Structural and functional perspectives on interactions between synthetic cathinones and monoamine transporters

Abstract

Synthetic cathinone derivatives comprise a family of psychoactive compounds structurally related to amphetamine. Over the last decade, clandestine chemists have synthesized a consistent stream of innovative cathinone derivatives to outpace governmental regulatory restrictions. Many of these unregulated substances are produced and distributed as designer drugs. Two of the principal chemical scaffolds exploited to expand the synthetic cathinone family are methcathinone and α-pyrrolidinopentiophenone (or α-pyrrolidinovalerophenone, α-PVP). These compounds’ main physiological targets are monoamine transporters, where they promote addiction by potentiating dopaminergic neurotransmission. This chapter describes techniques used to study the pharmacodynamic properties of cathinones at monoamine transporters in vitro. Biochemical techniques described include uptake inhibition and release assays in rat brain synaptosomes and in mammalian expression systems. Electrophysiological techniques include current measurements using the voltage clamp technique. We describe a Ca²⁺ mobilization assay wherein voltage-gated Ca²⁺ channels function as reporters to study the action of synthetic cathinones at monoamine transporters. We discuss results from systematic structure-activity relationship studies on simple and complex cathinones at monoamine transporters with an emphasis on identifying structural moieties that modulate potency and selectivity at these transporters. Moreover, different profiles of selectivity at monoamine transporters directly predict compounds associated with behavioral and subjective effects within animals and humans. In conclusion, clarification of the structural aspects of compounds which modulate potency and selectivity at monoamine transporters is critical to identify and predict potential addictive drugs. This knowledge may allow prompt allocation of resources toward drugs that represent the greatest threats after drugs are identified by forensic laboratories.

Introduction

Cathinone (Fig. 1) is a sympathomimetic aromatic alkaloid derived from the angiosperm Catha edulis, an ethnobotanical chewed for its stimulant, decongestive, and anorexigenic effects by the peoples of the Horn of Africa and the Arabian Peninsula. Cathinone represents the β-keto analog of amphetamine, which itself was synthesized during attempts to generate analogs of ephedrine, derived from the gymnosperm Ephedra distachya. The biosynthetic pathway giving rise to cathinone involves the deamination of L-phenylalanine to cinnamate, the subsequent β oxidation of cinnamate, and the condensation of cinnamate with pyruvate, giving rise to 1-phenyl-1,2-propanedione (Gruesorensen & Spenser, 1994). Transamination of 1-phenyl-1,2-propanedione gives rise to cathinone, the skeleton of which affords extensive subsequent modification. Botanically, stereoselective reduction of cathinone gives rise to norpseudoephedrine and norephedrine, and N-methylation of each gives rise to pseudoephedrine and ephedrine. Synthetically, the addition of functional groups to cathinone can be exploited to generate a remarkable breadth of cathinone derivatives. In particular, synthetic strategies have given rise to the N-alkylated cathinones, the pyrrolidinophenone derivatives, the 3,4-methylenedioxy ring-substituted derivatives, and the mixed derivatives (Almeida, Silva, Pinho, Remião, & Fernandes, 2022) (Fig. 1). Therapeutically, bupropion, an N-alkylated and 3-chlorinated cathinone derivative, is a dopamine and norepinephrine reuptake inhibitor commonly prescribed as an antidepressant and smoking cessation agent, while methylone (Fig. 1), a 3,4-methylenedioxy ring-substituted cathinone, is under investigation as a rapidly acting antidepressant and therapeutic for post-traumatic stress disorder (Warner-Schmidt et al., 2022).

Figure 1.

Chemical structure of some members of the amphetamine and cathinone families of compounds.

Cathinone and its derivatives exert their pharmacological effects, pathological and therapeutic, by interaction with the monoamine transporters, which are 12-transmembrane helical integral membrane proteins expressed by presynaptic catecholaminergic and serotonergic projection neurons (Glennon & Dukat, 2017; Torres, Gainetdinov, & Caron, 2003). Critical functions of monoamine transporters are to recycle aminergic neurotransmitters (Fig. 2) into the presynaptic neurons and to tune synaptic dynamics by evacuating monoamine neuromodulators after electrical activity (Benoit-Marand, Jaber, & Gonon, 2000). The particular behavioral and neurological effects of a given chemical arise in dependence on its relative affinity for the dopamine, norepinephrine, and serotonin transporters (DAT, NET, and SERT, respectively) and the compound’s mode of interaction at these transporters. Specifically, a compound can function as a monoamine transporter substrate or an inhibitor (Rothman & Baumann, 2003). Substrates permeate the transporter to access the intraneuronal environment and thus behave as the endogenous substrate of a given transporter. Substrates induce monoamine efflux from vesicular stores, concentrating monoamines within the cytosol and culminating in the retro-operation of monoamine transporters, ultimately flooding the synaptic space. On the other hand, inhibitors bind to the transporter and obstruct the transporter’s native function, resulting in accumulation of monoamines within the synaptic space. Thus, substrates and inhibitors ultimately lead to enhanced monoamine signaling, and can be conceptualized as indirect monoamine receptor agonists (Fleckenstein, Volz, Riddle, Gibb, & Hanson, 2007; Sulzer, Sonders, Poulsen, & Galli, 2005; Torres et al., 2003). The classical stimulants like amphetamine (Fig. 1) and cocaine possess high relative affinities for the dopamine and norepinephrine transporters and engender euphoria, enhanced vigilance, enhanced task salience, insomnia, locomotor hyperactivity, and general sympathomimetic stimulation (Kirkpatrick et al., 2012; Rothman & Baumann, 2003). The entactogens like 3,4-methylenedioxymethamphetamine (MDMA, Fig. 1) are substrates at the monoamine transporters and possess a higher relative affinity for the serotonin transporter, and their unique pharmacodynamics are associated with additional effects of tactile intensification and enhancement of empathy and sociability (Kirkpatrick et al., 2012; Oberlender & Nichols, 1988). Excessive and repeated doses of such compounds, or the administration of compounds with an extreme affinity for a given transporter, can cause cardiovascular damage, compulsive behavior, agitation, delirium, paranoia, hyperthermia, hyperreflexia, rhabdomyolysis, and death (Michael White, 2014). Therefore, understanding the particular pharmacodynamic profile of a compound is critical in predicting its particular neurological and cardiovascular consequences.

Figure 2.

Chemical structure of dopamine, norepinephrine, and serotonin.

