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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/01/20/jpet.115.229765.DC1

1521-0103/357/1/134–144$25.00 http://dx.doi.org/10.1124/jpet.115.229765 THE JOURNAL OF AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:134–144, April 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

In Vitro Characterization of Psychoactive Substances at Rat, Mouse, and Human Trace -Associated Receptor 1 s

Linda D. Simmler, Danièle Buchy, Sylvie Chaboz, Marius C. Hoener, and Matthias E. Liechti Division of Clinical Pharmacology and Toxicology, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland (L.D.S., M.E.L.); and Neuroscience Research, Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland (D.B., S.C., M.C.H) Received October 5, 2015; accepted January 19, 2016

ABSTRACT Downloaded from -associated receptor 1 (TAAR1) has been implicated Most , , and aminoindanes in the behavioral effects of -type in exhibited potentially physiologically relevant rat and mouse rodents. TAAR1 has also been suggested as a target for novel TAAR1 activation (EC50 , 5 mM)andshowedfullorpartial medications to treat psychostimulant addiction. We previously (Emax , 80%) properties. derivatives, reported that binding affinities at TAAR1 can differ between including and methylenedioxypyrovalerone, structural analogs of psychostimulants, and species differences exhibited weak (EC50 5 5–10 mM) to negligible (EC50 . 10 mM) jpet.aspetjournals.org have been observed. In this study, we complement our previous binding properties at TAAR1. Pipradrols, including methylphe- findings with additional substances and the determination of nidate, exhibited no affinity for TAAR1. We found considerable functional activation potencies. In summary, we present here species differences in activity at TAAR1 among the highly active pharmacological in vitro profiles of 101 psychoactive substances ligands, with a rank order of rat . mouse . human. This at human, rat, and mouse TAAR1. p-, b-phenylethylamine, characterization provides information about the pharmacologi- and were included as endogenous comparator cal profile of psychoactive substances. The species differences compounds. Functional cAMP measurements and radioligand emphasize the relevance of clinical studies to translationally displacement assays were conducted with human embryonic complement rodent studies on the role of TAAR1 activity for at ASPET Journals on September 23, 2021 kidney 293 cells that expressed human, rat, or mouse TAAR1. psychoactive substances.

Introduction phenethylamine derivatives, also bind to TAAR1 (Bunzow et al., 2001; Wainscott et al., 2007; Simmler et al., 2013; Reese Trace amine-associated receptor 1 (TAAR1) is a relatively et al., 2014). The activation of TAAR1 results in elevations in – recently discovered G protein coupled receptor (Borowsky intracellular cAMP (Bunzow et al., 2001; Xie and Miller, et al., 2001; Bunzow et al., 2001), which is expressed in 2007). brain regions and throughout the limbic Amphetamines have structural similarity to the endoge- system (Borowsky et al., 2001; Lindemann et al., 2008; nous b-PEA and were initially identified as TAAR1 Espinoza et al., 2015). TAAR1 is thought to play a role in ligands (Bunzow et al., 2001). We previously reported that regulating the limbic network, reward circuits, cognitive many novel psychoactive substances are also ligands of rat processes, and mood states and has been proposed as a and mouse TAAR1 (Simmler et al., 2013, 2014a,b; Rickli et al., pharmacological target for the treatment of mental disorders 2015a,b,c). However, several novel psychoactive substances do (Wolinsky et al., 2007; Lindemann et al., 2008; Miller, 2011; not bind to TAAR1, and little has been reported on the Revel et al., 2013) and psychostimulant dependence (Di Cara activation of human TAAR1. The pharmacological and toxi- et al., 2011; Pei et al., 2014; Cotter et al., 2015; Jing and Li, cological actions of novel psychoactive substances are also of 2015). TAAR1 is stimulated by endogenous ligands, including interest because of the emergence of hundreds of these b b -phenylethylamine ( -PEA), p-tyramine, tryptamine, and substances, referred to as “legal highs” or “research chem- 3-iodothyronamine (Scanlan et al., 2004; Zucchi et al., 2006). icals.” These chemical compounds are recreationally used but Many psychoactive compounds, including amphetamine and have poorly known pharmacological properties. TAAR1 is implicated in the control of neuronal firing frequency and is thus likely to contributetopsychoactive This research was supported by the Federal Office of Public Health [Grant and abuse-related effects. Ex vivo electrophysiology 13.006497] and F. Hoffmann-La Roche Ltd. and the University of Basel experiments that used slices from TAAR1 knockout (KO) [Translational Medicine Hub Innovation Fund]. dx.doi.org/10.1124/jpet.115.229765. mice (Lindemann et al., 2008) or pharmacological TAAR1 s This article has supplemental material available at jpet.aspetjournals.org. blockade (Bradaia et al., 2009) suggest that TAAR1 is

ABBREVIATIONS: b-PEA, b-phenylethylamine; DA, ; 5-HT, 5-hydroxytryptamine (); KO, knockout; MDMA, 3,4-methylenedioxymethamphetamine; PBS, phosphate-buffered ; RO5166017, (S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol- 2-ylamine; RO5203648, (S)-4-(3,4-dichlorophenyl)-4,5-dihydrooxazol-2-amine dihydrochloride; TAAR1, trace amine-associated receptor 1; WT, wild type.

134 Psychoactive Substances and TAAR1 135 constitutively active to control dopamine (DA) and serotonin (Zürich, Switzerland). and MDAI were synthesized in [5-hydroxytryptamine (5-HT)] tone. our laboratory as reported previously (Simmler et al., 2013, 2014b). 3 Compared with wild-type (WT) mice, TAAR1 KO mice Radiochemicals ( H-isotopes) were purchased from PerkinElmer 3 were shown to consume more ethanol and be more suscepti- (Schwerzenbach, Switzerland), with the exception of [ H]RO5166017 ble to its sedating effects (Lynch et al., 2013). The TAAR1 [(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine], which was synthesized at Roche (Basel, Switzerland). RO5203648 [(S)-4-(3,4-dichlorophenyl)-4,5- Cell Culture and Membrane Preparation. Human embryonic dihydrooxazol-2-amine dihydrochloride] reduced self- kidney 293 cells that stably expressed human, rat, or mouse TAAR1 administration and cocaine-induced hyperlocomotion in rats were used as described previously (Revel et al., 2011). All of the cell

