamine neurotransmitter transporters (MATs) have been shown to rescue cognition in patients with neurological disorders and improve cognitive abilities in healthy subjects at low doses. Here, we examined the effects upon cognition of a chemically synthesized novel MAT inhibiting compound 2-(benzhydrylsulfinylmethyl)-4-methylthiazole (named as CE-104). The efficacy of CE-104 in blocking MAT [dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter] was determined using in vitro neurotransmitter uptake assay. The effect of the drug at low doses (1 and 10 mg/kg) on spatial memory was studied in male rats in the radial arm maze (RAM). Furthermore, the dopamine receptor and transporter complex levels of frontal cortex (FC) tissue of trained and untrained animals treated either with the drug or vehicle were quantified on blue native PAGE (BN-PAGE). The drug inhibited dopamine (IC50: 27.88 μM) and norepinephrine uptake (IC50: 160.40 μM), but had a negligible effect on SERT. In the RAM, both drug-dose groups improved spatial working memory during the performance phase of RAM as compared to vehicle. BN-PAGE Western blot quantification of dopamine receptor and transporter complexes revealed that D1, D2, D3, and DAT complexes were modulated due to training and by drug effects. The drug's ability to block DAT and its influence on DAT and receptor complex levels in the FC is proposed as a possible mechanism for the observed learning and memory enhancement in the RAM.
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http://dx.doi.org/10.3389/fnbeh.2016.00020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761905PMC
March 2016

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

Angew Chem Int Ed Engl 2016 01 22;55(5):1719-22. Epub 2015 Dec 22.

Institute for Biophysics, Johannes Kepler University Linz, Gruberstrasse 40, 4020, Linz, Austria.

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+) -containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.
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http://dx.doi.org/10.1002/anie.201508755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932834PMC
January 2016

Binding Mode Selection Determines the Action of Ecstasy Homologs at Monoamine Transporters.

Mol Pharmacol 2016 Jan 30;89(1):165-75. Epub 2015 Oct 30.

Institute of Pharmacology, Center for Physiology and Pharmacology (W.S., T.Sto., P.S.H., M.H., T.Ste., H.H.S.) and Center for Addiction Research and Science (H.H.S.), Medical University of Vienna, Vienna, Austria; Designer Drug Research Unit (J.S.P., M.H.B.) and Medicinal Chemistry Section (J.J.C., A.H.N.), Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Baltimore, Maryland; Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria (A.S., G.F.E.); and Department of Pharmacology, Yale University, New Haven, Connecticut (Y.-W.Z., G.R.)

Determining the structural elements that define substrates and inhibitors at the monoamine transporters is critical to elucidating the mechanisms underlying these disparate functions. In this study, we addressed this question directly by generating a series of N-substituted 3,4-methylenedioxyamphetamine analogs that differ only in the number of methyl substituents on the terminal amine group. Starting with 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N,N-dimethylamphetamine (MDDMA) and 3,4-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) were prepared. We evaluated the functional activities of the compounds at all three monoamine transporters in native brain tissue and cells expressing the transporters. In addition, we used ligand docking to generate models of the respective protein-ligand complexes, which allowed us to relate the experimental findings to available structural information. Our results suggest that the 3,4-methylenedioxyamphetamine analogs bind at the monoamine transporter orthosteric binding site by adopting one of two mutually exclusive binding modes. 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy-N-methylamphetamine adopt a high-affinity binding mode consistent with a transportable substrate, whereas MDDMA and MDTMA adopt a low-affinity binding mode consistent with an inhibitor, in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes, whereas MDTMA exclusively binds to the low-affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation.
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http://dx.doi.org/10.1124/mol.115.101394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702095PMC
January 2016

A heterocyclic compound CE-103 inhibits dopamine reuptake and modulates dopamine transporter and dopamine D1-D3 containing receptor complexes.

Neuropharmacology 2016 Mar 25;102:186-96. Epub 2015 Sep 25.

Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria. Electronic address:

A series of compounds have been reported to enhance memory via the DA system and herein a heterocyclic compound was tested for working memory (WM) enhancement. 2-((benzhydrylsulfinyl)methyl)thiazole (CE-103) was synthesized in a six-step synthesis. Binding of CE-103 to the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters and dopamine reuptake inhibition was tested as well as blood brain permeation and a screen for GPCR targets. 60 male Sprague Dawley rats were divided into six groups: CE-103 treated 1-10 mg/kg body weight, trained (TDI) and yoked (YDI) and vehicle treated, trained (TVI) and yoked (YVI) rats. Daily single intraperitoneal injections for a period of 10 days were administered and rats were tested in a radial arm maze (RAM). Hippocampi were taken 6 h following the last day of training and complexes containing the unphosphorylated or phosphorylated dopamine transporter (DAT) and complexes containing the D1-3 dopamine receptor subunits were determined. CE-103 was binding to the DAT but insignificantly to SERT or NET and dopamine reuptake was blocked specifically (IC50 = 14.73 μM). From day eight the compound was decreasing WM errors in the RAM significantly at both doses tested as compared to the vehicle controls. In the trained CE-103-treated group levels of the complex containing the phosphorylated dopamine transporter (pDAT) as well as D1R were decreased while levels of complexes containing D2R and D3R were significantly increased. CE-103 was shown to enhance spatial WM and DA reuptake inhibition with subsequent modulation of D1-3 receptors is proposed as a possible mechanism of action.
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http://dx.doi.org/10.1016/j.neuropharm.2015.07.039DOI Listing
March 2016

A binding mode hypothesis of tiagabine confirms liothyronine effect on γ-aminobutyric acid transporter 1 (GAT1).