The emergence of a zoo of cathinone derivatives has represented a global public health scourge for more than two decades (Glennon & Dukat, 2017). MDMA and methamphetamine (Fig. 1) have been universally regarded as illicit drugs of abuse; however, entrepreneurs took advantage of the lack of definitive laws regarding the manufacture and sale of β-keto analogs of these chemicals. Sold brazenly in gas stations and head shops or via the Internet in vaguely branded packages marked as “bath salts” or “plant food”, methylone (the β-keto analog of MDMA), mephedrone (the 4-methyl β-keto analog of methamphetamine), and MDPV (the 3,4-methylenedioxy ring-substituted analog of α-pyrrolidinovalerophenone, α-PVP, Fig. 1) were introduced to the market in the United States beginning around 2009 (Glennon, 2014; Madras, 2017). MDPV is an inhibitor of DAT and NET (Baumann et al., 2017; Baumann et al., 2013; Cameron, Kolanos, Vekariya, De Felice, & Glennon, 2013a), while mephedrone and methylone are releasers with affinity for DAT, NET and SERT (Glennon & Dukat, 2017). MDPV possesses a potency at DAT greater than that of cocaine by an order of magnitude, and electrophysiological studies revealed that MDPV is exceptional in its propensity to bind DAT for a protracted interval of time even after washout (Cameron, Kolanos, Solis, Glennon, & De Felice, 2013b). Unrestricted access to these compounds generated a glut of media attention, and numerous grisly, cannibalistic murders and episodes of violent behavior were attributed to their intoxicating effects (Slomski, 2012). The lurid tone of this media attention may be attributable to a particular syndrome of extreme agitation, paranoia, and delirium that emerges as a consequence of the unique pharmacodynamics of these compounds, especially MDPV. MDPV was established as conferring outsized risk of medical emergency, with 14 of 18 patients presenting for bath salts intoxication at two American poison control centers manifesting MDPV positivity on gas chromatography/mass spectrometry analysis of urine and/or blood (Spiller, Ryan, Weston, & Jansen, 2011). MDPV was further associated with numerous fatalities during this time (Murray, Murphy, & Beuhler, 2012; Penders, Gestring, & Vilensky, 2012; Ross, Reisfield, Watson, Chronister, & Goldberger, 2012; Wright et al., 2013). Similarly, α-PVP possesses pharmacological properties at monoamine transporters comparable to those of MDPV, and it has been associated with numerous human fatalities (Karila, Lafaye, Scocard, Cottencin, & Benyamina, 2018; Kolesnikova, Khatsko, Demin, Shevyrin, & Kalueff, 2019; Nadal-Gratacos et al., 2022). Meanwhile, data from the US Drug Enforcement Agency portray a staggering upward trend in the incidence of methylone-associated seizures during this era, culminating in a zenith of 10,944 cases in 2013, the same year methylone was added to Schedule I of the United States Controlled Substances Act (Federal register 78 FR 21818). Mephedrone (Federal register 76 FR 65371), MDPV (Federal Register 76 FR 65371), and a vast diversity of synthetic cannabinoids had been added to Schedule I the prior year via passage of the 2012 Synthetic Drug Abuse Prevention Act.

Despite extensive legal measures to restrict the usage of synthetic cathinones, the inexorable demand-side pressure for stimulant drugs has catalyzed the continued innovation and synthesis of novel generations of cathinone analogs. Since 2013, the European Monitoring Centre for Drugs and Drug Addiction has detected 113 novel structural formulations of synthetic cathinones. These new generations of cathinone derivatives have been traced to clandestine laboratories disproportionately located in Asia. Advancing sophistication in dark web marketplaces and private cryptocurrencies has aided their distribution within Europe and the United States. In 2020, of the 3,306 kilograms of synthetic cathinones seized by authorities of European Union member states, 84% of this quantity consisted of N-ethylhexedrone, 3-chloro-methcathinone and 3-methyl-methcathinone, representing a stark illustration of the pattern of novel derivations of synthetic cathinones arising to supplant former generations (EMCDDA, 2022). Meanwhile, in the United States, the most commonly reported synthetic cathinone has been eutylone (Fig. 1), reported 8,379 times in 2021 and registered as the seventh most commonly reported illicit drug nationwide (NFLIS-DRUG 2021 midyear report). In addition, eutylone was associated with 343 fatalities in 2020, 75.5% of which arose in Florida and Maryland (Gladden, Chavez-Gray, O’Donnell, & Goldberger, 2022). Although novel synthetic cathinones may be inherently illegal in the United States under the Federal Analog Act, their novel structures nonetheless facilitate procurement of the precursor chemicals required for mass synthesis. Because the diversity of synthetic cathinones is indefinite and novel compounds emerge more rapidly than legal architecture can react to control their distribution, techniques for screening, non-ambiguous identification and subsequent formal communication of these chemicals is necessary to inform global medical systems in real time.

Monoamine transporters are the main neurobiological targets of cathinones

Organic compounds from a variety of drug classes that are self-administered by animals in experimental studies and/or abused by humans directly or indirectly potentiate mesolimbic dopaminergic neurotransmission. In the case of psychostimulant members of the cathinone or amphetamine families, their rewarding and reinforcing effects are mediated by altering DAT function directly (Corre et al., 2018; Ikemoto & Bonci, 2014; S. W. Johnson & North, 1992; Lerner et al., 2015; Negus & Miller, 2014; Wise, 2008). For example, the commonly abused psychostimulant methamphetamine, and its β-keto version, methcathinone, are potent substrates at DAT (Glennon, Yousif, Naiman, & Kalix, 1987). In contrast, the structurally related agent fenfluramine (Fig. 1), a selective SERT substrate, is not self-administered by experimental animals and shows low abuse liability in humans (Oswald, Lewis, Dunleavy, Brezinova, & Briggs, 1971; Woods & Tessel, 1974). Similarly, DAT inhibitors like α-PVP or MDPV are abused (Beck, Franzen, Backberg, Signell, & Helander, 2015; Glennon & Young, 2016; Odoardi, Romolo, & Strano-Rossi, 2016), but selective serotonin reuptake inhibitors like fluoxetine are used clinically as antidepressants, may attenuate amphetamine self-administration in rats (D. S. Yu, Smith, Smith, & Lyness, 1986), and have no abuse concern in humans (Zerbe, 1987).

Most monoamine transporter substrates active at DAT show similar or even higher potency at NET (Davies et al., 2020; Rickli, Hoener, & Liechti, 2015; Rothman & Baumann, 2003; Solis et al., 2017). Methamphetamine has one chiral center, existing as two stereoisomers: S(+)methamphetamine and R(−)methamphetamine. S(+)Methamphetamine is at least ~15-fold more potent than R(−)methamphetamine in mediating dopamine release thorough DAT in vitro and in vivo, whereas both stereoisomers are equipotent substrates at NET (Kuczenski, Segal, Cho, & Melega, 1995; Melega, Cho, Schmitz, Kuczenski, & Segal, 1999; Rothman & Baumann, 2003; Rothman et al., 2001). This stereoselectivity profile of methamphetamine correlates with the much higher abuse potential and psychostimulant effect of the S(+) over the R(−) enantiomer (Mendelson et al., 2006). In fact, S(+)methamphetamine is usually the stereoisomer traded in the illicit market (Cunningham et al., 2013), whereas R(−)methamphetamine has comprised of the active ingredient of nasal decongestants (Kohut, Bergman, & Blough, 2016). In addition, prescription drugs such as desipramine and atomoxetine that are selective NET inhibitors do not have abuse concern in humans. Altogether, these examples highlight the pivotal role of DAT modulation in reward and addiction of psychostimulants.

The neurochemical action of monoamine transporter ligands is not limited to dopamine signaling, and modification of specific chemical substitutions may alter an agent’s selectivity pattern at DAT, NET, or SERT, thus resulting in differential modulation of dopaminergic, noradrenergic and serotonergic signaling after administration (Negus & Miller, 2014). In addition, chemical modifications of a known active scaffold can transform a compound from a substrate to a blocker at a given transporter (Battisti et al., 2018; Solis et al., 2017), and a single compound may even exhibit hybrid activity, behaving as a substrate in one (or two) monoamine transporter(s) and as an inhibitor in another (Battisti et al., 2018; Blough et al., 2014; Solis et al., 2017). The constellation of potential changes in selectivity, efficacy, and substrate versus inhibitor activity at DAT, NET, and SERT constitute a multidimensional space, where a single modification of a known compound can reshape its neurochemical profile, resulting in unpredictable rewarding, subjective, and/or therapeutic effects.