(Revel et al., 2012b). Both selective TAAR1 partial lines were maintained at 37°C and 5% CO2 in high-glucose Dulbecco’s and selective TAAR1 full agonists reduced cocaine self- modified Eagle’s medium that contained 10% fetal calf serum (heat- administration and the reinstatement of drug-seeking behavior inactivated for 30 minutes at 56°C), 1% penicillin/streptomycin, and in rats (Pei et al., 2014, 2015) and decreased cocaine-mediated 375 mg/ml Geneticin (Gibco, Zug, Switzerland). For membrane intracranial self-stimulation (Pei et al., 2015). Reductions of preparation, the cells were released from culture flasks using hyperlocomotion, self-administration, and reinstatement by trypsin/EDTA, harvested, washed twice with ice-cold phosphate- 21 21 Â TAAR1 partial agonism have also been reported for metham- buffered saline (PBS; without Ca and Mg ), pelleted at 1000 g for 5 minutes at 4°C, frozen, and stored at 280°C. Frozen pellets were phetamine (Cotter et al., 2015; Jing and Li, 2015). These studies suspended in buffer A [20 ml HEPES-NaOH (20 mM, pH 7.4) that established TAAR1 as a promising target for therapeutics to

contained 10 mM EDTA] and homogenized with a Polytron (PT 6000; Downloaded from treat substance use disorders, regardless of the TAAR1 Kinematica, Luzern, Switzerland) at 14,000 rpm for 20 seconds. The binding properties of the abused substances themselves. By homogenate was centrifuged for 30 minutes at 48,000 Â g at 4°C. The directly interacting with TAAR1, psychoactive substances supernatant was removed and discarded, and the pellet was resus- may also modulate their own pharmacological effects. For pended in buffer A using the Polytron (20 seconds at 14,000 rpm). The example, amphetamine induces markedly more striatal centrifugation and removal of the supernatant was repeated, and monoamine release in TAAR1 KO mice than in WT mice the final pellet was resuspended in buffer A and homogenized using (Lindemann et al., 2008). and amphet- the Polytron. Typically, 2-ml aliquots of membrane portions were jpet.aspetjournals.org 2 amine increase locomotor activity to a greater extent in stored at 80°C. With each new membrane batch, the dissociation constant (K ) was determined by a saturation curve. TAAR1 KO mice compared with WT mice (Achat-Mendes d Radioligand Binding Assay. For the competitive binding assays, et al., 2012). TAAR1 also plays a role in contingent oral the TAAR1 agonist [3H]RO5166017 was used as a TAAR1 radioligand methamphetamine intake (Harkness et al., 2015). Similar to at a concentration equal to Kd values, which was usually around 0.7 nM amphetamine and methamphetamine, 3,4-methylenedioxy- (mouse TAAR1) and 2.3 nM (rat TAAR1). Nonspecific binding was methamphetamine (MDMA) significantly increased extra- defined as the amount of radioligand bound in the presence of 10 mM cellular striatal DA and 5-HT levels to a greater extent in RO5166017. Compounds were tested at a broad range of concentrations at ASPET Journals on September 23, 2021 TAAR1 KO mice compared with WT mice (Di Cara et al., (10 pM to 10 mM) in duplicate. Compounds (20 ml/well) were transferred 2011). TAAR1 KO mice are hypersensitive to psychoactive to a 96-deep-well plate (TreffLab, Degersheim, Switzerland), and 180 ml substances that are also TAAR1 ligands. By contrast, TAAR1 binding buffer (20 mM HEPES-NaOH, 10 mM MgCl2, and 2 mM CaCl2, m m overexpression in mice reduced locomotor activity in pH 7.4), 300 l radioligand, and 500 l membranes (resuspended at 60 mg protein/ml) were added. The plates were incubated at 4°C for response to amphetamine (Revel et al., 2012a). Because 90 minutes. Incubations were terminated by rapid filtration through MDMA, methamphetamine, and amphetamine are TAAR1 Unifilter-96 plates (Packard Instrument Company, PerkinElmer) and ligands, they possibly autoinhibit their own effects on neuro- glass filters GF/C (PerkinElmer) presoaked for 1 hour in polyethylenimine transmitter release. Di Cara et al. (2011) supported the (0.3%) and washed three times with 1 ml cold binding buffer. After the concept of the autoregulation of TAAR1-activating psychostim- addition of 45 ml Microscint 40 (PerkinElmer), the Unifilter-96 plate ulants, showing that the TAAR1 ligand o-phenyl-3-iodotyramine was sealed. After 1 hour, radioactivity was counted using a TopCount decreased the DA release response to p-chloroamphetamine, Microplate Scintillation Counter (Packard Instrument Company). which is a psychostimulant that is inactive at TAAR1, in WT IC50 values were determined by calculating nonlinear regression mice but not in TAAR KO mice. curves for a one-site model using at least three independent 10-point Because TAAR1 might be significantly involved in the mode concentration-response curves, run in duplicate, for each compound. K (affinity) values, which correspond to the dissociation constants, of action of many psychoactive drugs, we determined the i were determined using the Cheng–Prusoff equation. Ki values are TAAR1 binding and activation properties of a series of mostly presented as means 6 S.D. (in micromoles). For reasons of integrity, novel substances and found considerable differences in Table 1 includes several Ki values that we have previously published TAAR1 binding properties within and between substance as indicated by the references in the table. classes. Our data set provides evidence of significant species Functional TAAR1 Activity. Substances were tested for binding differences in ligand/receptor interactions between rodent and affinity at rat and mouse TAAR1 as described above. If relevant human TAAR1. binding was observed, then we also determined potencies for receptor activation and maximal efficacy at rat, mouse, and human TAAR1 to

characterize the compounds as full or partial agonists (Emax , 80%). b Materials and Methods The endogenous TAAR1 ligands -PEA, p-tyramine, and tryptamine served as reference substances for comparisons of affinity values and Chemicals. The compounds were purchased from Lipomed functional potency and efficacy. cAMP measurements were performed (Arlesheim, Switzerland) or Cayman Chemicals (Ann Arbor, MI) as described previously (Revel et al., 2011). In brief, cells that expressed as racemic mixtures, with the exception of D-amphetamine, rat or mouse TAAR1 were plated on 96-well plates (BIOCOAT 6640; D-methamphetamine, (1)-, and (2)-ephedrine. A list of Becton Dickinson, Allschwil, Switzerland) and incubated for 20 hours generic or full chemical names is provided in (Supplemental Table 1). at 37°C. Prior to stimulation of the cells with a broad concentra- 5-EAPB, diclophensine, diphenidine, , methoxphenidine, tion range of agonists for 30 minutes at 37°C, the cells were and N-methyl-2-AI were obtained from the Forensic Institute Zürich washed with PBS and preincubated with PBS that contained 1 mM TABLE 1 136 Binding affinities, activation potencies, and efficacy of psychoactive substances and endogenous comparator compounds at rat, mouse, and human TAAR1

