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N-Alkylated Analogs of 4-Methylamphetamine (4-MA) Differentially Affect Monoamine Transporters and Abuse Liability

Ernesto Solis Jr1,John S Partilla2,Farhana Sakloth3,Iwona Ruchala4,Kathryn L Schwienteck5,Louis J De Felice4,Jose M Eltit4,Richard A Glennon3,S Stevens Negus5,Michael H Baumann2,*
1In Vivo Electrophysiology Unit, Behavioral Neuroscience Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
2Designer Drug Research Unit, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
3Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, USA
4Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA
5Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA
*

Designer Drug Research Unit (DDRU), IRP, NIDA, NIH, 333 Cassell Drive, Suite 4400, Baltimore, MD 21224, USA, Tel: +1 443 740 2660, Fax: +1 443 740 2241, E-mail:mbaumann@mail.nih.gov

Received 2016 Nov 23; Revised 2017 May 4; Accepted 2017 May 12; Prepublished 2017 May 22; Issue date 2017 Sep.

Copyright © 2017 American College of Neuropsychopharmacology
PMCID: PMC5561352  PMID:28530234

Abstract

Clandestine chemists synthesize novel stimulant drugs by exploiting structural templates known to target monoamine transporters for dopamine, norepinephrine, and serotonin (DAT, NET, and SERT, respectively). 4-Methylamphetamine (4-MA) is an emerging drug of abuse that interacts with transporters, but limited structure–activity data are available for its analogs. Here we employed uptake and release assays in rat brain synaptosomes, voltage-clamp current measurements in cells expressing transporters, and calcium flux assays in cells coexpressing transporters and calcium channels to study the effects of increasingN-alkyl chain length of 4-MA on interactions at DAT, NET, and SERT. In addition, we performed intracranial self-stimulation in rats to understand how the chemical modifications affect abuse liability. All 4-MA analogs inhibited uptake at DAT, NET, and SERT, but lengthening the amine substituent from methyl to ethyl, propyl, and butyl produced a stepwise decrease in potency.N-methyl 4-MA was an efficacious substrate-type releaser at DAT that evoked an inward depolarizing current and calcium influx, whereas other analogs did not exhibit these effects. N-methyl andN-ethyl 4-MA were substrates at NET, whereasN-propyl andN-butyl 4-MA were not. All analogs acted as SERT substrates, thoughN-butyl 4-MA had very weak effects. Intracranial self-stimulation in rats showed that elongating theN-alkyl chain decreased abuse-related effectsin vivo that appeared to parallel reductions in DAT activity. Overall, converging lines of evidence show that lengthening theN-alkyl substituent of 4-MA reduces potency to inhibit transporters, eliminates substrate activity at DAT and NET, and decreases abuse liability of the compounds.

Introduction

New psychoactive substances (NPSs) are synthetic alternatives to more traditional drugs of abuse that are engineered to circumvent drug control laws. Most NPSs that produce stimulant-like behavioral effects are derived from the structure of amphetamine or itsβ-keto analog cathinone (Baumannet al, 2013a;De Feliceet al, 2014;Simmleret al, 2014). Subtle chemical modifications to these structural templates have created a vast number of potentially dangerous stimulants sold via the internet, in brick-and-mortar shops, and by street drug dealers (Baumannet al, 2014b). Like traditional stimulants, newly emerging stimulants interact with plasma membrane transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT) to increase synaptic concentrations of these neurotransmitters in the brain. Drugs targeting transporters can be divided into two types based on their mode of action: (1) ‘inhibitors’ bind to the orthosteric site on the transporter to block neurotransmitter uptake, whereas (2) ‘substrates’ bind to the orthosteric site, are subsequently transported into cells, and evoke release of neurotransmitters by reverse transport (Kahliget al, 2005;Khoshboueiet al, 2003;Rothman and Baumann, 2006;Scholzeet al, 2000). Importantly, transporter substrates and inhibitors can be discriminated based on their electrophysiological signatures, whereby substrates induce transporter-mediated inward sodium currents and inhibitors induce an apparent outward current (Cameronet al, 2013;Solis, 2017;Soliset al, 2012). Evidence shows that transporter-mediated inward currents and subsequent accumulation of substrate-type drugs in cells may be responsible for producing intracellular deficits in monoamine neurons such as inhibition of neurotransmitter synthesis and disruption of vesicular storage, leading to long-term neurotransmitter depletions (Baumannet al, 2014a;Fleckensteinet al, 2007).

4-Methylamphetamine (4-MA) is an example of a stimulant-like NPS that was resurrected from older biomedical literature. In the 1950s, 4-MA was investigated as a potential anorectic medication, but its development was abandoned before reaching the clinical market. 4-MA remained in relative obscurity until 2009, when it first appeared on the European street drug market in powders mixed with amphetamine and caffeine, being sold as ‘speed’ (EMCDDA, 2012). Since that time, 4-MA has been associated with many serious intoxications and fatalities in European countries (Blanckaertet al, 2013;EMCDDA, 2014). Preclinical studies from our laboratory and others show that 4-MA is a nonselective transporter substrate capable of releasing dopamine and serotonin from neuronsin vitro andin vivo (Baumannet al, 2011;Weeet al, 2005). When compared with the parent drug amphetamine, 4-MA has much greater potency as a substrate at SERT, and a recent study confirms that locomotor and hypothermic effects produced by 4-MA in rats depend upon serotonergic systems (Rubioet al, 2015). Older literature indicates that 4-MA is also a potent inhibitor of monoamine oxidase (MAO) A, the principal enzyme responsible for the breakdown of monoamine neurotransmitters (Rosset al, 1977).