In vitro methods to study the pharmacology of monoamine transporters

Ligands at monoamine transporters can be categorized functionally as substrates or inhibitors. Substrates are typically small molecules that are taken up by the transporter. Examples of substrates are the endogenous neurotransmitters (dopamine, norepinephrine, and serotonin, Fig. 2) and unsubstituted amphetamine and cathinone (Glennon & Dukat, 2017; Rickli et al., 2015; Rothman & Baumann, 2003). N-Methyl substitution of these scaffolds (methamphetamine and methcathinone) preserves substrate activity at these transporters (Rothman & Baumann, 2003; Rothman et al., 2003; Walther, Shalabi, Baumann, & Glennon, 2019). As described in more detail later in the chapter, substrates are co-transported with Na⁺ ions that provide the main driving force to concentrate the transported substrate within the intracellular space (Torres et al., 2003). Thus, the Na⁺ gradient across the plasma membrane is necessary to maintain a normal transporter-mediated substrate influx in cells (Rudnick, 1977). In addition, non-endogenous substrates, when transported inside cells, can reach intracellular targets and produce additional effects. These effects include disruption of internal stores of neurotransmitters and the stimulation of reverse transport or non-vesicular release of the neurotransmitters through the monoamine transporter (Fleckenstein et al., 2007; Sulzer et al., 2005). For the latter reason, substrates may be referred to as releasers. Inhibitors, on the other hand, are molecules that bind the transporter without being transported, thus hindering the uptake of a substrate (Seeman & Madras, 1998). Commonly known inhibitors of monoamine transporters are cocaine, methylphenidate, fluoxetine and, in general, a family of therapeutic drugs called reuptake inhibitors of monoaminergic neurotransmitters (Mandrioli, Mercolini, Saracino, & Raggi, 2012; Seeman & Madras, 1998; Wilens, Morrison, & Prince, 2011). Bulkier cathinones such as α-PVP (flakka), having a pyrrolidine ring and a propyl alkyl chain both bound the α-carbon of the cathinone scaffold, are monoamine transporter inhibitors (Glennon & Dukat, 2017; Kolanos, Sakloth, et al., 2015). Several groups are or have studied the pharmacology of cathinones at monoamine transporters via in vitro approaches.

Rat Brain Synaptosomes

Measurement of monoamine transporter activity in rat brain synaptosomes has represented a critical assay toward the elucidation of general “rules” relating chemical structure to the activity of compounds at these transporters (Glennon & Dukat, 2017). The use of synaptosomes is a biochemical method in which brain membranes are homogenized and isolated using centrifugation, yielding closed membrane vesicles retaining all components normally present at neuronal synapses. For the study of DAT, synaptosomes are prepared from the rat caudate nucleus where DAT is abundant. For the study of SERT, synaptosomes are prepared from the whole brain minus the cerebellum, while the whole brain minus the caudate is used for the study of NET. Highly selective inhibitors are included within the synaptosome preparations to nullify the contribution of transporters that are not of interest, thus allowing assessment of the activity of a single monoamine transporter in isolation (Rothman et al., 1993; Rothman et al., 2000). The activity of the transporters can be measured using radioactive tracers. Two protocols have been developed: one measures the ability of a test compound to inhibit the uptake of external radiolabeled substrate, and another measures the release of pre-loaded, internal radiolabeled substrate following exposure to the test compound (Rothman & Baumann, 2003; Rothman et al., 2000) (Fig. 3). By means of these methods, it is possible to study the potency at which compounds compete with endogenous uptake at the three transporters. In addition, test compounds that work as substrates at transporters are identified because substrates (but not inhibitors) cause release of the preloaded radiolabeled substrate from synaptosomes (Baumann et al., 2012; Rothman & Baumann, 2003; Solis et al., 2017).

Figure 3.

Illustration describing the use of synaptosomes to study the pharmacology of monoamine transporters. The uptake of a tritiated substrate is blocked by an inhibitor or competed by another non-tritiated substrate (cold substrate). By varying the concentration of the test compounds their potency to decrease uptake can be determined. Note that both cold substrates and inhibitors decrease the uptake of the tritiated substrate. In the release assay synaptosomes are pre-loaded with a tritiated substrate and these synaptosomes are exposed to a test compound. Transporter inhibitors do not induce release of the pre-loaded substrate, but other substrates produce the release of the preloaded substrate by reverse transport. By varying the concentration of the test compound potency and efficacy of release can be determined. Note that blockers may show small levels of release, but this is attributed to a non-specific leak not mediated by the monoamine transporters.

Expression of transporters in mammalian cell lines

Similar approaches have been developed using cells lines (usually HEK293 cells) expressing monoamine transporters. The use of HEK293 cells is advantageous because these cells do not express monoamine transporter natively (Cameron, Solis, Ruchala, De Felice, & Eltit, 2015; Galli, DeFelice, Duke, Moore, & Blakely, 1995; Solis et al., 2012). Monoamine transporters can be transiently expressed through transfection or permanently expressed by producing stable cell lines (Galli et al., 1995; Ruchala et al., 2014; Tatsumi, Groshan, Blakely, & Richelson, 1997). In expression systems, it is possible to use human cDNA sequences thus conferring translational value, although the primary sequence identity between human and rat transporter isoforms is very high (>90%) (Bruss, Porzgen, Bryan-Lluka, & Bonisch, 1997; Chang et al., 1996; Giros et al., 1992). Because arbitrary sequences of cDNA can be expressed within expression systems, mutagenesis approaches can be used to investigate mechanisms of transporter-ligand interactions (Steele et al., 2021). The level of expression of the transporter may differ in the expression system compared to the native system. This differential expression may influence inter-transporter quaternary structure, or oligomerization, which in turn can affect the trafficking, pharmacology, and biophysics of transporters (Ingram et al., 2021; Jayaraman et al., 2021; Ramsey & DeFelice, 2002; Siciliano et al., 2018). Another disadvantage is that partners or neighboring modulatory proteins expressed in the native system (e.g., in the brain) may not be present in HEK293 cells, conceivably resulting in divergent transporter behavior compared to the native system (Quick, 2003; Sager & Torres, 2011). Thus, experiments performed in synaptosomes and expression systems should be considered complementary and compared to attain more robust conclusions.

Through the use of mammalian expression systems (HEK293 cells), uptake inhibition and release protocols have been widely reported using radio-ligands, in analogy to the synaptosome studies described above. One caveat of using HEK293 cells for release studies is that the preloaded substrate is not released as effectively by releasers compared to the synaptosome preparation. Thus, researchers in some studies have used a Na⁺ ionophore (monensin) to dissipate the Na⁺ gradient, producing a more effective release of the preloaded radioligand when cells are exposed to a substrate (Mayer et al., 2018).

Another technique to study the pharmacology of monoamine transporter in expression systems is tracing the activity of the transporters using fluorescent substrates. 1-Methyl-4-phenylpyridinium (MPP⁺), the active metabolite of the dopaminergic neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), is a potent, non-fluorescent substrate of monoamine transporters. Tritiated MPP⁺ is commonly used as a radioligand in synaptosomal release assays. APP⁺ (4-(4-(dimethylamino)phenyl)-1-methylpyridinium or IDT307), a fluorescent analog of MPP⁺, has been widely used in live cell microscopy and in automated fluorometry to study the pharmacology of monoamine transporters (Ingram et al., 2021; Iyer et al., 2019; Ruchala et al., 2021; Solis et al., 2012). APP⁺ has the advantage over ASP⁺ (4-(4-(dimethylamino)styryl)-N-methylpyridinium, a previously developed fluorescent substrate) in functioning as a substrate at all three monoamine transporters. APP⁺ is not fluorescent in solution. However, with cellular uptake, the interaction of APP⁺ with intracellular components engenders a biplanar molecular configuration enabling its fluorescent properties (Solis et al., 2012).