Values are given as means 6 S.D. Ki values of human TAAR1 were not determined because of the lack of a reliable radioligand needed for the in vitro assay. EC50 was determined for substances with relevant binding (Ki value , 10 mM). Ki values from our previous publications are included and referenced. The generic or full chemical names for abbreviated substances are listed in Supplemental Table 1. ime tal. et Simmler Rat TAAR1 Mouse TAAR1 Human TAAR1 Substance Receptor Activation Receptor Activation Emax Activation Potency EC50 Emax Emax Binding Ki Potency EC50 Binding Ki Potency EC50 mM% mM%mM% Endogenous ligands b-PEA 0.24 6 0.12 0.11 6 0.08 100 6 11 0.31 6 0.15 0.20 6 0.09 102 6 8 0.26 6 0.09 104 6 10 p-Tyramine 0.059 6 0.020 0.03 6 0.021 94 6 11 0.38 6 0.13 0.28 6 0.17 88 6 9 0.99 6 0.29 91 6 8 Tryptamine 0.13 6 0.05 0.41 6 0.15 91 6 81.46 0.4 2.7 6 0.5 117 6 2216 13 73 6 16 25B-NB2OMe 0.28 6 0.002a 1.2 6 0.4 37 6 19 4.5 6 1.7a 6.1 6 2.5 47 6 4 .10 25C-NB2OMe 0.52 6 0.10a 1.6 6 0.4 29 6 5156 2a 6.7 6 1.6 48 6 6 .30 25D-NB2OMe 0.81 6 0.10a 1.5 6 0.4 34 6 4136 4a 4.0 6 0.6 67 6 12 .30 25E-NB2OMe 0.26 6 0.03a 0.65 6 0.39 37 6 15 1.1 6 0.3a 1.8 6 0.3 46 6 10 .10 25H-NB2OMe 1.5 6 0.2a 3.0 6 1.4 37 6 11 .20a 6.1 6 2.6 53 6 10 .10 25I-NB2OMe 0.44 6 0.07a 1.8 6 1.0 32 6 17 3.4 6 0.9a 5.2 6 2.2 17 6 9 .10 25N-NB2OMe 2.2 6 0.14a 1.5 6 0.3 34 6 9 .20a .30 .10 25P-NB2OMe 0.055 6 0.004a 0.51 6 0.20 34 6 17 0.24 6 0.03a 1.3 6 0.2 40 6 26 .10 25T2-NB2OMe 0.35 6 0.02a 0.93 6 0.38 24 6 14 4.2 6 0.6a 2.9 6 0.6 30 6 23 .10 25T4-NB2OMe 0.12 6 0.02a 1.1 6 0.6 31 6 12 1.5 6 0.4a 4.7 6 2.1 33 6 12 .10 25T7-NB2OMe 0.088 6 0.032a 0.55 6 0.17 52 6 23 1.0 6 0.2a 2.1 6 0.5 68 6 23 .10 -B 0.079 6 0.008a 0.24 6 0.16 57 6 16 2.2 6 0.3a 2.3 6 0.4 69 6 13 3.3 6 0.9 10 6 2 2C-B-Fly 0.029 6 0.008b 0.27 6 0.16 48 6 1 0.71 6 0.23b 1.8 6 0.7 49 6 6 .30 2C-C 0.11 6 0.02a 0.34 6 0.15 51 6 14 4.1 6 0.3a 2.3 6 1.5 57 6 23 .10 2C-D 0.15 6 0.03a 0.49 6 0.14 55 6 10 3.5 6 0.1a 2.0 6 0.2 61 6 19 .10 2C-E 0.066 6 0.009a 0.18 6 0.14 72 6 13 1.2 6 0.1a 1.1 6 0.2 64 6 23 .10 2C-H 0.9 6 0.16a 1.5 6 0.7 80 6 7116 2a 7.5 6 3.3 56 6 14 6.5 6 0.7 53 6 5 2C-I 0.12 6 0.02a 0.19 6 0.11 50 6 19 3.3 6 0.1a 2.4 6 0.8 51 6 21 .10 2C-N 0.34 6 0.02a 0.25 6 0.12 59 6 16 .20a 15 6 12 28 6 13 .10 2C-P 0.020 6 0.005a 0.030 6 0.022 84 6 8 0.28 6 0.03a 0.56 6 0.23 91 6 27 4.2 6 0.5 72 6 11 2C-T2 0.043 6 0.006a 0.096 6 0.051 86 6 17 2.2 6 0.6a 4.3 6 2.8 54 6 14 .10 2C-T4 0.053 6 0.008a 0.083 6 0.050 67 6 11 4.5 6 0.9a 3.7 6 2.2 51 6 21 .10 2C-T7 0.033 6 0.005a 0.079 6 0.034 83 6 11 0.56 6 0.12a 0.91 6 0.67 67 6 4 .10 Mescaline 3.3 6 0.5a 3.7 6 1.8 37 6 18 11 6 4a 4.8 6 3.7 25 6 20 .10 Mescaline-NB2OMe 13 6 6a .30 .20a .30 .10 Amphetamines Amphetamine 0.23 6 0.18c 0.66 6 0.61 91 6 20 0.089 6 0.059c 0.53 6 0.67 90 6 30 2.8 6 0.8 91 6 15 4-APB 0.11 6 0.02b 0.16 6 0.09 75 6 92.16 0.1b 0.85 6 0.78 72 6 12 4.1 6 2.1 50 6 21 5-APB 0.042 6 0.006b 0.067 6 0.040 88 6 12 0.11 6 0.002b 0.13 6 0.07 67 6 11 6.1 6 2.3 43 6 16 6-APB 0.052 6 0.017b 0.042 6 0.029 90 6 14 0.056 6 0.015b 0.067 6 0.039 93 6 13 7.2 6 0.3 47 6 7 7-APB 0.066 6 0.006b 0.058 6 0.033 109 6 13 0.13 6 0.02b 0.11 6 0.07 95 6 17 0.63 6 0.13 89 6 4 5-APDB 0.49 6 0.05b 1.4 6 0.7 93 6 20 0.77 6 0.06b 1.5 6 1.2 64 6 14 .10 6-APDB 1.0 6 0.04b 1.0 6 0.97 83 6 17 0.21 6 0.04b 0.51 6 0.27 95 6 19 .10 5-EAPB 0.81 6 0.08b 1.1 6 0.6 39 6 10 .15b 13 6 7266 11 .10 N-Ethylamphetamine 2.5 6 1.4d 0.88 6 0.05 62 6 10 .10d .10 .10 4-Fluoroamphetamine 0.081 6 0.041e 0.069 6 0.004 78 6 12 0.32 6 0.1e 0.13 6 0.02 77 6 12 3.5 6 0.6 67 6 9 4-Fluoromethamphetamine 0.24 6 0.09e 0.16 6 0.02 76 6 11 1.7 6 0.9e 0.46 6 0.05 69 6 6 6.2 6 2.2 44 6 11 5-IT 0.15 6 0.02 0.20 6 0.01 66 6 4 0.36 6 0.15 0.34 6 0.07 63 6 5 .30 5-MAPDB 0.67 6 0.09b 1.1 6 0.9 65 6 11 3.5 6 0.1b 4.3 6 3.0 59 6 11 .10 MBDB 1.2 6 0.1c 1.7 6 1.2 75 6 83.66 1.1c 4.1 6 1.1 34 6 28 .30 MDA 0.25 6 0.04b 0.74 6 0.16 86 6 5 0.16 6 0.01b 0.58 6 0.08 102 6 11 3.6 6 0.4 11 6 4 MDEA 2.7 6 1.0c 1.5 6 0.9 66 6 24 6.6 6 3.1c 6.2 6 3.4 35 6 33 .30 MDMA 0.37 6 0.12c 1.0 6 0.7 56 6 10 2.4 6 1.1c 4.0 6 1.0 71 6 16 35 6 21 26 6 8

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Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 23, 2021 23, September on Journals ASPET at TABLE 1—Continued