To date, few structure–activity studies have examined the pharmacology of 4-MA analogs. In the present investigation, we examined the effects of lengthening the amine substituent of 4-MA on drug interactions at DAT, NET, and SERT. We focused on the amine group because it is a primary site for modification of NPS based on the amphetamine and cathinone templates (Rothmanet al, 2012;Sahaet al, 2015;Simmleret al, 2014). In a representative study,Sahaet al (2015) showed that elongating the amine substituent of 4-methyl-N-methylcathinone (mephedrone) by one carbon, which forms 4-methyl-N-ethylcathinone (4-MEC), produces marked changes in pharmacology. Whereas mephedrone is a nonselective transporter substrate, 4-MEC is an inhibitor at DAT but a substrate at SERT. Thus, 4-MEC displays so-called ‘hybrid’ transporter activity, characterized by a different molecular mechanism at DATvs SERT. Here we synthesizedN-methyl,N-ethyl,N-propyl, andN-butyl analogs of 4-MA (seeSupplementary Figure 1), and hypothesized that increasingN-alkyl chain length would generate hybrid transporter ligands (Bloughet al, 2014;Sahaet al, 2015). The mechanism of transporter activity for 4-MA analogs was evaluated using a variety ofin vitro methods including uptake and release assays in rat brain synaptosomes, measurement of ionic currents in cells expressing monoamine transporters, and detection of calcium influx in cells coexpressing transporters and calcium channels. The effects of 4-MA analogs were also testedin vivo using intracranial self-stimulation (ICSS) in rats to measure abuse-related and abuse-limiting effects of drugs (Negus and Miller, 2014). Overall, our findings demonstrate that increasing the length of theN-alkyl substituent of 4-MA causes a decrease in potency to inhibit monoamine transporters, a loss of substrate activity at DAT and NET, and a profound reduction in the rewarding effects of the compounds.

Materials and methods

Drugs and Reagents

N-methyl 4-MA was prepared as its hydrobromide salt, whereasN-ethyl,N-propyl, andN-butyl analogs of 4-MA were synthesized as their hydrochloride salts and analytically characterized to ensure purity. Their syntheses and characterization are provided in theSupplementary Methods section. [3H]5-HT (specific activity=30 Ci/mmol) was purchased from Perkin Elmer (Shelton, CT). [3H]1-Methyl-4-phenylpyridinium ([3H]MPP+, specific activity=85 Ci/mmol) was purchased from American Radiolabeled Chemicals (St Louis, MO). All other chemicals and reagents were acquired from Sigma-Aldrich (St Louis, MO) unless otherwise noted. For the ICSS studies, compounds were dissolved in 0.9% sterile saline and administered intraperitoneally (i.p.) at a volume of 1 ml/kg. Drug doses are expressed as the salt forms mentioned above.

Animals

Animal facilities were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, and all animal procedures were carried out in accordance with the Institutional Animal Care and Use Committees and the National Institutes of Health guidelines on care and use of animal subjects (National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals.et al, 2011).

Adult male Sprague-Dawley rats (Harlan, Frederick, MD) were used for the synaptosome assays (rats weighing 250–350 g) and for the ICSS procedures (rats weighing 313–360 g at the time of surgery). Rats were group housed with free access to food and water, under a 12 h light/dark cycle with lights on from 0700 to 1900 h. Oocytes for the electrophysiology experiments were harvested and prepared from adult femaleXenopus laevis females following standard procedures (Hatcher-Soliset al, 2014;Korenet al, 1990).

Transporter Uptake and Release in Rat Brain Synaptosomes

Rats were killed by CO2 narcosis, and synaptosomes were prepared from brains as previously described (Rothmanet al, 2003). Synaptosomes were prepared from caudate tissue for DAT assays, whereas synaptosomes were prepared from whole brain minus caudate and cerebellum for NET and SERT assays. For uptake inhibition assays, 5 nM [3H]dopamine, 10 nM [3H]norepinephrine, and 5 nM [3H]5-HT were used for DAT, NET, and SERT, respectively. To optimize uptake for a single transporter, unlabeled blockers were included that prevented uptake of [3H]transmitter by competing transporters. Uptake inhibition was initiated by incubating synaptosomes with test compound and [3H]transmitter in Krebs-phosphate buffer. Uptake assays were terminated by rapid vacuum filtration and retained radioactivity was quantified with liquid scintillation counting (Baumannet al, 2013b). For release assays, 9 nM [3H]MPP+ was used as the radiolabeled substrate for DAT and NET, whereas 5 nM [3H]5-HT was used for SERT. All buffers used in the release assay contained 1 μM reserpine to block vesicular uptake of substrates. The selectivity of release assays was optimized for a single transporter by including unlabeled blockers to prevent the uptake of [3H]MPP+ or [3H]5-HT by competing transporters. Synaptosomes were preloaded with radiolabeled substrate in Krebs-phosphate buffer for 1 h. Release assays were initiated by incubating preloaded synaptosomes and test drug. Release was terminated by vacuum filtration and retained radioactivity was quantified by liquid scintillation counting. Effects of test drugs on release were expressed as % maximum release, with maximum release (ie, 100% Emax) defined as the release produced by tyramine at doses that evoke the efflux of all ‘releasable’ tritium by synaptosomes (10 nM tyramine for DAT and NET assay conditions, and 100 nM tyramine for SERT assay conditions). Effects of test drugs on uptake inhibition and release were analyzed by nonlinear regression using GraphPad Prism 6 (GraphPad Scientific, San Diego, CA). Dose–response values for the uptake inhibition and release were fit to the equation,Y(x)=Ymin+(Ymax –Ymin) / (1+ 10exp[(logP50 – logx)] ×n), wherex is the concentration of the compound tested,Y(x) is the response measured,Ymax is the maximal response,P50 is either IC50 (the concentration that yields half-maximal uptake inhibition response) or EC50 (the concentration that yields half-maximal release), andn is the Hill slope parameter.