Using electrophysiological recordings to study monoamine transporter pharmacology

Monoamine transporters employ a secondary active transport mechanism to concentrate neurotransmitters inside cells. These transporters are Na⁺- and Cl⁻- dependent symporters, and the large difference in electrochemical potential across the plasma membrane for Na⁺ provides significant free energy to drive the influx of these neurotransmitters. The model of alternating access has been proposed to explain the process whereby translocation of substrate from the extracellular milieu to the cytosol is accomplished. In this model, transporters are envisioned to undergo structural transitions from an outward-facing conformation where a charged substrate molecule (positively charged at physiological pH) binds to the transporter along with Na⁺ and Cl⁻. This dual binding stimulates transition through an intermediate, occluded state to an inward-facing conformation in which the substrate and ions are released into the intracellular compartment (Cheng & Bahar, 2019; Rudnick & Nelson, 1978). Subsequently, in the case of DAT and NET, the transporter returns empty to the starting outward-facing conformation, while in the case of SERT, a K⁺ cation binds to the inward-facing conformation, expediting the return to the initial outward-facing conformation (Rudnick & Nelson, 1978). All transporters are coupled to one positive substrate molecule and one Cl⁻ ion per cycle; however, the stoichiometry of transport is different for each transporter. NET and SERT transport one Na⁺ ion per cycle, while DAT transport is coupled to two Na⁺ ions per cycle, and only SERT exchanges one K⁺ per cycle (Gu, Wall, & Rudnick, 1994). This stoichiometry renders the transport electrogenic for DAT and NET and electroneutral for SERT. The electroneutrality of SERT transport is supported by experimentation: while the rate of DAT- and NET- mediated transport changes as a function of membrane potential, the rate of SERT-mediated transport is not sensitive to changes in membrane potential (Galli, Blakely, & DeFelice, 1996; Galli et al., 1995; Mager et al., 1994; Sonders, Zhu, Zahniser, Kavanaugh, & Amara, 1997). Early experiments expressing SERT in Xenopus oocytes showed that despite the electroneutrality of SERT transport, a clear inward current, called the substrate-induced current (Mager et al., 1994), was observed during transport. In addition, a second type of current was described, a constitutive leak current that was observed as an apparent outward current following SERT blockade by pharmacological inhibitors (Mager et al., 1994). Quantitative estimation of the amount of electrical charge carried during transport and the amount of substrate taken up in the same cell showed that the electrical charge carried during transport exceeds the expectation of the alternating access model described above, and that charge versus transport ratio changes with membrane potential (Galli et al., 1996; Galli et al., 1995; Mager et al., 1994; Sonders et al., 1997). These observations suggested either the existence of an uncoupled ionic current through the transporter during transport or variable coupling within the transport mechanism (Carvelli, McDonald, Blakely, & DeFelice, 2004; Galli et al., 1996; Lin, Lester, & Mager, 1996). For further discussion about models of transport for monoamine transporters please see (S. V. Adams & DeFelice, 2002, 2003; De Felice, 2016; DeFelice, Adams, & Ypey, 2001; Erreger, Grewer, Javitch, & Galli, 2008; Schicker et al., 2012; Su, Mager, Mayo, & Lester, 1996). Regardless of the precise mechanism, monoamine transporters produce electrical responses that can be used to study their pharmacology. DAT and SERT can be expressed in Xenopus oocytes and ionic currents studied using two electrode voltage clamp (Mager et al., 1994; Sonders et al., 1997) (Fig. 4). For unknown reasons, NET does not express in frog oocytes. All three transporters are well-expressed in mammalian cells such as HEK293 cells, and electrical currents can be measured using single electrode voltage clamp (Bhat et al., 2017; Cameron et al., 2015; Galli et al., 1995). Compounds that generate an inward current are considered substrates of the transporter and compounds that produce an outward current (inhibition of the constitutive leak) and/or hinder the substrate-induced current are considered inhibitors of the transporter (Mager et al., 1994; Sonders et al., 1997). These electrophysiological signatures are useful in classifying ligands as substrates or inhibitors and can determine both potency and efficacy of compounds at monoamine transporters (Iyer et al., 2019; Solis et al., 2016).

Figure 4.

Cartoon representation of two electrode voltage clamp technique within Xenopus oocytes for the study of monoamine transporter pharmacology. cRNA encoding a monoamine transporter of interest is injected into oocytes. Electric current across the membrane is measured using two electrode voltage clamp technique at −60 mV holding potential in this example. Experiments are performed under constant perfusion of a saline buffer and by using valves the oocyte can be exposed to a test compound for a few seconds. Exposure to monoamine transporter substrates induces an inward current, while exposure to monoamine transporter inhibitors induces an outward current. The outward current represents the inhibition of a monoamine transporter-mediated leak inward current. This differential signature response can be used to classify new monoamine transporters ligands, and metrics of potency and efficacy can be established by systematically increasing the concentration of compound of interest. Recordings were kindly provided by Dr. Ernesto Solis Jr.

Voltage-gated Ca²⁺ channels as sensors of monoamine transport activity

A few years ago, we discovered that the substrate-induced current of monoamine transporters is strong enough to activate voltage-gated Ca²⁺ channels in intact mammalian cells (Ruchala et al., 2014). The measurement of intracellular Ca²⁺ concentration in cells is possible owing to the availability of extremely bright and sensitive fluorescent Ca²⁺ indicators (Tsien, Rink, & Poenie, 1985). Thus, by measuring intracellular Ca²⁺ concentration in cells co-expressing transporters and voltage-gated Ca²⁺channels, we designed protocols to determine potency and efficacy of ligands at monoamine transporters (Steele & Eltit, 2019) (Fig. 5). For these experiments, we employed a modified version of HEK293 cells, the Flp-InT-REx 293 system. These cells, originally developed by Invitrogen, can be used to efficiently develop stable and inducible cell lines to express genes of interest via a targeted recombination method. Using this system, cell lines expressing DAT, SERT, or NET were generated and subsequently transiently transfected with voltage-gated Ca²⁺ channels. Intracellular Ca²⁺ signals were measured using either Fluo4, Fura2, or the genetically encoded GCamP6s sensors visualized using an automatic fluorimeter for screening, e.g. FlexStation 3 (Ruchala et al., 2021), or a fluorescent microscope for quantitative measurements (Battisti et al., 2018; Cameron et al., 2015; Ruchala et al., 2014; Solis et al., 2017). The fluorescent microscope setup included computer-controlled electronic valves that provided a precise timing of ligand exposure in a constant perfusion environment maintained at 35°C. For these experiments, temperature control is critical, as at physiological temperature transporters produce larger substrate-induced currents favoring the initial membrane depolarization that activates the Ca²⁺ channel (Steele & Eltit, 2019). Three voltage-gated Ca²⁺ channel isoforms were tested in these experiments: Ca_V1.3, Ca_V1.2, and Ca_V2.2, which require smaller, intermediate, and larger depolarizations for activation, respectively (Cameron et al., 2015). When the cells were exposed to the respective neurotransmitter or a known monoamine transporter substrate (such as amphetamine or MDMA), cells responded by increasing Ca²⁺ immediately, and the Ca²⁺ levels returned to baseline rapidly after washout, as expected for an electrical response (Cameron et al., 2015; Ruchala et al., 2014). These signals were observed for the L-type Ca²⁺ channels (Ca_V1.3 and Ca_V1.2) but not with the N-type channel Ca_V2.2 (Cameron et al., 2015; Ruchala et al., 2014). Because the threshold of activation of Ca_V2.2 is ~−15 mV and Ca_V1.2 is ~−30 mV, the overall conductance of the transporter at saturation should depolarize the membrane by approximately 20 to 30 mV in intact cells, assuming a resting potential of −50 mV (Cameron et al., 2015; Ruchala et al., 2014). Thus, the initial depolarization resulting from the transporter activation reaches only intermediate levels, sufficient for L-type voltage-gated Ca²⁺ channel activation [see (Cameron et al., 2015; Ruchala et al., 2014; Steele & Eltit, 2019) for more details].

Figure 5.