Rat TAAR1 Mouse TAAR1 Human TAAR1 Substance Receptor Activation Receptor Activation Emax Activation Potency EC50 Emax Emax Binding Ki Potency EC50 Binding Ki Potency EC50 Methamphetamine 0.35 6 0.12c 0.85 6 0.38 73 6 10 0.55 6 0.24c 0.73 6 0.47 78 6 7 5.3 6 2.3 70 6 21 4-Methylamphetamine 0.10 6 0.01 0.11 6 0.02 93 6 17 0.15 6 0.07 0.071 6 0.013 94 6 8 .30 4-MTA 0.28 6 0.04d 0.26 6 0.03 56 6 10 0.043 6 0.007d 0.089 6 0.016 79 6 19 .10 PMA 0.66 6 0.02d 0.34 6 0.13 83 6 8 0.14 6 0.10d 0.24 6 0.14 91 6 5 .30 PMMA 1.3 6 0.2d 0.63 6 0.29 75 6 15 0.26 6 0.11d 1.0 6 0.1 82 6 13 .30 4-Bromomethcathinone 1.8 6 0.1e 5.2 6 2.5 28 6 12 13 6 3e 15 6 7506 12 .30 .10d .10d .20 .20 .20c .20c Cathinone 2.2 6 0.7c 1.2 6 0.3 28 6 82.16 0.7c 1.2 6 0.3 66 6 31 6.9 6 3.2 53 6 16 N,N-Dimethylcathinone .10d .10d .10d .10d 4-Ethylmethcathinone .20e .20e .20c .20c 5.4 6 1.7c 15 6 3466 8 .10c .20 .30 3-Fluoromethcathinone .10d .10d MDPBP .20e .50e MDPPP 16 6 7e .20e MDPV 7.2 6 1.1c 5.9 6 2.7 62 6 14 .10c .30 .30 Mephedrone 4.3 6 2c 9.0 6 4.3 52 6 3 .10c 20 6 7876 16 .30 4.1 6 1.2c 8.2 6 2.5 41 6 10 .10c 6.8 6 2.7 64 6 16 .30 18 6 4d .20d 4.8 6 0.9b 5.7 6 0.9 53 6 20 6.5 6 2.8b 7.8 6 2.4 57 6 2 .30 3-Methylmethcathinone 5.7 6 1.4 .10 11 6 13.86 0.04 25 6 7 .10 4-Methylethcathinone .20d .20d .13c .10c Naphyrone .20c .20c .10d .10d .10d .10d a-PVP 16 6 6e .20e .13c .10c scocieSbtne n TAAR1 and Substances Psychoactive (2)-Ephedrine 3.7 6 0.9 2.5 6 0.7 42 6 5 .15 14 6 2316 7 .10 (+)-Ephedrine 5.2 6 1.7 10 6 6296 9 .15 19 6 10 21 6 16 .10 4-Fluoroephedrine 2.6 6 1.2e 2.2 6 0.8 40 6 18 18 6 8e 23 6 81006 10 .30 5-MeO-aMT 1.1 6 0.2 2.3 6 0.2 38 6 64.86 0.9 3.7 6 1.6 55 6 5 .10 4-HO-DiPT .15 .15 4-HO-MET 3.1 6 0.2 2.1 6 0.3 71 6 9126 32.56 1.1 78 6 4 .10 5-MeO-MiPT .15 .15 DiPT .15 .15 N,N-DMT 2.2 6 0.2 1.5 6 0.1 81 6 15 3.3 6 0.4 1.2 6 0.2 73 6 1 .10 Psilocin 1.4 6 0.2 0.92 6 0.58 85 6 7176 22.76 2.5 80 6 9 .30 Aminoindanes 2-AI 0.31 6 0.095f 0.11 6 0.04 90 6 52.16 0.4f 0.33 6 0.06 54 6 15 1.5 6 0.1 110 6 5 N-Methyl-2-AI 0.53 6 0.04 0.37 6 0.21 63 6 52.66 0.1 0.94 6 0.09 108 6 14 3.3 6 0.2 54 6 8 5-IAI 0.030 6 0.007f 0.033 6 0.013 96 6 24 1.1 6 0.4f 0.41 6 0.002 36 6 10 3.2 6 0.8 33 6 5 MDAI 0.57 6 0.19f 0.22 6 0.13 95 6 15 1.8 6 0.1f 0.52 6 0.24 99 6 14 4.1 6 0.5 30 6 7 .20f .20f m-CPP 0.054 6 0.010f 0.15 6 0.11 60 6 13 6.6 6 1.1f 3.2 6 1.2 40 6 20 .30 137

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Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 23, 2021 23, September on Journals ASPET at 138 Simmler et al.

3-isobutyl-1-methylxanthine for 10 minutes at 37°C and 5% CO2. Stimulation with 0.2% dimethylsulfoxide was set as the basal level,

max and the effect of 30 mM b-PEA was set as the maximal response. E Subsequently, the cells were lysed, and cAMP assays were performed according to the manufacturer’s instructions (cAMP kit; Upstate/ Millipore, Schaffhausen, Switzerland). Finally, the plates were read with a luminometer (1420 Multilabel Counter; PerkinElmer), and the 50 amount of cAMP was calculated. The results were obtained from at 30 20 30 30 10 least three independent experiments. Experiments were run in . . . . . duplicate or triplicate. EC50 values are presented as means 6 S.D. Activation

Potency EC (in micromoles). The Emax value for the functional activity data at TAAR1 describes the degree of functional activity compared with 100% for the endogenous ligand and full agonist b-PEA. 4 4 15 6 6 6 max E Results

The binding affinity values (Ki), receptor activation poten- cies (EC50), and maximal efficacy (Emax) of 104 substances are Downloaded from 50 summarized in Table 1. These substances represent the collection of compounds in our laboratory that have been used 0.2 44 3.5 13 344 to characterize the in vitro pharmacology of novel designer 30 30 6 6 6 drugs (for review, see Liechti, 2015). The substances for these . . 29

3.8 9.7 studies were chosen based on the availability of pure chemical compounds and the reported abuse of these substances. For a jpet.aspetjournals.org

Activation Potency EC subset of compounds, we have previously published rat and mouse TAAR1 affinities as indicated by references in Table 1, but no human, rat, and mouse activity data. All substances were grouped according to their basic chemical structure i f K a f f f c (Fig. 1) as phenethylamines, amphetamines, cathinones, 0.6 3 0.7 15 10 10 30 15 15 15 15 10 6 6 6 ephedrines, tryptamines, aminoindanes, pipradrols, and ...... 9 Receptor .

Binding piperazines. A few psychoactive substances, such as cocaine

and lysergic acid diethylamide, were added but not classi- at ASPET Journals on September 23, 2021 fied because of the lack of common basic structures. TAAR1 Binding and Functional TAAR1 Activation. 16 2.3 810 9 16 We found marked differences in affinities at TAAR1 and 6 6 6 6 max the functional activation of TAAR1 among the various E

30 psychoactive substances. The ligand properties varied con- siderably within substance classes, with the exception of cathinones and pipradrols, which generally exhibited none to very weak binding to TAAR1. We also observed species 50 0.18 59 0.4 29 10 58 0.1 differences in TAAR1 activation. At the human TAAR1, only 30 6 6 6 6 19 substances had EC50 values that indicated functional . 11 15 , m , m Activation 1.4 activation ( 10 M), whereas the EC50 values were 10 M 0.75 Potency EC

Rat TAAR1 Mouse TAAR1for 52 and 68 substances Human TAAR1 at the mouse and rat TAAR1, respectively. Therefore, below we present the properties of the psychoactive compounds compared with the endogenous

f a TAAR1 ligands b-PEA, p-tyramine, and tryptamine sepa- i K f f f c 0.06 0.1 0.05 rately for each species. 10 10 15 15 15 15 15 15 10 6 6 6 Human TAAR1. b-PEA and p-tyramine activated the ...... Receptor