Transporter-Mediated Ionic Currents in DAT- or SERT-Expressing Oocytes

AdultX. laevis females were anesthetized with 1.5 mg/ml tricaine and 1.5 mg/ml NaHCO3. Oocytes were removed and digested at 22 °C with collagenase type 2 (4 mg/ml, Worthington Biochemical Corporation, Lakewood, NJ), and dissolved in OR2 solution (in mM: 85 NaCl, 5 HEPES, 5 KCl, 5 NaOH, 1 MgCl2-6H2O, pH 7.4) until oocytes were defolliculated. Stage V–VI oocytes were selected for transporter cRNA injection within 24 h of isolation. The cRNA was transcribed from the pOTV vector with the mMessage Machine T7 kit (Ambion, Austin, TX) and oocytes were injected with 18–41.4 ng of either hDAT or hSERT cRNA (Nanoject AutoOocyteInjector, Drummond Scientific, Broomall, PA) and incubated at 18 °C for 4–12 days in Ringers solution supplemented with NaPyruvate (550 μg/ml), streptomycin (100 μg/ml), tetracycline (50 μg/ml), and 5% dialyzed horse serum. All two-electrode voltage-clamp (TEVC) recordings were performed at room temperature (23–25 °C). Electrodes with resistances from 1 to 5 MΩ were filled with 3 M KCl. As previously reported (Rodriguez-Menchacaet al, 2012;Soliset al, 2012),X. laevis oocytes expressing hSERT or hDAT were voltage-clamped to –60 mV and the holding current was recorded using a GeneClamp 500 TEVC amplifier (Axon Instruments, Sunnyvale, CA) and the Clampex 10 software (Molecular Devices, Sunnyvale, CA). External solution consisted of (in mM): 120 NaCl, 7.5 HEPES, 5.4 K Gluconate, 1.2 Ca Gluconate, pH 7.4. Compounds were perfused for durations indicated by solid horizontal lines on traces.

Intracellular Ca2+ Flux in Cells Coexpressing Monoamine Transporters and Cav Channels

The stable HEK cell lines expressing hDAT or hSERT were previously described (Cameronet al, 2015;Ruchalaet al, 2014), whereas cells expressing hNET (accession numberM65105.1) were created using the Flp-In T-REx 293 expression system (Life Technologies, Carlsbad, CA). Cells expressing hDAT, hNET, or hSERT were plated in 96-well imaging plates and were cotransfected with the cardiac isoform of the voltage-gated Ca2+ channel, CaV1.2, its auxiliary subunits, and the EGFP expression plasmid (as the transfection marker) in the following proportion: α1:β3:α2δ1:EGFP=1:1:1:0.2. Ca2+ measurements were performed 3 days after induction. Acquisition of intracellular Ca2+ signals was performed using the Ca2+-sensitive dye Fura-2AM and visualized in an epifluorescence microscope following procedures described previously (Cameronet al, 2015;Ruchalaet al, 2014). In all experiments, a 5 s pulse of dopamine, norepinephrine, and 5HT at a saturating concentration (5–10 μM) was used as the internal positive control for DAT, NET, and SERT, respectively. Peak Ca2+ signals induced by test compounds that behaved as substrates were normalized to the peak signal of the positive control. For blockers, the peak signal induced by the transporter’s endogenous transmitter mixed with the test drug was normalized to the peak value of the positive control signal in the absence of the test drug. Dose–response values for drugs eliciting transporter-mediated Ca2+ signals and for drugs inhibiting Ca2+-evoked responses by transmitters were fit to the equationY(x)=Ymin+(Ymax –Ymin) / (1+ 10exp[(logP50 – logx)] ×n) using GraphPad Prism 6, wherex is the concentration of the compound tested,Y(x) is the response measured,Ymax is the maximal response,P50 is either EC50 (the concentration that yields half-maximal Ca2+ response) or IC50 (the concentration yielding half-maximal inhibition of the transmitter-elicited Ca2+ signal), andn is the Hill slope parameter. All Ca2+ data are presented as mean±SEM, except in Figures 3c–e that are presented as mean±SD.

Intracranial Self-Stimulation in Rats

ICSS studies were conducted as described previously (Bonanoet al, 2015; Negus and Miller, 2014). Briefly, rats were anesthetized with 3% isoflurane (Webster Veterinary, Phoenix, AZ) and stainless steel electrodes (Plastics One, Roanoke, VA) were stereotaxically implanted into the left medial forebrain bundle and fixed to the skull (Bonanoet al, 2015; Negus and Miller, 2014). Ketoprofen (5 mg/kg) was used for postoperative analgesia, and animals were single-housed after surgery for at least 7 days of recovery. After recovery, rats were trained to press a lever for pulses of electrical brain stimulation under a frequency-rate procedure (frequencies of 1.75–2.2 log Hz, in ten 0.05 log increments). The drugs and doses tested wereN-methyl 4-MA (0.1–3.2 mg/kg),N-ethyl 4-MA (0.32–3.2 mg/kg),N-propyl 4-MA (3.2–32 mg/kg), andN-butyl 4-MA (0.32–10 mg/kg). The effects of vehicle (0.9% sterile saline) were determined before and after each drug. Raw reinforcement rates during availability of each brain stimulation frequency were normalized to percent maximum control rate (%MCR) within each rat on each test day, and then averaged across rats. Group mean frequency-rate curves were analyzed by repeated measures two-way ANOVA with ICSS frequency and drug dose as factors. A significant ANOVA was followed by Holm–Sidak multiple comparisonspost hoc test, and the criterion for statistical significance wasP<0.05. As an additional summary measure of drug effects on ICSS, the total number of stimulations delivered across all frequencies was also calculated and normalized to baseline data for each drug dose in each rat, and then averaged across rats in each experimental condition.