Representation of the co-expression of monoamine transporters and voltage-gated Ca²⁺ channels in HEK293 cells to study substrates and inhibitors of these transporters.

Control experiments using cells expressing the L- type voltage-gated Ca²⁺ channel alone or the monoamine transporter alone did not produce Ca²⁺ signals in response to monoamine transporter substrates (Cameron et al., 2015; Ruchala et al., 2014). Similarly, selective Ca²⁺ channel inhibitors or monoamine transporter inhibitors also blocked the Ca²⁺ signals induced by monoamine transporter substrates (Cameron et al., 2015; Ruchala et al., 2021; Solis et al., 2017; Steele & Eltit, 2019). Collectively, these experiments clearly showed that transport through monoamine transporters is coupled to voltage-gated Ca²⁺ channel opening upon co-expression of these two membrane proteins in cells.

In principle, as mentioned above, the depolarization mediated by the transporter substrate-induced current depolarizes the plasma membrane to the level of L-type channel activation. Although unlikely, another possible mechanism that could explain the opening of L-type Ca²⁺ channels by transporter activation involves a direct mechanical coupling between the proteins. To assess the precise spatial distribution of voltage-gated Ca²⁺ channels and monoamine transporters within our expression system, we took advantage of techniques in super-resolution microscopy. Cells co-expressing DAT and a version of Ca_V1.2 tagged with a fluorescent protein (mVenus-Ca_V1.2) and auxiliary subunits of this channel (subunits β3 and α2δ) were subjected to immunofluorescence staining and visualized using structured illumination microscopy (SIM) (Fig. 6A–6D). This modality extracts extra information from a sample by shaping structure into the illuminating light itself, ultimately surpassing the optical diffraction limit of conventional confocal microscopy and improving resolution by a factor of 2, thus allowing visualization of the fine-scale super-structure of membrane proteins. mVenus-Ca_V1.2 demonstrated a plasma membrane localization with a distinct punctate pattern (Fig. 6A and 6a), while DAT was also expressed in the plasma membrane and demonstrated a more diffuse pattern (Fig. 6B and 6b). Interestingly, the degree of colocalization was only partial (yellow pixels in Fig. 6D and 6d), suggesting that each protein occupies separate membrane sub-domains. To further evaluate proximity between these two membrane proteins, fluorescence resonance energy transfer (FRET) studies were performed between a mVenus–Ca_V1.2 (donor) and tetracysteine–tagged (tC) DAT constructs (acceptor when stained with ReAsH). A tC-tag was inserted alternatively into the N-terminus (tC-DAT), in one intracellular loop, or at the proximal portion of the C-terminus (DAT-tC) of DAT (Fig. 6E). The tC tag reacts with ReAsH generating a fluorescent adduct working as energy acceptor in the FRET experiment (S. R. Adams et al., 2002; Mahalingam, Perez, & Fessenden, 2016) (insert in Fig. 6E). The FRET efficiency (FE) was estimated by assessing the emission recovery of the donor after acceptor photobleaching (Fig. 6F). Immunostaining visualized via confocal microscopy showed that two constructs, tC-DAT and DAT-tC, were expressed in the plasma membrane (Fig. 6G) and were conducive to FRET analysis; however, the construct that has the tC-tag in an intracellular segment of DAT was clearly trapped within the endoplasmic reticulum (not shown); thus it was not further evaluated. The FE (mean ± SEM) for the negative control mVenus-Ca_V1.2 and the positive control tC-mVenus-Ca_V1.2 (that has the tC tag fused to the N-terminus of the mVenus that is also fused to Ca_V1.2) were 0.004 ± 0.0069 (n=104 cells) and 0.192 ± 0.0075 (n=111), respectively (Fig. 6H). The stable cell lines expressing tC-DAT or DAT-tC were transfected with the mVenus-Ca_V1.2, β3, and α2δ plasmids, and the tagged-DAT proteins were stained with ReAsH (Thermo Fisher). The FE of DAT (negative control), tC-DAT, and DAT-tC was 0.0076 ± 0.0098 (n=94), 0.0319 ± 0.0090 (n=91), and 0.0237 ± 0.0087 (n=95), respectively (Fig. 6H). The FE of the test conditions tC-DAT and DAT-tC was not different from the FE of the negative control using untagged DAT (one-way ANOVA) indicating that DAT and Ca_V1.2 do not interact at FRET critical distance (less than 10 nm). In summary, L-type Ca²⁺ channels and monoamine transporters display different staining patterns and only partially colocalize in the plasma membrane as revealed by SIM. Moreover, the assessment of spatial proximity between the two proteins via FRET studies did not produce signal (Fig. 6H); thus, a physical interaction is unlikely to exist. Thus far, all empirical evidence indicates that monoamine transporters activate L-type Ca²⁺ channels by means of membrane depolarization.

Figure 6.

Co-expression of monoamine transporters and voltage-gated Ca²⁺ channels evaluated using SIM microscopy and the study of their proximity via FRET. Cells expressing hDAT and co-transfected with a mVenus-tagged Ca_V1.2 construct, β3 and α2δ were visualized via structured illumination microscopy (SIM). Because the mVenus fluorophore resisted the fixation process, it was visualized directly (A). The red signal corresponds to hDAT stained using a primary anti-DAT antibody and a secondary antibody conjugated to Alexa-555 (B). The nucleus was visualized via DAPI staining (C). The merging of the three channels is shown in D. The scale bar corresponds to 5 μm. The panels labeled with lower case correspond to a zoom of the panels A, B, C and D (a, b, c, and d, respectively). For FRET studies stable and inducible cell lines expressing wild type hDAT (DAT), tetracysteine-tagged in the N-terminus of hDAT (tC-hDAT), or C-terminus of hDAT (DAT-tC), and control cells where generated using sequences shown in (E). The ReAsH reagent produces an adduct with the tC tag constituting a fluorophore used as FRET acceptor and mVenus was used as energy donor. FRET between hDAT and CaV1.2 was evaluated by the increase in donor fluorescence upon acceptor photobleaching (F). The membrane expression of these hDAT variants was evaluated by immunostaining using confocal microscopy (G). The FRET efficiency was evaluated as [FRET efficiency = 1 – (F_prebleach/F_postbleach)]. Control constructs without the tC-tag are shown in open bars defining the basal signal of the FRET measurements and constructs with tC-tag are the test conditions shown as black bars. No statistical significance was reached between the control cells expressing hDAT and cells expressing tC-hDAT or hDAT-tC (one-way ANOVA, Tukey’s posttest) suggesting that no significant FRET occurs between hDAT and Ca_V1.2 (H).

Additional evidence for this notion derives from studies examining the constitutive “leak” current through transporters using Li⁺. This ion can constitutively permeate cells through monoamine transporters in the absence of a substrate (S. V. Adams & DeFelice, 2003; Borre, Andreassen, Shi, Weinstein, & Gether, 2014; Mager et al., 1994). Indeed, it has been reported that a brief exposure to an external solution in which Na⁺ was substituted by Li⁺ resulted in Ca²⁺ signals in cells co-expressing DAT and L-type Ca²⁺ channels. However, when the channels were expressed alone (in the absence of the monoamine transporter transporters), Li⁺ substitution failed to produce such signals (Cameron et al., 2015), indicating that current crossing through the transporter (Li⁺ in this case) and resulting depolarization of the plasma membrane is the mechanism underlying the coupling between monoamine transporters and L-type Ca²⁺ channels.