Binding m 1.3 human TAAR1 with EC50 values of 0.26 and 0.99 M, 0.38 0.45 respectively, and showed full agonistic properties (Emax 5 104% and 91%, respectively). Amphetamine, 7-APB, and 2-AI had potency (EC50 5 0.6–2.8 mM) and agonist efficacy (Emax . 89%) that were comparable to b-PEA and p-tyramine at human TAAR1. Methamphetamine exhibited an EC50 of 5.3 mM and 70% efficacy. The endogenous ligand tryptamine exhibited weak activation of human TAAR1, with an EC50 of 21 mM(Emax 5 73%). Generally, most of the psychoactive compounds that were tested were weak human TAAR1 Continued

— ligands, and none of them were more potent than the b values for rat and mouse TAAR1 previously published in Simmler et al. (2014a). values for rat and mouse TAAR1 previously published in Rickli et al. (2015c).

values for rat and mouse TAAR1 previously published in Rickli et al. (2015b). endogenous ligand -PEA. values for rat and mouse TAAR1 previously publishedvalues in for Simmler rat et and al. mouse (2013). TAAR1 previously published in Rickli et al. (2015a). values for rat and mouse TAAR1 previously published in Simmler et al. (2014b). i i i i i i 2-DPMP Cocaine TFMPP D2PM Ethylphenidate Methoxphenidine LSD Diphenidine K K K

K K b m K Rat TAAR1. At the rat TAAR1, -PEA had a K of 0.24 M a b c d e f i Pipradrols Others Substance

TABLE 1 and showed full agonism, with an EC50 of 0.11 mM, whereas Psychoactive Substances and TAAR1 139 Downloaded from

Fig. 1. Chemical structures of basic compounds (in bold) and location for derivatization (indicated by “R”) for compounds included in the data set. Residues for each compound are specified in Fig. 2 and Supplemental Table 1. Structural analogs were grouped according to the basic chemical structures for presentation of the data in tables and heat maps (Fig. 2). The generic or full chemical names for abbreviated substances are listed in Supplemental Table 1. jpet.aspetjournals.org