Results

Uptake and Release Assays in Rat Brain Synaptosomes

Figure 1 depicts the dose–response effects ofN-substituted 4-MA analogs on uptake inhibition and release at DAT, NET, and SERT in rat brain synaptosomes.Table 1 summarizes drug potencies for uptake inhibition (IC50 values) and release (EC50 values). In general, the potency forN-substituted 4-MA analogs to inhibit uptake at DAT, NET, and SERT decreased in a stepwise manner asN-alkyl chain length increased beyond a methyl group (Figures 1a–c). The potency forN-butyl 4-MA at SERT was so weak that an IC50 value could not be determined. The potency for 4-MA analogs to promote release also decreased as theN-substitution was elongated. However, the most dramatic effect of increasingN-alkyl chain length on releasing activity was reduction in efficacy (ie, flattening of the dose–response curves), especially at DAT and NET (Figures 1d–f). AlthoughN-methyl 4-MA elicited maximal release efficacy at DAT, the release efficacies ofN-ethyl,N-propyl, andN-butyl analogs at DAT were 58%, 20%, and 25% of maximal release, respectively. In a similar manner,N-methyl andN-ethyl 4-MA produced maximal release at NET, butN-propyl andN-butyl displayed reduced efficacies of 57% and 23% of maximal, respectively. Finally, all of the analogs produced fully efficacious release at SERT, with the exception ofN-butyl 4-MA that produced no observable effects, perhaps because of its low potency at SERT. The data from synaptosomes provided evidence that theN-ethyl andN-propyl analogs of 4-MA might display hybrid transporter activity, acting as inhibitors at DAT but substrates at SERT. Definitive determination of inhibitorvs substrate activity of 4-MA analogs required additional approaches examining transporters in cells.

Figure 1.

Figure 1

Effects ofN-substituted 4-MA analogs on monoamine uptake and release in rat brain synaptosomes. Top panels (a–c) show drug effects on uptake inhibition. Synaptosomes were incubated with different concentrations of theN-substituted 4-MA analogs in the presence of 5 nM [3H]DA (a, for DAT), 10 nM [3H]NE (b, for NET), or 5 nM [3H]5HT (c, for SERT) and the effect on uptake was plotted as a percent of maximal uptake. Bottom panels (d–f) show drug effects on monoamine release. Synaptosomes were preloaded with 9 nM [3H]MPP+ for DAT (d) and NET (e) or 5 nM [3H]5HT for SERT (f), followed by incubation with different concentrations of theN-substituted 4-MA analogs to stimulate release that was plotted as a percent of maximal release elicited by tyramine. EC50 and IC50 values determined from these curves are reported inTable 1. All points show mean±SEM of three experiments.

Table 1. IC50 and EC50 Values (nM) forN-Substituted 4-MA Analogs to Inhibit Uptake or Stimulate Release at Monoamine Transporters in Rat Brain Synaptosomes or to Block Monoamine-Induced Ca2+ Flux or Stimulate Ca2+ Flux in Flp-In T-REx 293 Cells.

 N-methyl 4-MAN-ethyl 4-MAN-propyl 4-MAN-butyl 4-MA
Rat brain synaptosomes
 DAT uptake, IC50910±38795±713064±20813 660±2050
 NET uptake, IC50438±24685±452916±1836009±701
 SERT uptake, IC50171±23166±17984±124*
 DAT release, EC50 (%)41±3.1 (100)550±75 (58)– (20)– (29)
 NET release, EC50 (%)67±4.8 (97)182±11 (90)742±98 (57)– (23)
 SERT release, EC50 (%)67±7.8 (100)102±9.0 (92)650±51 (86)– (0)
     
Cells coexpressing transporters/channels
 DAT blockade, IC50 4353±61218 260±360060 960±12 640
 NET blockade, IC50  14 350±356030 280±11 710
 SERT blockade, IC50   *
 DAT influx, EC50208±21.0   
 NET influx, EC50252±39.6728±184  
 SERT influx, EC50479±104683±902444±388 

All IC50 and EC50 values were derived from data shown inFigures 1 and3. *Indicates an IC50 that could not be calculated. The lower limit of release efficacy is considered 30% of maximal, and no EC50 values were obtained for efficacies below this level (marked as ‘–’). For the transporter/Ca2+ channel assay, either EC50 or IC50 values were calculated for each compound depending on their action on the transporter.

Transporter-Mediated Ionic Currents in Transporter-Expressing Oocytes

We evaluated the electrophysiological effect of the test compounds at hDAT and hSERT inX. laevis oocytes expressing these transporters and voltage-clamped to –60 mV. As noted previously, transporter substrates induce inward depolarizing currents whereas inhibitors induce outward currents (Solis, 2017;Soliset al, 2012).Figure 2 shows the effects of 4-MA analogs on transporter-mediated currents. When examining effects at hDAT,N-methyl 4-MA induced an inward depolarizing current indicative of substrate activity, whereasN-ethyl,N-propyl, andN-butyl analogs induced outward currents consistent with nontransported inhibitor activity (Figures 2a–d). All analogs of 4-MA induced inward currents at SERT, but the magnitude of response varied (Figures 2e–h). SERT-mediated currents forN-methyl 4-MA andN-ethyl 4-MA were much larger than the effect of the endogenous substrate 5-HT, whereasN-propyl 4-MA induced a current similar in magnitude to the 5-HT-induced current.N-butyl 4-MA induced a small but detectable inward current at SERT, characteristic of a substrate. These results show that elongating theN-alkyl chain of 4-MA changes drug mechanism at DAT from substrate to inhibitor, whereas all analogs behave as SERT substrates regardless ofN-alkyl chain length.

Figure 2.

Figure 2

Effect ofN-substituted 4-MA analogs on membrane currents in oocytes transfected with hDAT or hSERT. In voltage-clamped (Vcom=–60 mV) hDAT-expressing oocytes 10 μMN-methyl 4-MA produces an inward current (a), and 10 μMN-ethyl 4-MA (b),N-propyl 4-MA (c), andN-butyl 4-MA (d) display a block of the endogenous hDAT leak current. For comparison, individual traces are adjusted to match the inward current in response to hDAT endogenous substrate DA (5 μM, shown before application of theN-substituted 4-MA analogs) (a–d). In voltage-clamped (Vcom=–60 mV) hSERT-expressing oocytes, allN-substituted 4-MA analogs produce inward currents (e–h); however, 10 μMN-methyl 4-MA (e) andN-ethyl 4-MA (f) display large responses,N-propyl 4-MA displays a moderate response (g), andN-butyl 4-MA (h) produces a small response. For comparison, individual traces are adjusted to match an inward current in response to hSERT endogenous substrate 5HT (5 μM, shown before application of theN-substituted 4-MA analogs) (e–h). Horizontal dotted lines along the baseline (ie, holding current) for hDAT and hSERT recordings are added to compare compound responses.