Pharmacology of cathinones at monoamine transporters using in vitro assays

Methcathinone scaffold

In vitro assays have been extremely useful to decipher selectivity patterns of ligands at monoamine transporters. As described above, electrophysiological recordings (including the use of Ca²⁺ channels as sensors) and release assays using radioligands have been utilized to discriminate substrate versus inhibitor activity of compounds at monoamine transporters. By using these methods, researchers have studied simple and complex cathinone scaffolds, describing how different substitutions alter the activity of the resulting compounds at monoamine transporters. For a historical and thorough description of studies about structure-activity relationship of cathinones at monoamine transporters, we refer the interested reader to the review of (Glennon & Dukat, 2017). In this section, we will summarize studies where systematic substitutions at synthetic cathinone scaffolds produce changes in the pharmacological activity of these compounds at transporters in vitro. Methcathinone, the N-methyl version of cathinone, is a potent substrate at DAT and NET, and demonstrates potency similar to methamphetamine at these transporters in synaptosome release assays and very weak releaser activity at SERT (Rothman et al., 2003; Walther et al., 2019). This pattern of selectivity for methcathinone has also been demonstrated via comparison of transport inhibition in different preparations expressing DAT, NET, and SERT (Cozzi, Sievert, Shulgin, Jacob, & Ruoho, 1999) and using transporter overexpression systems in HEK293 cells (Eshleman et al., 2017; Rickli et al., 2015; Walther et al., 2019). In experiments co-expressing transporters and Ca²⁺ channels, methcathinone produced Ca²⁺ signal at the three transporters that were much more potent at DAT and NET than at SERT (Davies et al., 2020). Altogether, these observations suggest that methcathinone is a releasing agent (or substrate) at the three transporters possessing a strong selectivity for DAT and NET over SERT. Stereoisomers of methcathinone (Fig. 7) were studied using the Ca²⁺ assay, and the S(−) enantiomer was marginally more potent than the R(+) enantiomer at DAT and both enantiomers were equipotent at NET. In comparison, S(−) methcathinone was very weak at SERT and the R(−) enantiomer was virtually inactive at this transporter (Davies et al., 2020). In the latter study, modifications at the α-carbon of the methcathinone scaffold were studied using the Ca²⁺ mobilization assay showing that an additional methyl group (α-methyl methcathinone, Fig. 7) reduced by approximately half the potency at DAT, while maintaining equal potency at NET and the same low potency at SERT (Davies et al., 2020). α-des-Methylation of methcathinone (Fig. 7) resulted in a seven-fold reduction of potency at DAT, maintained the same high potency at NET, and decreased the potency even more at SERT (Davies et al., 2020). All these compounds produced Ca²⁺ signals, a feature associated with monoamine transporter substrates. Altogether, these studies suggest that the methyl group bound to the α-carbon in methcathinone is integral in maintaining its high potency at DAT while being dispensable for its high potency at NET. Moreover, although methcathinone is a very weak substrate at SERT, the α-methyl group facilitates interaction with the transporter.

Figure 7,

Methcathinone (MCAT) and its modifications in the α-position.

In a different study using rat brain synaptosomes, the extension of the α-methyl in methcathinone to α-ethyl (PAL-429, Fig. 7) decreased potency at DAT as a releaser agent by 8 fold (SERT and NET data were not reported in the study) (Reith et al., 2015). In addition, the extension of the α-methyl group in methcathinone to an α-n-propyl (molecule known as pentedrone, Fig. 7) blunted the releasing action at all three transporters, transforming the compound to a reuptake inhibitor with much higher potency at DAT and NET than at SERT (Eshleman et al., 2017).

Several studies have described the activity of methcathinone analogs with modified aryl moieties at monoamine transporters. Modifications of the 4-position of methcathinone (Fig. 8) generate analogs less potent at DAT depending on the substitution: −Cl, −Br, and −CH₃ substitutions reduce potency ~4-fold; −F reduces potency ~7-fold; −OCH₃ reduces potency ~40-fold; and −CF₃ reduces potency ~216-fold as measured by stimulation of release in rat synaptosomes (Sakloth et al., 2015). Interestingly, these modifications of the 4- position of methcathinone produce a reverse effect at SERT, resulting in enhanced releaser potency: −F increases potency ~3-fold; −CF₃ increases potency ~20-fold; −OCH₃, CH₃, and −Cl increase potency ~30-fold, and −Br increases potency ~60-fold. Quantitative structure-activity relationship (qSAR) analysis suggested that smaller substituents at the 4-position are associated with superior binding at DAT while bulkier substituents in that position are associated with superior binding at SERT. In addition, hydrophobicity at this position plays a role in binding at SERT (Sakloth et al., 2015). Similar trends were observed in HEK293 cells when the high DAT versus SERT selectivity of methcathinone was abrogated after similar modifications were introduced at the aryl 4-position (Eshleman et al., 2017; Niello et al., 2019; Rickli et al., 2015). Similarly, the 3,4-methylenedioxy substitution in methcathinone (i.e. methylone) results in a compound with decreased releaser potency at DAT and NET but with enhanced releaser potency at SERT, generating a compound that is much less selective for DAT compared to unsubstituted methcathinone (Baumann et al., 2012; Cozzi et al., 1999; Simmler et al., 2013).

Figure 8.

Methcathinone and its modifications in the aryl 4- position.

Modifications of the 3-positon of the aryl moiety of methcathinone showed that −Cl, −Br and −CH₃ substitutions maintained the high potency activity at DAT and NET using the synaptosome release assay; these modifications improved the potency of the compounds at SERT by ~30-fold (Walther et al., 2019). The −OCH₃ substitution slightly decreased activity at DAT and NET (~5-fold) and improved activity at SERT ~8-fold compared to the -aryl unsubstituted methcathinone (Walther et al., 2019). The −CF₃ substitution in the 3-position decreased potency at DAT (34-fold decrease) and NET (17-fold decrease), and improved potency at SERT (21-fold increase) (Walther et al., 2019). Similar modifications of the 2-position of methcathinone were tested using the synaptosome release assay; overall, these modifications decreased potency at all transporters, with the exception of the −CH₃ substitution, which produced a small decrease in potency at DAT and NET, but a 5-fold increase in potency at SERT (Walther et al., 2019).

The extension of the N-methyl moiety of methcathinone to N-ethyl (Fig. 9) blunts its activity as a releaser and transforms it into a reuptake inhibitor at DAT, (H. Yu, Rothman, Dersch, Partilla, & Rice, 2000) while maintaining its activity as a releaser at NET and its weak releaser activity at SERT (H. Yu et al., 2000). Similarly, 4-methyl methcathinone (a.k.a. mephedrone, Fig. 8) demonstrates robust releaser activity at DAT and SERT. However, extension of the N-alkyl chain by one carbon generates N-ethyl 4-methyl- cathinone (Fig. 9), which maintains robust releaser activity at SERT yet transforms into an inhibitor at DAT (Saha et al., 2015). This trend of transition from substrate to inhibitor by N-methyl to N-ethyl extension observed at DAT, but not at NET or SERT, was also reported for a similar series of compounds based on the 4 methyl-amphetamine scaffolds (Battisti et al., 2018; Solis et al., 2017). Bupropion (Fig. 1) is a reuptake inhibitor at DAT and NET, possessing very weak activity at SERT. Bupropion is an analog of methcathinone characterized by an N-tert-butyl moiety and a 3-Cl substitution. A deconstruction study showed that the N-methyl counterpart of bupropion functions as a releaser at all three monoamine transporters but that the N-isopropyl substitution transforms the compound into an inhibitor at DAT and NET and releaser at SERT (Shalabi, Walther, Baumann, & Glennon, 2017). These findings suggest that extension beyond N-methyl in the methcathinone scaffold transforms the compound’s activity from a substrate to an inhibitor at DAT.

Figure 9.

Chemical structure of N-Ethylcathinone and two of its analogs.