p-tyramine was more potent, with a Ki of 0.06 mM and EC50 of p-tyramine, and many phenethylamines (25E-NB2OMe, 0.03 mM, with full agonist properties (Emax 5 94%). The third 25P-NB2OMe, 25T7-NB2OMe, 2C-E, and 2C-T7), amphet- endogenous ligand tested, tryptamine, showed similar affinity (4-APB, 4-fluoroamphetamine, 5-IT, methamphet- (Ki 5 0.13 mM) to b-PEA and p-tyramine and slightly lower amine, and PMMA), and aminoindanes (2-AI, N-methyl-2-AI, functional activity (EC50 5 0.41 mM). Several of the screened and MDAI) were similarly active as b-PEA and p-tyramine, amphetamines (5-APB, 6-APB, 7-APB, and 4-fluoroamphetamine) although all of them were partial agonists (Emax 5 36%–78%), at ASPET Journals on September 23, 2021 and phenethylamines (2C-P, 2C-T2, 2C-T4, and 2C-T7) and with the exception of PMMA and MDAI (Emax 5 82% and the aminoindane 5-IAI had affinities and EC50 values that 99%, respectively). Interestingly, binding affinity did not were comparable to p-tyramine, the most potent of the three always predict functional potency, such as with the amino- endogenous ligands at rat TAAR1, but none of these had a indanes, which showed functional activities similar to am- more potent EC50 than p-tyramine. The majority of these phetamine but exhibited much lower binding affinities than potent novel psychoactive substances exhibited full agonist amphetamine. properties (Emax . 80%) at rat TAAR1. The Emax values of Differences in Activation Potencies at Human versus 4-fluoroamphetamine and 2C-T4 were 78% and 67%, suggesting Rat and Mouse TAAR1. Our results suggest significant partial agonism. species differences in TAAR1 affinities and activation The m-CPP and several amphetamines (4-APB, potencies for most of the substances with relevant binding 4-fluoromethamphetamine, 5-IT, 4-methylamphetamine, and properties in the rat. Importantly, although the endogenous 4-MTA), phenethylamines (2C-B, 2C-B-Fly, 2C-C, 2C-E, 2C-I, ligands p-tyramine and tryptamine activated TAAR1 with a and 2C-N), and aminoindanes (2-AI, N-methyl-2-AI, and potency rank order of rat . mouse . human, like many MDAI) were comparable to the less potent endogenous ligands psychostimulant compounds, b-PEA had similar EC50 b-PEA and tryptamine in their affinities and functional values across the three species. This is relevant because a potencies at rat TAAR1. The majority of these compounds comparison of activation potencies across species with in were partial agonists. Amphetamine and the well known vitro assays could be biased by assay-specific variables, such amphetamine derivatives methamphetamine, MDMA, and as expression levels of the transporters in the respective cell MDA were slightly less active than the structurally related lines. However, b-PEA can serve as a reference compound endogenous ligand b-PEA. for species comparisons. Wainscott et al. (2007) also report- Mouse TAAR1. b-PEA and p-tyramine had equal affini- ed comparable EC50 values for b-PEA at rat and human ties at mouse TAAR1, with Ki values of 0.31 and 0.38 mM, TAAR1 and species differences for other compounds. To respectively, and full agonist properties, with EC50 values of quantify the extent of species differences in TAAR1 activa- 0.2 and 0.28 mM, respectively. Various amphetamines (am- tion potencies, we calculated human/rat EC50 ratios and phetamine, 6-APDB, 4-fluoroamphetamine, 5-IT, MDA, and human/mouse EC50 ratios for substances with low to PMA) and one phenethylamine (2C-P) showed similar binding submicromolar (,10 mM) potencies for human TAAR1 affinities and functional potencies to these endogenous activation. The human/rat ratios ranged from 171 to 2.4, TAAR1 ligands, whereas some amphetamines were even more demonstrating the lower activity of the compounds at hu- potent (5-APB, 6-APB, 7-APB, 4-methylamphetamine, and 4- man versus rat TAAR1 (Table 2). This broad range of ratios MTA), with mostly full agonist properties. The endogenous showed that the extent of species differences varied sub- ligand tryptamine was slightly weaker than b-PEA and stantially between compounds. The endogenous ligand 140 Simmler et al. tryptamine exhibited a high human/rat ratio (51). Metham- Discussion phetamine and amphetamine presented relatively low For this in vitro study, we determined the binding affinities human/rat ratios of 6.2 and 4.2, respectively, whereas the and activation potencies of a large set of psychoactive human/rat ratio for MDMA was significantly higher (35). The compounds at the human, rat, and mouse TAAR1 in heterol- human/mouse ratios were lower than their respective human/ ogous expression systems. We also characterized the ligands rat ratios for all substances, with human/rat ratios $ 8.9. as full or partial agonists. None of the active compounds had Calculations of ratios for substances that were inactive at full antagonist properties. As indicated by our previous human TAAR1 (EC . 10 mM) were not meaningful, but 50 studies (Simmler et al., 2013, 2014a), cathinone derivatives substantial differences between human/rat and human/mouse stood out as poor TAAR1 ligands. Most of the other psychoac- ratios were observed among the substances that were active at tive compounds were potent to moderate rat and mouse rat and mouse TAAR1. TAAR1 agonists but exhibited generally weak or no activity Species differences and differences across substances in at human TAAR1. The active compounds showed full or TAAR1 activation potencies are presented as a heat map in Fig. 2, in which the substances were sorted according to partial TAAR1 agonist properties, with generally no distinct patterns related to their chemical structure. their EC50 values for the activation of rat TAAR1. Clearly, there was an overall rank order of rat . mouse . human To our knowledge, our screening is the most extensive across the psychoactive substances. Figure 2 also shows that published data set to date, which included 101 psychoactive TAAR1 binding and activation was greater for certain sub- substances and 3 endogenous ligands as comparator com- Downloaded from stance classes than for others. Amphetamines, cathinones, pounds. The in vitro pharmacology of comparator com- and phenethylamines represented the three largest substance pounds and some psychoactive substances (amphetamine, classes in our screen. Amphetamines and phenethylamines methamphetamine, MDMA) were previously determined by were well represented among the potent TAAR1 ligands, us and others (Borowsky et al., 2001; Bunzow et al., 2001; Reese et al., 2007; Wainscott et al., 2007; Lindemann et al., with EC50 values within a range that could be physiologi- cally relevant (30 nM to 5 mM), whereas the activity of 2008; Lewin et al., 2011). The replication of those data jpet.aspetjournals.org for this study was an important validation of our assays. cathinone derivatives was low (EC50 . 5 mM, except for cathinone). None of the pipradrols exhibited significant Furthermore, because we determined binding affinities for binding properties. Interestingly, tryptamine derivatives some novel psychoactive substances in earlier studies, we were weak agonists or did not bind to TAAR1 at all, although included these data in this study. As a result, all data tryptamine itself is an endogenous rat and mouse TAAR1 determined by our laboratory are conveniently summarized ligand, with activation potency that is comparable to b-PEA here in Table 1. and full efficacy. Species differences in TAAR1 activation between rodent at ASPET Journals on September 23, 2021 and human receptors have been reported previously for phenethylamine analogs (Wainscott et al., 2007), p-tyramine, TABLE 2 and methamphetamine (Reese et al., 2007). These compounds , m EC50 ratios calculated for all substances with EC50 values 10 M for have consistently exhibited lower potencies for human TAAR1 the human TAAR1 activation than for rodent TAAR1 activation. Structure- Ratios are ranked according to the human/rat ratio. Values . 1 indicate lower b potency at the human TAAR1 versus the rat or mouse TAAR1. activity correlations for -PEA derivatives with regard to human TAAR1 activation have shown that bulky residues on EC Ratio 50 the amine or phenyl ring reduced ligand potency (Lewin et al., Substance Structure Class Human/ Human/ 2008). Reduced human TAAR1 activity could be expected for Rata Mouse novel psychoactive substances for which substantial derivati- 6-APB 171 107 Amphetamines zation is typical. Together with previous reports on species 2C-P 140 7.5 Phenethylamines 5-IAI 97 7.8 Aminoindanes differences, the data provide evidence that many psychoactive 5-APB 91 47 Amphetamines substances are considerably less potent at human TAAR1 Tryptamine 51 7.8 Endogenous than at mouse or rat TAAR1. In rodents, psychoactive ligandsb 4-Fluoroamphetamine 51 27 Amphetamines compounds could reduce neuronal firing via TAAR1 activation, 4-Fluoromethamphetamine 39 13 Amphetamines comparable to the endogenous TAAR1 ligand p-tyramine, MDMA 35 8.6 Amphetamines which has been shown to reduce the DA firing p-Tyramine 33 3.5 Endogenous ligandsc rate (Bradaia et al., 2009). Consequently, the psychoactive 4-APB 26 4.8 Amphetamines TAAR1 ligands likely exert autoregulatory effects on their MDAI 19 7.9 Aminoindanes TAAR1-independent effects, such as reducing drug-induced 2C-B 14 1.4 Phenethylamines DA release in the striatum (Di Cara et al., 2011). Given that 2-AI 14 4.5 Aminoindanes 7-APB 11 5.7 Amphetamines studies in rodents have reported autoregulatory effects of the N-Methyl-2-AI 8.9 3.5 Aminoindanes psychostimulant TAAR1 ligands amphetamine, methamphet- Methamphetamine 6.2 7.3 Amphetamines amine, and MDMA (Lindemann et al., 2008; Di Cara et al., Cathinone 5.8 5.8 Cathinones MDA 4.9 6.2 Amphetamines 2011; Achat-Mendes et al., 2012; Harkness et al., 2015), these 2C-H 4.3 0.9 Phenethylamines species differences at TAAR1 could be relevant to the trans- Amphetamine 4.2 5.3 Amphetamines lational validity of preclinical studies. Particularly for novel b-PEA 2.4 1.3 Endogenous ligandsc psychoactive substances with large TAAR1 species differ- ences, the abuse liability that is evaluated in rodent models aSubstances are sorted according to human/rat ratios. bStructure class: tryptamines. may actually underestimate the risk for addiction that is cStructure class: phenethylamines. posed by the drugs in humans. Psychoactive Substances and TAAR1 141 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 2. Heat map illustrating the diversity of TAAR1 activity between individual substances and between human, rat, and mouse TAAR1. The substances are sorted according to their rat TAAR1 activity (EC50 values). The compounds were split into the 52 more active (A) and the 51 less active (B) rat TAAR1 ligands. Next to the substance names, the respective substance classes are specified by color code and basic chemical structures are defined by numbers. The residues R2–R11 refer to the chemical structures presented in Fig. 1. Underlined residues indicate ring structures between two locations for derivatization. The generic or full chemical names for abbreviated substances are listed in Supplemental Table 1.

Species differences in TAAR1/ligand interactions have been Importantly, TAAR1 is a promising target for therapeutic predicted from sequence alignment studies that compared the drugs for the treatment of substance use disorders, regardless critical residues for the binding of b-PEA, showing that amino of species differences in the direct TAAR1 agonism properties acids that correspond to the critical residues differ between of psychoactive substances. TAAR1 agonists that have been rat, mouse, and human TAAR1 (Kratochwil et al., 2011). Site- reported in the literature are similarly potent at both human directed mutagenesis studies have identified two locations in and rat TAAR1. Furthermore, the efficacy that has been TAAR1 transmembrane domains 6 and 7, where reported in animal models is comparable to the efficacy that substitutions markedly reduce or increase methamphetamine has been reported in in vitro expression systems, which may TAAR1 activation potencies in the rat and mouse TAAR1 provide a basis for predicting effective doses in humans. The (Reese et al., 2014). Docking studies with a homology model for TAAR1 partial agonist RO5203648 effectively reduced cocaine the human TAAR1 (Cichero et al., 2013, 2014) could serve to self-administration and relapse to drug-seeking behavior in further elucidate the essential amino acids that are required rats (Revel et al., 2012b; Pei et al., 2014), although cocaine for ligand binding and discover structural determinants for is not a TAAR1 ligand itself. TAAR1 partial agonism mark- the TAAR1 activity or inactivity of psychoactive substances. edly reduced cocaine-induced DA overflow in the nucleus 142 Simmler et al. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 23, 2021