J Med Chem 2015 Mar 26;58(5):2149-58. Epub 2015 Feb 26.

University of Vienna , Department of Pharmaceutical Chemistry, Division of Drug Design and Medicinal Chemistry, Althanstraße 14, 1090 Vienna, Austria.

Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand-transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure-activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone.
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http://dx.doi.org/10.1021/jm5015428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4360375PMC
March 2015

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

Neuropsychopharmacology 2015 May 15;40(6):1321-31. Epub 2014 Dec 15.

Designer Drug Research Unit (DDRU), Intramural Research Program (IRP), NIDA, NIH, Baltimore, MD, USA.

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.
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http://dx.doi.org/10.1038/npp.2014.325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397398PMC
May 2015

Missense dopamine transporter mutations associate with adult parkinsonism and ADHD.

J Clin Invest 2014 Jul 9;124(7):3107-20. Epub 2014 Jun 9.

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.
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http://dx.doi.org/10.1172/JCI73778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071392PMC
July 2014

Aminorex, a metabolite of the cocaine adulterant levamisole, exerts amphetamine like actions at monoamine transporters.

Neurochem Int 2014 Jul 1;73:32-41. Epub 2013 Dec 1.

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringerstrasse 13A, 1090 Vienna, Austria.

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 30μM, 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".
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http://dx.doi.org/10.1016/j.neuint.2013.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077236PMC
July 2014

Probing the Selectivity of Monoamine Transporter Substrates by Means of Molecular Modeling.

Mol Inform 2013 Jun 12;32(5-6):409-413. Epub 2013 Jun 12.

University of Vienna, Department of Medicinal Chemistry, Pharmacoinformatics Research Group Vienna, Austria.

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http://dx.doi.org/10.1002/minf.201300013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743209PMC
June 2013

Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.

Proc Natl Acad Sci U S A 2013 Jul 24;110(28):11642-7. Epub 2013 Jun 24.

Center of Physiology and Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
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http://dx.doi.org/10.1073/pnas.1220552110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710838PMC
July 2013

A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content.

ACS Chem Neurosci 2013 Jan 3;4(1):182-90. Epub 2012 Dec 3.

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Waehringer Strasse 13a, A-1090 Vienna, Austria.

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.
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http://dx.doi.org/10.1021/cn3001763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3547486PMC
January 2013

Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a principal constituent of psychoactive 'bath salts' products.

Neuropsychopharmacology 2013 Mar 17;38(4):552-62. Epub 2012 Oct 17.

Medicinal Chemistry Section of the Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA.

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 [(3)H]dopamine (IC(50)=4.1 nM) and [(3)H]norepinephrine (IC(50)=26 nM) with high potency but has weak effects on uptake of [(3)H]serotonin (IC(50)=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.
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http://dx.doi.org/10.1038/npp.2012.204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3572453PMC
March 2013

Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux.

J Biol Chem 2012 Aug 21;287(35):29702-12. Epub 2012 Jun 21.

Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037, USA.

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 Thr(53). In this report, we use mass spectrometry and a newly developed phospho-specific antibody to positively identify DAT phosphorylation at Thr(53) in rodent striatal tissue and heterologous expression systems. Basal phosphorylation of Thr(53) 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 Thr(53) to prevent phosphorylation led to reduced dopamine transport V(max) and total apparent loss of amphetamine-stimulated substrate efflux, supporting a major role for this residue in the transport kinetic mechanism.
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http://dx.doi.org/10.1074/jbc.M112.367706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436161PMC
August 2012

Probing binding pocket of serotonin transporter by single molecular force spectroscopy on living cells.

J Biol Chem 2012 Jan 27;287(1):105-113. Epub 2011 Oct 27.

Institute for Biophysics, Johannes Kepler University Linz, Altenbergerstrasse 69, 4040 Linz, Austria; Center for Advanced Bioanalysis, Scharitzerstrasse 6-8, 4020 Linz, Austria. Electronic address:

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.
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http://dx.doi.org/10.1074/jbc.M111.304873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249061PMC
January 2012

The N terminus of monoamine transporters is a lever required for the action of amphetamines.

J Biol Chem 2010 Apr 29;285(14):10924-38. Epub 2010 Jan 29.

Center of Biomolecular Medicine and Pharmacology, Institute of Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.

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 SERT(T81A) and SERT(T81D), 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 SERT(T81A) in the inward conformation, its reduced turnover number, and a larger distance between its N and C termini) were verified. Most importantly, SERT(T81A) (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 SERT(T81A) were comparable with those through the wild type transporter. Both abundant Na(+) entry and accumulation of SERT(T81A) 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.
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http://dx.doi.org/10.1074/jbc.M109.083154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856298PMC
April 2010