The length of the α-alkyl chain was varied in the N-ethyl-cathinone scaffold; the stepwise lengthening of this aliphatic chain from methyl to n-butyl (N-ethylhexedrone, Fig. 9) progressively increased potency in an uptake inhibition assay at DAT (Nadal-Gratacos et al., 2023). Importantly, this result demonstrates that lengthening the α-alkyl chain in the context of a bulkier N-ethyl background of the cathinone scaffold increases inhibitor potency at DAT. These results show that modifications of simple cathinones via variants possessing both extended N-alkyl and α-alkyl substitutions can produce potent DAT versus SERT selective inhibitors. In the next section, we will discuss one of these scaffolds: the pyrrolidinophenone class of psychostimulants.

Pyrrolidinophenone scaffold

Clandestine chemists have modified the chemical scaffold of the pyrrolidinophenone class of psychostimulants to generate novel “designer drugs” for distribution within underground markets and via the Internet. In this section, we will describe results from systematic structure-activity relationship studies; α-PVP (Fig. 1) will be considered the starting scaffold, and we will discuss the consequences of alterations of its moieties in modulating activity at monoamine transporters. α-PVP is a very potent ligand at DAT and NET and is virtually inactive at SERT (Meltzer, Butler, Deschamps, & Madras, 2006). Modifications of its aryl ring with 4-CH₃, 4-halogen, or 3,4-methylendioxy substitutions produce only slight variations in the high DAT potency of these compounds (Kolanos, Solis, Sakloth, De Felice, & Glennon, 2013; Marusich et al., 2014; Meltzer et al., 2006; Rickli et al., 2015), with the exception of 4-CF₃ substitution, which strongly decreases DAT potency (Davies, Nguyen, Eltit, & Glennon, 2023).

The 3,4-methylendioxy substituted α-PVP (MDPV, Fig. 1) produced an apparent outward current in oocytes expressing DAT. In contrast, methcathinone (Fig. 1) or mephedrone (Fig. 8) produced the inward currents typical of substrates (Cameron et al., 2013a; Cameron et al., 2013b; Kolanos et al., 2013). MDPV and α-PVP did not generate Ca²⁺ signals in cells expressing DAT within the Ca²⁺ flux assay, but blocked the signal induced by dopamine (Steele et al., 2021). Moreover, these compounds were inactive when tested in a release assay using synaptosomes, but strongly inhibited uptake through DAT and NET (Marusich et al., 2014). Altogether, these studies suggest that these pyrrolidinophenone compounds are inhibitors at monoamine transporters.

Because α-PVP and MDPV (Fig. 1) are bulky ligands relative to methcathinone, possessing an α-propyl chain and the tertiary amine portion of the pyrrolidine moiety, it is not surprising that they behave as inhibitors at DAT and NET (Marusich et al., 2014). Stereoisomers of MDPV (Fig. 10) and α-PVP were studied and tested in DAT and NET using synaptosome uptake assays. The S(+) configuration is at least 20-fold more potent than the R(−) enantiomer; thus these compounds are stereoselective, and the S(+) enantiomer mediates most of the activity.(Kolanos, Partilla, et al., 2015; Schindler et al., 2020).

Figure 10.

Chemical structure of MDPV and the relative potency at DAT of related compounds. Deconstruction of MDPV was done in (Kolanos et al., 2013).

An elegant deconstruction study of MDPV showed that both the pyrrolidine moiety and the α-propyl substitutions are important for the high DAT potency of MDPV in HEK293 cell uptake studies. Interestingly, the N,N-dimethyl deconstructed variant of MDPV (Fig. 10) presented less than full but still significant activity at DAT, suggesting that tertiary amines (usually weak at DAT) in combination with the α-propyl substitution are sufficient to attain significant activity at DAT (Kolanos et al., 2013). Several studies have shown that shortening the α-propyl chain in α-PVP or MDPV attenuates potency at DAT in various functional studies (Eshleman et al., 2017; Kolaczynska, Thomann, Hoener, & Liechti, 2021; Kolanos, Sakloth, et al., 2015; Kolanos et al., 2013; Marusich et al., 2014; Rickli et al., 2015; Steele et al., 2021) (Fig. 10). On the other hand, the α-alkyl chain of α-PVP was studied by systematically extending it one carbon at a time, generating the series: -butyl, -cyclopentyl, and -cyclohexanyl. These substitutions were well-tolerated and demonstrated high potency DAT inhibition in synaptosome uptake assays (Kolanos, Sakloth, et al., 2015). In the same study, the expansion of the pyrrolidine moiety in α-PVP to a piperidine decreased DAT inhibition potency ~7-fold (Kolanos, Sakloth, et al., 2015). These studies suggest that rings larger than pyrrolidine are not well-tolerated, yet bulkier hydrophobic extensions of the α-alkyl chain are well-tolerated by pyrrolidinophenones to preserve DAT inhibition. Figure 11 summarizes the main findings about structure-activity relationship studies of cathinones at monoamine transporters described above.

Figure 11.

The effects of structural modifications of “simple” and “complex” cathinones at inhibiting DAT.

Recently, a series of benzoylpiperidines, a cathinone-like version of methylphenidate or a constrained version of pentedrone (Jones, Eltit, & Dukat, 2023), were studied via DAT uptake inhibition assay using APP⁺ as the substrate. Although the parental unsubstituted molecule was active but weak at DAT (weaker than cocaine), the 3,4-dichloro substitution increased potency ~20-fold, producing an inhibitor more potent than cocaine and threo-methylphenidate (Yadav-Samudrala, Eltit, & Glennon, 2019). The 3,4-dichloro substituted benzoylpiperidine was weak at SERT, thus showing very high DAT versus SERT selectivity (Jones et al., 2023). Via the use of the Ca²⁺ flux assay, it was determined that the members of this class of compounds are likely DAT inhibitors, as they did not produce Ca²⁺ signals in cells co-expressing DAT and voltage-gated Ca²⁺ channels (Jones et al., 2023; Yadav-Samudrala et al., 2019). These compounds are part of a new class of selective DAT cathinones that warrant further investigation.

DAT versus SERT in vitro selectivity as a predictor of abuse liability of cathinones in vivo

There exists a remarkable diversity within the classes of organic molecules active at the monoamine transporters. The interacting profile of compounds at DAT, SERT, and NET varies along two dimensions: the relative potency of a compound at each transporter, and whether it is a substrate or inhibitor of one, two or all (Blough et al., 2014; Rothman et al., 2003; Solis et al., 2017). As shown in the previous section, minor chemical modifications of known active small molecules can generate classes of compounds with divergent profiles at these transporters (Battisti et al., 2018; Bonano et al., 2015; Sakloth et al., 2015; Solis et al., 2017). Monoamine transporter substrates such as methamphetamine and methcathinone are psychostimulants with high abuse liability, and although some psychostimulants are used as attention deficit and hyperactivity disorder medication, they are common recreational drugs of abuse (Negus & Miller, 2014; Rothman & Baumann, 2003; UNODC, 2019). The rewarding effect of these compounds is primarily responsible for their high abuse potential and is principally driven by interference with DAT function (Di Chiara & Imperato, 1988; Giros, Jaber, Jones, Wightman, & Caron, 1996; Lyness, Friedle, & Moore, 1979; Negus & Miller, 2014). The administration of methcathinone or other selective DAT substrates produced a robust increase in extracellular dopamine in the nucleus accumbens (NA) and a minor increase in serotonin after administration to rats evaluated using microdialysis, thus demonstrating a high dopamine over serotonin selectivity in vivo (Johnson, Banks, Selley, & Negus, 2018; Suyama et al., 2016). These findings directly recapitulate the high DAT over SERT selectivity of this type of psychostimulant observed in vitro (Sakloth et al., 2015). The administration of 4-substituted (−F, −Cl, −Br, −CH₃, or −OCH₃) versions of methcathinone—which, as discussed earlier in the chapter, are associated with a gradual decrease in DAT versus SERT selectivity in vitro—produced elevations of both extracellular dopamine and serotonin in the NA as determined by in vivo microdialysis experiments, which recapitulated the progressive decrease in dopamine and increase in serotonin responses according to their DAT versus SERT selectivity in vitro (Suyama et al., 2016). On the other extreme, fenfluramine, a selective SERT substrate in vitro (Rothman & Baumann, 2003), gives rise to elevated serotonin but not dopamine in the NA after in vivo administration (Suyama et al., 2016). Taken together, these results suggest that the in vitro DAT versus SERT selectivity of monoamine transporter substrates (structurally related to methcathinone) can be used as a predictor of the dopamine versus serotonin selectivity profile of a compound and its propensity to increase these neurotransmitters in vivo. Specifically, more DAT selective compounds give rise to increased dopamine, and more SERT selective compounds give rise to increased serotonin.