Transporter-Mediated Depolarization in Cells Coexpressing Transporters and Ca2+ Channels

To further explore the nature of the interactions of 4-MA analogs with monoamine transporters, we employed HEK cells expressing hDAT, hNET, or hSERT in combination with the voltage-gated Ca2+ channel Cav1.2 (Cameronet al, 2015;Ruchalaet al, 2014). In this assay, transporter substrates elicit transporter-mediated depolarizing currents that activate Ca2+ channels, allowing Ca2+ influx and generation of a traceable Ca2+ signal. As transporter inhibitors lack transporter-mediated depolarizing currents, Ca2+ channels will not be activated and no Ca2+ signal will be produced by inhibitors.Figure 3a depicts representative raw data showingN-methyl 4-MA elicits a dose-dependent Ca2+ signal similar to the effects of dopamine that confirms its activity as a DAT substrate.Figure 3b shows thatN-ethyl 4-MA is unable to produce a Ca2+ signal at hDAT, indicating its lack of activity as a substrate, agreeing with the electrophysiological results showing this drug is a nontransported inhibitor at hDAT. Importantly,Figure 3b also demonstrates thatN-ethyl 4-MA is able to inhibit the Ca2+signal produced by dopamine in a dose-dependent manner. Using this assay, we determined whether theN-substituted 4-MA analogs behaved as substrates or inhibitors at the three monoamine transporters. As summarized inFigures 3c–e, transporter substrates produced dose-dependent depolarization-induced Ca2+ signals that are depicted as upward curves, whereas inhibitors produced dose-dependent reductions in Ca2+ signals elicited by endogenous transmitters, and are depicted as downward curves (raw data are shown inSupplementary Figure 2 and potencies are shown inTable 1). For hDAT experiments, elongating theN-alkyl chain greater than anN-methyl group converted the drug mechanism from substrate to inhibitor. Similarly, for hNET, elongating theN-alkyl chain greater than anN-ethyl flipped the drug mechanism from substrate to inhibitor. Importantly,N-alkyl chain length did not alter substrate activity of compounds at SERT. Similar to the hSERT currents observed in oocytes, lengthening theN-alkyl chain of 4-MA only diminished the magnitude of the Ca2+ response.N-butyl 4-MA acted as a substrate but was so weak that a potency measure could not be acquired (Supplementary Figure 2l). It is noteworthy that 4-MA analogs that acted as low-efficacy partial releasers at DAT and NET in rat brain synaptosomes acted as inhibitors in the Ca2+ assays shown here.

Figure 3.

Figure 3

Effects ofN-substituted 4-MA analogs on hDAT-, hNET-, and hSERT-mediated L-type Ca2+ signals in Flp-In T-REx 293 cells. Intracellular Ca2+ levels were determined by fluorescence microscopy using the Ca2+-sensitive dye Fura2AM in Flp-hDAT, Flp-hNET, or Flp-hSERT cells that were cotransfected with Cav1.2 and the β3, α2δ, and EGFP plasmids 3 days before each experiment. (a) In cells coexpressing hDAT and Cav1.2 channels, a Ca2+ signal is produced in response to cell depolarization induced by a depolarizing hDAT-mediated current (produced with 10 μM DA).N-methyl 4-MA (0.03–10 μM) elicits Ca2+ signals of increasing magnitude. (b) In cells coexpressing hDAT and Cav1.2 channels,N-ethyl 4-MA does not produce a Ca2+ signal regardless of concentration, butN-ethyl 4-MA (0.01–30 μM) is able to inhibit DA-induced Ca2+ signals in a dose-dependent manner. (c–e) ForN-substituted 4-MA analogs that behaved as substrates, the dose responses to produce Ca2+ signals were obtained (N-methyl 4-MA at hDAT;N-methyl andN-ethyl 4-MA at hNET; all 4 analogs at hSERT); for analogs that behaved as inhibitors (N-methyl,N-ethyl, andN-propyl 4-MA at hDAT;N-methyl andN-ethyl 4-MA at hNET), the dose responses to inhibit Ca2+ signals induced by the endogenous transmitter were obtained. EC50 values were determined from a two-pulse protocol in which exposure to a fixed concentration of the endogenous transmitter for each transporter (DA, NE, and 5HT for hDAT, hNET, and hSERT, respectively) was followed by a brief exposure to increasing concentrations of anN-substituted 4-MA analog. IC50 values were determined from a two-pulse protocol in which brief exposure of a fixed concentration of the endogenous transmitter for each transporter was followed by exposure to increasing concentrations of theN-substituted 4-MA analogs for 30 s (yielding no response) and coapplication of the endogenous transmitter. Dose–response curves were obtained for both depolarizing and inhibition induced by theN-substituted 4-MA analogs at hDAT, hNET, and hSERT by fitting the responses to the equation described in methods. EC50 and IC50 values for all drug–transporter interactions are summarized inTable 1. The data shown in (a) and (b) are mean±SEM, and in (c–e) are mean±SD.

We next wished to correlate the findings from Ca2+ assays in cells with the data obtained from rat brain synaptosomes. To this end, we first classified 4-MA analogs as either transporter inhibitors or substrates based on the findings from the Ca2+ flux assays; it is noteworthy that using this classification scheme, low-efficacy partial releasers in the synaptosome assays (eg,N-ethyl 4-MA at DAT) are considered transporter inhibitors. For those compounds that behaved as inhibitors or low-efficacy releasers in the rat synaptosome assays and inhibitors in the Ca2+ assays, we found a significant correlation between the IC50 values obtained from both methods (Figure 4a). Similarly, for the compounds that behaved as fully efficacious substrates in the rat synaptosome and Ca2+ assays, we found a significant correlation between the EC50 values obtained from both methods (Figure 4b).

Figure 4.