Fig. 2. Continued.

accumbens (Pei et al., 2014). Because TAAR1 is involved in the firing rates in WT mice (Bradaia et al., 2009; Revel et al., constitutive regulation of neuronal firing (Bradaia et al., 2012b), whereas full agonists like p-tyramine decreased firing 2009), pharmacological TAAR1 activation with a therapeutic rates (Revel et al., 2011, 2012b). However, both full and partial drug may regulate neuronal firing and result in hyposensi- agonists have been shown to be protective against the re- tivity to drugs, similar to the overexpression of TAAR1 in a warding and reinforcing effects of the psychostimulant cocaine transgenic mouse model (Revel et al., 2012a). Moreover, the (Pei et al., 2015), but the opposing effects of full and partial efficacy of these potentially therapeutic compounds could be agonists on firing rates suggest that psychoactive substances even greater in humans than in rodents. In rodents, but not or that are full agonists would exert effects that are different less so in humans, TAAR1-mediated negative feedback that from partial agonists. Data on the full or partial agonist is induced by the abused substances could be present and properties of TAAR1 ligands are thus important. Whereas full attenuate the extent of therapeutic drug effects. agonists such as amphetamine induce negative feedback to Ex vivo electrophysiology experiments in the ventral teg- blunt their own effect on DA and 5-HT systems (Lindemann mental area and dorsal raphe nuclei showed that both partial et al., 2008; Di Cara et al., 2011), partial agonists might agonists and antagonists enhanced DA and 5-HT neuron increase their effect on neuronal signal transmission by Psychoactive Substances and TAAR1 143 increasing firing rates via TAAR1. This is an assumption that identification of a family of mammalian G protein-coupled receptors. Proc Natl Acad Sci USA 98:8966–8971. would be based on findings with selective TAAR1 ligands and Bradaia A, Trube G, Stalder H, Norcross RD, Ozmen L, Wettstein JG, Pinard A, requires further investigation. Buchy D, Gassmann M, and Hoener MC, et al. (2009) The selective antagonist EPPTB reveals TAAR1-mediated regulatory mechanisms in In our data set and based on data reported previously by two of the mesolimbic system. Proc Natl Acad Sci USA 106:20081–20086. different laboratories (Reese et al., 2007; Wainscott et al., Bunzow JR, Sonders MS, Arttamangkul S, Harrison LM, Zhang G, Quigley DI, b Darland T, Suchland KL, Pasumamula S, and Kennedy JL, et al. (2001) Am- 2007), the activation potencies of -PEA at rat, human, and phetamine, 3,4-methylenedioxymethamphetamine, lysergic acid diethylamide, and mouse TAAR1 exhibited similar EC50 values between species, metabolites of the are agonists of a rat trace whereas p-tyramine was more potent at rat TAAR1, followed amine receptor. Mol Pharmacol 60:1181–1188. Cichero E, Espinoza S, Franchini S, Guariento S, Brasili L, Gainetdinov RR, by mouse and human TAAR1. Given that these data were and Fossa P (2014) Further insights into the pharmacology of the human trace generated in independent laboratories that used different amine-associated receptors: discovery of novel ligands for TAAR1 by a virtual screening approach. Chem Biol Drug Des 84:712–720. assay conditions and expression systems, the similarities of Cichero E, Espinoza S, Gainetdinov RR, Brasili L, and Fossa P (2013) Insights into the pharmacological profiles suggest good consistency of the the structure and pharmacology of the human trace amine-associated receptor 1 (hTAAR1): homology modelling and docking studies. Chem Biol Drug Des 81: data and support the validity of the comparisons between 509–516. species. Cisneros IE and Ghorpade A (2014) Methamphetamine and HIV-1-induced neuro- toxicity: role of trace amine associated receptor 1 cAMP signaling in . In this study, we simply determined activity at specific Neuropharmacology 85:499–507. targets, which is common with interpretations of in vitro data, Cotter R, Pei Y, Mus L, Harmeier A, Gainetdinov RR, Hoener MC, and Canales JJ and we did not take into account that the processes that allow (2015) The trace amine-associated receptor 1 modulates methamphetamine’s

and behavioral effects. Front Neurosci 9:39. Downloaded from a substance to interact with TAAR1 in vivo depend on more Di Cara B, Maggio R, Aloisi G, Rivet JM, Lundius EG, Yoshitake T, Svenningsson P, variables than solely substance/receptor interactions. The Brocco M, Gobert A, and De Groote L, et al. (2011) Genetic deletion of trace amine 1 receptors reveals their role in auto-inhibiting the actions of ecstasy (MDMA). location of TAAR1 expression is mostly intracellular in J Neurosci 31:16928–16940. neurons (Miller, 2011) and also in glial cells (Cisneros and Espinoza S, Lignani G, Caffino L, Maggi S, Sukhanov I, Leo D, Mus L, Emanuele M, Ronzitti G, and Harmeier A, et al. (2015) TAAR1 modulates cortical glutamate Ghorpade, 2014). Because the substances need to reach the NMDA receptor function. Neuropsychopharmacology 40:2217–2227. location of expression of TAAR1 to bind to the receptor, the Harkness JH, Shi X, Janowsky A, and Phillips TJ (2015) Trace amine-associated