The behavioral correlates of the neurochemical effects of cathinones have been studied using various assays. One of these approaches is intracranial self-stimulation in rats, where methcathinone facilitates intracranial self-stimulation (ICSS) in a manner like other DAT selective psychostimulants such as amphetamine and methamphetamine (Negus & Miller, 2014). ICSS facilitation is considered a behavioral signature of abuse liability (Negus & Miller, 2014). By testing unsubstituted and 4-substituted methcathinones having different degrees of DAT versus SERT selectivity, it was possible to categorize different degrees of abuse potential for these drugs. The highly DAT selective methcathinone progressively decreases the expression of abuse related effects (i.e., decrease in ICSS facilitation) as substitution at the 4 position gradually increases selectivity towards SERT (Bonano et al., 2015; Suyama et al., 2016).

Methamphetamine (N-methyl amphetamine) and MDMA (Fig. 1) are structurally similar drugs that differ in their DAT versus SERT pattern of selectivity. Methamphetamine is DAT selective while MDMA (as other aryl substituted amphetamines) is a non-selective monoamine transporter substrate (both are potent substrates at NET) (Rothman & Baumann, 2003). These two drugs are widely used recreationally, but their patterns of abuse by humans are different (Kamilar-Britt & Bedi, 2015; Kirkpatrick et al., 2012). Methamphetamine is an archetypical psychostimulant, engendering euphoria, as well as enhanced alertness and cognition, and stimulating compulsive re-dosing (Kirkpatrick et al., 2012). MDMA, as other psychostimulants, gives rise to euphoria and general metrics of stimulation, but in contrast to “pure” psychostimulants, is associated with additional effects of tactile intensification and enhancement of pro-social behavior and empathy. Indeed, it has been proposed that MDMA-like drugs constitute a class unto themselves: the empathogens (or entactogens) (Hysek et al., 2014; Kamilar-Britt & Bedi, 2015; Nichols, 2022; Oberlender & Nichols, 1988). Furthermore, MDMA may acutely increase negative subjective effects, such as mental fatigue and apathy, that may explain its lesser abuse potential when compared to methamphetamine (Kirkpatrick et al., 2012), accordingly, non-selective monoamine transporters substrates such as MDMA show less reinforcing efficacy in experimental animals when compared to classical psychostimulants (Aarde & Taffe, 2017; Lile, Ross, & Nader, 2005).

A behavioral approach to categorize similarities (or divergence) of subjective effects of agents is drug-discrimination studies in experimental animals (Young, 2009). For instance, in squirrel monkeys trained to discriminate methamphetamine, MDMA did not substitute for methamphetamine (Wakeford et al., 2021). However, in MDMA-trained squirrel monkeys, methamphetamine partially substituted MDMA. This asymmetric discriminatory response is consistent with differences in the neurochemical mechanisms of these two drugs. In the same study, methcathinone, methylone (3,4-methylenedioxy methcathinone), and mephedrone (4-methyl methcathinone) were studied in methamphetamine- and MDMA- trained monkeys. Methcathinone completely substituted for methamphetamine, but substituted for MDMA at a level of 50%, which was the same pattern of substitution observed for methamphetamine. These results indicate that methamphetamine and methcathinone, both selective at DAT (versus SERT), have very similar subjective effects within these monkeys. Methylone completely substituted for MDMA and substituted for methamphetamine at a level of 76%, whereas mephedrone also completely substituted for MDMA but substituted for methamphetamine only at a level of 18% (Wakeford et al., 2021). The latter experimental approach indicates that knowing the DAT to SERT selectivity of a substrate of the cathinone family can be used to predict their effect on animals. In this paradigm, DAT selective substrates such as methcathinone produce subjective effects like the classical psychostimulant methamphetamine, while non-selective cathinone substrates (i.e., aryl substituted cathinones) produce effects more like the empathogen MDMA. Also in the latter experimental example, methylone is more similar to methamphetamine than mephedrone and MDMA, indicating that even compounds from the same subfamily are not identical and that subtle differences in selectivity at transporters and/or other pharmacological parameters may fine tune subjective perceptions [see (Gatch, Dolan, & Forster, 2020) for further discussion].

More complex cathinones having N-alkyl substitutions longer than methyl or tertiary amines, including long or bulky α-alkyl substitutions, are inhibitors at transporters and possess high DAT versus SERT selectivity. As discussed in the previous section, in general, simple substitutions in the aryl moiety of these complex cathinone scaffolds may modulate but not strongly alter their selectivity pattern at transporters (Davies et al., 2023; Kolaczynska et al., 2021). Members of this family of complex cathinones such as α-PVP and MDPV produced a robust increase in extracellular dopamine in the NA of rats. This response was selective, as extracellular serotonin levels in this brain region were unchanged by these cathinones (Johnson et al., 2018; Schindler et al., 2020). Like other psychostimulant inhibitors at monoamine transporters, complex cathinones produce robust and very potent facilitation of ICSS response, thus indicating high abuse potential (Johnson et al., 2018; Kolanos, Partilla, et al., 2015). Moreover, α-PVP and MDPV showed strong and persistent self-administration and were associated with even greater reinforcing potency than methamphetamine and cocaine in rhesus monkeys (Collins, Sulima, Rice, & France, 2019). In drug-discrimination studies done in squirrel monkeys, α-PVP and MDPV produced full substitution in methamphetamine-trained animals and partial substitution (at ~50 level) in MDMA-trained animals, recapitulating the pattern of substitution observed for methcathinone and methamphetamine in methamphetamine- and MDMA- trained animals (Wakeford et al., 2021). These results using behavioral studies in experimental animals demonstrate that these complex cathinones have high potency at DAT and are psychostimulants with high abuse potential, thus explaining their prevalence in the illicit market of synthetic drugs.

Conclusion

The study and identification of new compounds that interact with monoamine transporters is valuable from several perspectives: identifying molecules with diverse profiles of interaction at monoamine transporters can provide a resource to develop new, more efficacious drugs for use in personalized medicine. In addition, better understanding of the structural aspects of compounds dictating DAT activity and selectivity may constitute a tool for forensic scientists and policy makers regarding the early identification and scheduling of novel “designer drugs” often appearing within clandestine markets. Because DAT ligands may be associated with significant abuse liability, in some instances, DAT can be considered an “anti-target” for drug developers, and in vitro assays can help to flag DAT ligands early in the screening process.

Abbreviations:

APP⁺

4-(4-(dimethylamino)phenyl)-1-methylpyridinium

DAT

dopamine transporter

FRET

fluorescence resonance energy transfer

MCAT

methcathinone

MDMA

3,4-methylenedioxymethamphetamine

MDPV

methylenedioxypyrovalerone

MPP⁺

1-Methyl-4-phenylpyridinium

NET

norepinephrine transporter

α-PVP

α-pyrrolidinopentiophenone

SERT

serotonin transporter