Figure 4

Correlations of rat synaptosome and fluorometric Ca2+ channel assays. Data used for these correlations were obtained from experiments performed in the rat synaptosome and the fluorometric transporter-Ca2+ channel assays testing the effects ofN-substituted 4-MA analogs on transporters (Figures 1 and 3 andTable 1). (a) Log IC50 values from uptake inhibition in the synaptosome assay are correlated to log IC50 values from the Ca2+ assay. Only values from compound–transporter combinations that elicited clear inhibitor activity in the Ca2+ assay were used. (b) Log EC50 values obtained from the synaptosome release assay are correlated to log EC50 values from the transporter-Ca2+ channel assay. Values used were from compound–transporter combinations that promoted maximal or near-maximal release in the synaptosome assay and evoked prominent Ca2+ signals in response to transporter-mediated depolarization.

Intracranial Self-Stimulation in Rats

As a means to assess the abuse-related effects of 4-MA analogs, we tested these agents in a rat ICSS procedure.Figure 5 shows effects ofN-substituted 4-MA analogs on ICSS in rats. The average±SEM baseline MCR was 62.53±2.48 stimulations per trial, and the mean±SEM number of total baseline stimulations was 296.90±18.40 stimulations per component. After vehicle administration, electrical brain stimulation maintained a frequency-dependent increase in ICSS rates (Figures 5a and d, dotted lines), and frequency-rate curves after vehicle treatment were not different from baseline curves determined before vehicle (data not shown). All fourN-alkylated 4-MA analogs produced dose-dependent changes in ICSS, but both the potency and the extent of abuse-related and presumably dopamine-mediated facilitation of ICSS rates tended to decrease asN-alkyl chain length increased.Figures 5a and e show thatN-methyl 4-MA produced exclusive facilitation of ICSS rates (1.9–1.95 log Hz) at 0.32 mg/kg, whereas a higher dose of 1.0 mg/kg produced mixed effects consisting of facilitation of ICSS rates at low stimulation frequencies (1.8–1.95 log Hz) and depression of ICSS rates at high frequencies (2.15–2.2 log Hz). The highest dose of 3.2 mg/kgN-methyl 4-MA produced only depression of ICSS rates.Figures 5b and f show thatN-ethyl 4-MA produced mixed ICSS effects at 1.0 mg/kg (facilitation of ICSS rates at 1.9–1.95 log Hz, depression of ICSS rates at 2.2 log Hz) but only depression of ICSS rates at 3.2 mg/kg (2.0–2.2 log Hz). BothN-propyl (3.2–32 mg/kg;Figures 5c and g) andN-butyl (0.32–10 mg/kg;Figures 5d and h) analogs produced only dose-dependent depression of ICSS rates across a range of intermediate to high brain stimulation frequencies.

Figure 5.

Figure 5

Effects ofN-substituted 4-MA analogs on ICSS in rats. Left panels (a–d) show full ICSS frequency-rate curves. Abscissae indicate brain stimulation frequency in log Hz. Ordinates indicate percent maximum control response rate (%MCR). Filled points show drug effects significantly different from VEH treatment as determined by two-way ANOVA followed by Holm–Sidakpost hoc test (p<0.05). For all panels, there were significant main effects of frequency (p<0.001) and dose (p<0.001) (data not shown). Frequency–dose interactions were also significant (allp<0.001), and ANOVA results are as follows:N-methyl 4-MA, F(36, 180)=9.91,N-ethyl 4-MA, F(36, 180)=6.38,N-propyl 4-MA, F(36, 180)=7.01, andN-butyl 4-MA, F(36, 180)=2.93. Right panels (e–h) show summary data for the total number of stimulations delivered across all brain-stimulation frequencies. Abscissae indicate drug dose in mg/kg. Ordinates indicate total number of stimulations per component expressed as percent baseline (% baseline total stimulations). Upward/downward arrows indicate that ICSS rates were significantly increased/decreased for at least one brain-stimulation frequency as determined by analysis of full frequency-rate curves in the left panels. All points and bars show mean±SEM in six rats.

Discussion

The main purpose of the present investigation was to examine the effects of lengthening the amine substituent of 4-MA on drug interactions at monoamine transporters and the resulting behavioral consequences. We observed that increasing theN-alkyl chain length of 4-MA fromN-methyl toN-butyl decreased the potency to inhibit uptake at DAT, NET, and SERT in rat brain synaptosomes. Similarly, the potency of substrate activity at monoamine transporters decreased as theN-alkyl group was elongated, but the efficacy to promote release in synaptosomes differed markedly across the three transporters. In general, increasing theN-alkyl chain tended to convert DAT and NET substrates to inhibitors. For instance, when the amine substituent was lengthened fromN-methyl toN-ethyl, release efficacy at DAT dropped from ~100% to ~60% of maximal, respectively.N-propyl andN-butyl analogs displayed <30% releasing efficacy at DAT consistent with the effects of DAT inhibitors. In contrast,N-methyl,N-ethyl, andN-propyl 4-MA displayed maximal or near-maximal release efficacy (80–100% of maximal) at SERT consistent with the profile of substrates.N-butyl 4-MA appeared to display no releasing activity in synaptosomes, but this lack of effect was most likely because of its extremely low potency. The structure–activity findings shown here are consistent with prior data demonstrating that increasing the steric bulk of amine substituents on amphetamine analogs converts substrates at DAT and NET to nontransported inhibitors (Rothmanet al, 2012;Sandtneret al, 2016).