receptor 1 regulation of methamphetamine intake and related traits. Neuro- jpet.aspetjournals.org intracellular availability of the ligands is also relevant. 40:2175–2184. Certain psychoactive substances, such as amphetamine de- Jing L and Li JX (2015) Trace amine-associated receptor 1: a promising target for the treatment of psychostimulant addiction. Eur J Pharmacol 761:345–352. rivatives, are substrates of monoaminergic transporters and Kratochwil NA, Gatti-McArthur S, Hoener MC, Lindemann L, Christ AD, Green LG, carried into the cell (Zaczek et al., 1991). These substrate-type Guba W, Martin RE, Malherbe P, and Porter RH, et al. (2011) G protein-coupled substances, therefore, might be more likely available to receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey. Curr Top Med Chem 11:1902–1924. intracellular TAAR1 than substances that are not trans- Lewin AH, Miller GM, and Gilmour B (2011) Trace amine-associated receptor 1 is a porter substrates, including, for example, cocaine, MDPV, other stereoselective binding site for compounds in the amphetamine class. Bioorg Med Chem 19:7044–7048. pyrovalerone cathinones, methylphenidate, and other pipra- Lewin AH, Navarro HA, and Mascarella SW (2008) Structure-activity correlations for at ASPET Journals on September 23, 2021 drols (Simmler et al., 2013, 2014b). One limitation of our study beta-phenethylamines at human trace amine receptor 1. Bioorg Med Chem 16: 7415–7423. is that we did not consider stereoselectivity of the compounds Liechti M (2015) Novel psychoactive substances (designer drugs): overview and by screening racemic mixtures for most substances. As with pharmacology of modulators of monoamine signaling. Swiss Med Wkly 145: w14043. activity at other psychostimulant targets, such as monoamin- Lindemann L, Meyer CA, Jeanneau K, Bradaia A, Ozmen L, Bluethmann H, Bettler ergic reuptake transporters, TAAR1 has a stereoselective bind- B, Wettstein JG, Borroni E, and Moreau JL, et al. (2008) Trace amine-associated ing site, and the assessment of racemates could underestimate receptor 1 modulates dopaminergic activity. J Pharmacol Exp Ther 324:948–956. Lynch LJ, Sullivan KA, Vallender EJ, Rowlett JK, Platt DM, and Miller GM (2013) the activity of the more active isomer (Lewin et al., 2011). Trace amine associated receptor 1 modulates behavioral effects of ethanol. Subst In conclusion, we provide an extensive data set on the ligand Abuse 7:117–126. Miller GM (2011) The emerging role of trace amine-associated receptor 1 in the properties of psychoactive substances at TAAR1. With differ- functional regulation of monoamine transporters and dopaminergic activity. ences between activity at rodent and human TAAR1, we J Neurochem 116:164–176. Pei Y, Lee J, Leo D, Gainetdinov RR, Hoener MC, and Canales JJ (2014) Activation of provide evidence of significant species differences in interac- the trace amine-associated receptor 1 prevents relapse to cocaine seeking. Neuro- tions between TAAR1 and psychoactive drugs, which could be psychopharmacology 39:2299–2308. Pei Y, Mortas P, Hoener MC, and Canales JJ (2015) Selective activation of the trace relevant to the translational validity of preclinical studies to amine-associated receptor 1 decreases cocaine’s reinforcing efficacy and prevents clinical applications. cocaine-induced changes in brain reward thresholds. Prog Neuropsychopharmacol Biol Psychiatry 63:70–75. Reese EA, Bunzow JR, Arttamangkul S, Sonders MS, and Grandy DK (2007) Trace Acknowledgments amine-associated receptor 1 displays species-dependent stereoselectivity for iso- mers of methamphetamine, amphetamine, and para-hydroxyamphetamine. The authors thank Lipomed for providing the 2C and NBOMe drugs J Pharmacol Exp Ther 321:178–186. at no cost, Michael Arends for text editing, and Roger Norcross for Reese EA, Norimatsu Y, Grandy MS, Suchland KL, Bunzow JR, and Grandy DK helpful discussions. (2014) Exploring the determinants of trace amine-associated receptor 1’s functional selectivity for the stereoisomers of amphetamine and methamphetamine. J Med Chem 57:378–390. Authorship Contributions Revel FG, Meyer CA, Bradaia A, Jeanneau K, Calcagno E, André CB, Haenggi M, Miss MT, Galley G, and Norcross RD, et al. (2012a) Brain-specific overexpression of Participated in research design: Hoener, Liechti. trace amine-associated receptor 1 alters monoaminergic neurotransmission and Conducted experiments: Buchy, Chaboz. decreases sensitivity to amphetamine. Neuropsychopharmacology 37:2580–2592. Performed data analysis: Simmler, Buchy, Chaboz, Hoener. Revel FG, Moreau JL, Gainetdinov RR, Bradaia A, Sotnikova TD, Mory R, Durkin S, Zbinden KG, Norcross R, and Meyer CA, et al. (2011) TAAR1 activation modulates Wrote or contributed to the writing of the manuscript: Simmler, monoaminergic neurotransmission, preventing hyperdopaminergic and hypo- Hoener, Liechti. activity. Proc Natl Acad Sci USA 108:8485–8490. Revel FG, Moreau JL, Gainetdinov RR, Ferragud A, Velázquez-Sánchez C, Sotnikova TD, Morairty SR, Harmeier A, Groebke Zbinden K, and Norcross RD, et al. (2012b) References Trace amine-associated receptor 1 partial agonism reveals novel paradigm for Achat-Mendes C, Lynch LJ, Sullivan KA, Vallender EJ, and Miller GM (2012) neuropsychiatric therapeutics. Biol Psychiatry 72:934–942. Augmentation of methamphetamine-induced behaviors in transgenic mice lacking Revel FG, Moreau JL, Pouzet B, Mory R, Bradaia A, Buchy D, Metzler V, Chaboz S, the trace amine-associated receptor 1. Pharmacol Biochem Behav 101:201–207. Groebke Zbinden K, and Galley G, et al. (2013) A new perspective for : Borowsky B, Adham N, Jones KA, Raddatz R, Artymyshyn R, Ogozalek KL, Durkin TAAR1 agonists reveal antipsychotic- and -like activity, improve MM, Lakhlani PP, Bonini JA, and Pathirana S, et al. (2001) Trace amines: cognition and control body weight. Mol Psychiatry 18:543–556. 144 Simmler et al.

Rickli A, Hoener MC, and Liechti ME (2015a) and receptor Wainscott DB, Little SP, Yin T, Tu Y, Rocco VP, He JX, and Nelson DL (2007) interaction profiles of novel psychoactive substances: para-halogenated amphet- Pharmacologic characterization of the cloned human trace amine-associated re- amines and pyrovalerone cathinones. Eur Neuropsychopharmacol 25:365–376. ceptor1 (TAAR1) and evidence for species differences with the rat TAAR1. Rickli A, Kopf S, Hoener MC, and Liechti ME (2015b) Pharmacological profile of J Pharmacol Exp Ther 320:475–485. novel psychoactive benzofurans. Br J Pharmacol 172:3412–3425. Wolinsky TD, Swanson CJ, Smith KE, Zhong H, Borowsky B, Seeman P, Branchek T, Rickli A, Luethi D, Reinisch J, Buchy D, Hoener MC, and Liechti ME (2015c) Receptor and Gerald CP (2007) The trace amine 1 receptor knockout mouse: an animal interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy- model with relevance to schizophrenia. Genes Brain Behav 6:628–639. substituted phenethylamines (2C drugs). Neuropharmacology 99:546–553. Xie Z and Miller GM (2007) Trace amine-associated receptor 1 is a modulator of the Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S, . J Pharmacol Exp Ther 321:128–136. Crossley DA, Bunzow JR, and Ronca-Testoni S, et al. (2004) 3-Iodothyronamine is Zaczek R, Culp S, and De Souza EB (1991) Interactions of [3H]amphetamine with rat an endogenous and rapid-acting derivative of thyroid hormone. Nat Med 10:638–642. brain synaptosomes. II. Active transport. J Pharmacol Exp Ther 257:830–835. Simmler LD, Buser TA, Donzelli M, Schramm Y, Dieu LH, Huwyler J, Chaboz S, Zucchi R, Chiellini G, Scanlan TS, and Grandy DK (2006) Trace amine-associated Hoener MC, and Liechti ME (2013) Pharmacological characterization of designer receptors and their ligands. Br J Pharmacol 149:967–978. cathinones in vitro. Br J Pharmacol 168:458–470. Simmler LD, Rickli A, Hoener MC, and Liechti ME (2014a) Monoamine transporter and receptor interaction profiles of a new series of designer cathinones. Neuro- Address correspondence to: Dr. Matthias E. Liechti, Division of Clinical pharmacology 79:152–160. Pharmacology and Toxicology, Department of Biomedicine, University Hospi- Simmler LD, Rickli A, Schramm Y, Hoener MC, and Liechti ME (2014b) Pharma- tal Basel, Hebelstrasse 2, CH-4031 Basel, Switzerland. E-mail: matthias. cological profiles of aminoindanes, piperazines, and derivatives. Biochem [email protected] Pharmacol 88:237–244. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 23, 2021