Previous investigations in synaptosomes and transporter-expressing cells have shown that potent uptake inhibitors can induce low-efficacy efflux of preloaded [3H]MPP+ in release assays, in the range of 20–30% of maximal releasing response (Baumannet al, 2013b;Scholzeet al, 2000). Low-efficacy efflux of this magnitude, or ‘pseudoefflux’, is due to the diffusion of [3H]MPP+ out of synaptosomes that is unmasked in the presence of transporter blockade. Under these circumstances, the effects of transporter inhibitors in release assays could be erroneously interpreted asbona fide transporter-mediated reverse transport. Other studies in rat brain synaptosomes have identified transporter ligands exhibiting more substantial ‘partial releasing’ effects, in the range of 50–75% of maximal releasing response, similar to the effects ofN-ethyl 4-MA at DAT observed here (Rothmanet al, 2012;Sandtneret al, 2016). As a specific example, we demonstrated previously that 3,4-methylenedioxyethamphetamine (MDEA), theN-ethyl analog of 3,4-methylenedioxyamphetamine (MDA), induces partial release at DAT with ∼60% efficacy (Rothmanet al, 2012). The precise molecular underpinnings of transporter-mediated partial release are not fully understood, but our prior data provide compelling evidence that the blunted releasing efficacy of MDEA at DAT is because of a much slower rate of [3H]MPP+ efflux (ie, a slower rate of reverse transport) when compared with full-efficacy releasers like MDA. The present findings withN-ethyl 4-MA reveal an important caveat of the synaptosome assays: methods using rat brain tissue cannot definitively discriminate transporter inhibitors from substrates when drugs display partial releasing actions. To further explore the mechanism of action of 4-MA analogs at transporters, we employed additional methods that measure different aspects of transporter activity.

By using the TEVC technique inX. laevis oocytes overexpressing monoamine transporters, we previously showed that transportable substrates produce large Na+-dependent inward currents, whereas nontransported inhibitors do not. In fact, transporter inhibitors evoke an apparent outward current because of blockade of the endogenous transporter ‘leak’ current (Cameronet al, 2013;Rodriguez-Menchacaet al, 2012;Solis, 2017;Soliset al, 2012). As expected, compounds eliciting maximal release efficacy in synaptosomes (eg,N-methyl 4-MA at DAT) also produced large inward currents through hDAT. In contrast,N-ethyl 4-MA displayed outward currents at DAT consistent with a nontransported inhibitor, and elongating toN-propyl orN-butyl 4-MA induced sequentially smaller inhibitor-like currents at DAT. The inhibitor-like outward currents produced byN-ethyl 4-MA in DAT-expressing oocytes might seem at odds with the partial substrate actions of this compound at DAT in synaptosomes. As noted above, partial releasing properties ofN-ethyl analogs like MDEA in synaptosomes are characterized by a substantially slower rate of DAT-mediated efflux when compared with fully efficacious releasers. Thus, one possible explanation for the discordant findings withN-ethyl 4-MA in oocytesvs synaptosomes could be related to the different time courses for each assay. More specifically, the oocyte assays measure transporter-mediated currents on the timescale of seconds, whereas the synaptosome assays measure [3H]substrate efflux on the timescale of minutes. Because of the abbreviated time window of electrophysiological measurements, it seems feasible that only the inhibitor activity of 4-ethyl 4-MA at DAT is observed under the oocyte assay conditions. Other explanations for discrepancies in the data between oocytes and synaptosomes could be the existence of species differences in responsiveness to specific transporter ligands, or intermediate conformational states in transporter proteins that allow some reverse transport in the absence of an inward current. Further investigations are required to address these intriguing possibilities.

In contrast to the divergent effects of 4-MA analogs on DAT-mediated currents, all of the 4-MA analogs produced inward currents consistent with transporter substrates in hSERT-expressing oocytes. AlthoughN-butyl 4-MA displayed no discernable activity at SERT in the synaptosome release assay, it elicited a small inward current through hSERT in oocytes indicative of substrate activity. Although the TEVC technique provides detailed mechanistic information about the action of compounds on transporters, there are significant drawbacks. For instance, hSERT is efficiently expressed in oocytes, whereas hDAT shows significant expression variability among oocyte batches. Moreover, hNET activity is undetectable in oocytes because it does not properly incorporate into oocyte plasma membranes. Thus, the oocyte TEVC system cannot be used for comparison of drug effects across DAT, NET, and SERT.

To overcome limitations of the synaptosome assays and TEVC methods, we employed a fluorometric assay that takes advantage of the electrogenic nature of transporter proteins (Cameronet al, 2015;Ruchalaet al, 2014). This assay consists of HEK cell lines that coexpress either hDAT, hNET, or hSERT along with voltage-gated Ca2+ channels (VGCC) and uses the Fura2 dye to measure changes in Ca2+ influx into individual cells. In recent work, we demonstrated that transporter-mediated currents induced by substrates are strong enough to evoke opening of L-type Ca2+ channels in expression systems where transporters and Ca2+ channels are coexpressed (Cameronet al, 2015;Ruchalaet al, 2014). Because monoamine transporter currents are electrically coupled to the opening of voltage-gated Ca2+ channels, the magnitude of Ca2+ influx is proportional to the concentration of substrate drug applied, allowing for the generation of reliable dose–response curves and potencies of different drugs to be determined (Cameronet al, 2015). Transporter inhibitors do not induce depolarizing currents and do not produce a Ca2+ signal, but they are able to block Ca2+ signals induced by substrates (Cameronet al, 2015;Ruchalaet al, 2014). Using this assay, we first characterized the molecular mechanism of action forN-substituted 4-MA analogs at monoamine transporters, and used these results to classify the compounds as either transporter substrates or inhibitors. We then performed dose–response experiments in which we determined EC50 values for substrate-type drugs and IC50 values for inhibitor-type drugs. Importantly, the compounds that elicited maximal releasing efficacy in synaptosomes and inward currents in oocytes also elicited depolarization-induced Ca2+ responses in the fluorescence assay compounds. In contrast, the compounds that behaved as transporter inhibitors or partial releasers in synaptosomes, and behaved as blockers in the electrophysiological assay, did not elicit Ca2+ signals in the fluorescence assay.N-butyl 4-MA, which lacked measurable potency as a SERT substrate in synaptosomes, induced an inward current in oocytes and a positive response in the fluorometric Ca2+ assay. Importantly, the small outward hDAT current induced byN-butyl 4-MA in the TEVC experiments was substantiated by inhibition of dopamine-induced Ca2+ increases. These results highlight the sensitivity of the Ca2+ fluorescence assay to determine the molecular mechanism of action for compounds at monoamine transporters, even those with weak potency that cannot be effectively characterized in the synaptosome-based assays. It is worth mentioning that the differences in potency across the rat synaptosome and the transporter/Ca2+ channel fluorescence assays are likely because of technical differences in methods employed. For example, the IC50 value for a compound blocking a Ca2+ response elicited by a transmitter depends on the concentration of the transmitter being inhibited (ie, if a lower concentration of transmitter is competed off by a compound, the IC50 value would be more potent).

When we used the results from the transporter/Ca2+ channel assays to classify compounds as either transporter inhibitors or substrates at DAT, NET, and SERT, we observed remarkable correlations between the potencies obtained from the Ca2+ assays and rat synaptosome methods. More specifically, the IC50 values for transporter inhibitors in the Ca2+ fluorescence assays were positively correlated with the IC50 values for transporter inhibitors and partial substrates in the synaptosome uptake inhibition assays. Similarly, the EC50 values for transporter substrates in the Ca2+ fluorescence assays were positively correlated with fully efficacious substrates in the synaptosome assays. Such correlations validate the Ca2+ fluorescence assay as a suitable and complementary technique to study monoamine transporter pharmacology as compared with the established [3H]transmitter uptake inhibition and release assays. In addition, the fluorometric transporter-Ca2+ channel assay has the added advantage to accurately discern the mechanism of action of a compound, possesses high signal-to-noise ratio, and is amenable to studying dose–response effects for many compounds. From the correlation results, we can make the case that monoamine transporters from distinct mammalian species (ratsvs humans) interact similarly with substrates and blockers that differs from drug interactions with nonmammalian monoamine transporters. For example, cocaine has different actions at drosophila DAT and hDAT (Porzgenet al, 2001), and chicken SERT is much less sensitive to reuptake inhibitors than hSERT (Larsenet al, 2004).

Previous studies have identified DATvs SERT selectivity as one important determinant of abuse-related effects of monoamine transporter ligands in ICSS procedures (Baueret al, 2013;Bonanoet al, 2014,2015;Hutsellet al, 2016;Milleret al, 2015;Rosenberget al, 2013), as well as in other behavioral procedures, such as drug self-administration (Czotyet al, 2002;Neguset al, 2007;Rothmanet al, 2008;Wang and Woolverton, 2007). In general, transporter ligands with greater DAT selectivity are associated with abuse-related effects, whereas those with greater SERT selectivity tend to produce abuse-limiting effects. The behavioral effects ofN-substituted 4-MA analogs in the ICSS experiments were generally consistent with their function at DAT and SERT, and selectivity for DATvs SERT. In particular,N-methyl 4-MA produced the most potent and robust abuse-related facilitation of ICSS rates, whereas higher doses engendered rate-decreasing effects. This behavioral profile is consistent with the function ofN-methyl 4-MA as a nonselective transporter substrate with somewhat greater potency at DAT. The expression of abuse-related effects and overall drug potency declined with increasingN-alkyl chain length that was associated with: (1) loss of selectivity for DATvs SERT, (2) overall loss of potency at all transporters, and (3) change in function from DAT substrate to inhibitor. Thus, in comparison withN-methyl 4-MA,N-ethyl 4-MA produced weaker and less potent facilitation of ICSS rates, but displayed similar potency for depression of ICSS rates. This profile is consistent with the function ofN-ethyl 4-MA as a DAT blocker with lower DAT potency thanN-methyl 4-MA and its inverted selectivity for DAT<SERT.N-propyl 4-MA produced only low-potency depression of ICSS rates, agreeing with its low DAT potency and inverted DAT<SERT selectivity. Finally,N-butyl 4-MA also produced only depression of ICSS rates, consistent with its inactivity at DAT; however,N-butyl 4-MA also displayed very low potency at SERT. The finding thatN-butyl 4-MA was more potent at depressing ICSS thanN-propyl 4-MA, despite having weaker SERT potency, suggests thatN-butyl 4-MA induced depression of ICSS rates independent of SERT.

In summary, the converging lines of evidence presented here demonstrate that lengthening theN-alkyl chain of 4-MA decreases potency to inhibit monoamine transporters. Perhaps more importantly, elongating theN-alkyl chain greater than a methyl group converts DAT substrates to inhibitors, whereas elongating the chain greater than an ethyl group converts NET substrates to inhibitors. Thus, theN-ethyl,N-propyl, andN-butyl analogs of 4-MA display hybrid transporter activity characterized by differential mechanisms at DAT, NET, and SERT for the same drug molecule (Bloughet al, 2014;Sahaet al, 2015). It is important to note that elucidating the complex pharmacology of 4-MA compounds could not have been accomplished without the multipronged approach utilized in our study. Although synaptosome assays provide physiologically relevant data from native tissue, and can discriminate fully efficacious transporter releasers from transporter inhibitors, these methods cannot discern the precise mechanism of action for drugs acting as partial releasers. The electrophysiological and Ca2+ fluorescence assays employed here demonstrate that partial releasers in the synaptosome assays can function as pure transporter inhibitors in cells expressing human transporters. Determining the molecular mechanisms responsible for the apparent disparities between the effects of partial releasers in native tissue preparationsvs their effects in cell-based systems warrants further study. Thein vivo ICSS data corresponded well with thein vitro data as increasing alkyl chain length reduced abuse-related effects of drugs in parallel with decreased DAT potency. Overall, our findings indicate that theN-alkylated analogs of 4-MA display less abuse liability than the parent compound. Given the continued appearance of stimulant-like NPS in the street drug marketplace, future investigations are warranted to evaluate the pharmacology and toxicology of these substances using an experimental approach that employs complementaryin vitro andin vivo methods.

Funding and disclosure

This research was generously funded by NIH R01 DA033930 and by the National Institute on Drug Abuse, Intramural Research Program, Grant DA000523. The authors declare no conflict of interest.

Acknowledgments

This study is dedicated to Dr Louis J De Felice, our beloved friend, mentor, and colleague, who passed away on 14 November 2016. He will be fondly remembered.

Footnotes

Supplementary Information accompanies the paper on the Neuropsychopharmacology website (http://www.nature.com/npp)

Supplementary Material

Supplementary Figures
Supplementary Methods

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