Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/10/30/mol.115.101394.DC1 1521-0111/89/1/165–175$25.00 http://dx.doi.org/10.1124/mol.115.101394 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:165–175, January 2016 U.S. Government work not protected by U.S. copyright Binding Mode Selection Determines the Action of Ecstasy Homologs at Monoamine Transporters s Walter Sandtner,1 Thomas Stockner,1 Peter S. Hasenhuetl, John S. Partilla, Amir Seddik, Yuan-Wei Zhang, Jianjing Cao, Marion Holy, Thomas Steinkellner, Gary Rudnick, Michael H. Baumann, Gerhard F. Ecker, Amy Hauck Newman, and Harald H. Sitte Institute of Pharmacology, Center for Physiology and Pharmacology (W.S., T.Sto., P.S.H., M.H., T.Ste., H.H.S.) and Center for Addiction Research and Science (H.H.S.), Medical University of Vienna, Vienna, Austria; Designer Drug Research Unit (J.S.P., M.H.B.) and Medicinal Chemistry Section (J.J.C., A.H.N.), Molecular Targets and Medications Discovery Branch, National Downloaded from Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Baltimore, Maryland; Department of Pharmaceutical Chemistry, University of Vienna, Vienna, Austria (A.S., G.F.E.); and Department of Pharmacology, Yale University, New Haven, Connecticut (Y.-W.Z., G.R.) Received August 17, 2015; accepted October 23, 2015 molpharm.aspetjournals.org ABSTRACT Determining the structural elements that define substrates and findings to available structural information. Our results suggest inhibitors at the monoamine transporters is critical to elucidating that the 3,4-methylenedioxyamphetamine analogs bind at the the mechanisms underlying these disparate functions. In this monoamine transporter orthosteric binding site by adopting one study, we addressed this question directly by generating a series of two mutually exclusive binding modes. 3,4-methylenediox- of N-substituted 3,4-methylenedioxyamphetamine analogs that yamphetamine and 3,4-methylenedioxy-N-methylamphetamine differ only in the number of methyl substituents on the terminal adopt a high-affinity binding mode consistent with a transport- amine group. Starting with 3,4-methylenedioxy-N-methylamphet- able substrate, whereas MDDMA and MDTMA adopt a low- amine, 3,4-methylenedioxy-N,N-dimethylamphetamine (MDDMA) affinity binding mode consistent with an inhibitor, in which the and 3,4-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) ligand orientation is inverted. Importantly, MDDMA can alternate at ASPET Journals on September 26, 2021 were prepared. We evaluated the functional activities of the between both binding modes, whereas MDTMA exclusively compounds at all three monoamine transporters in native brain binds to the low-affinity mode. Our experimental results are tissue and cells expressing the transporters. In addition, we used consistent with the idea that the initial orientation of bound ligand docking to generate models of the respective protein- ligands is critical for subsequent interactions that lead to trans- ligand complexes, which allowed us to relate the experimental porter conformational changes and substrate translocation. Introduction neurotransmitter homeostasis because it inactivates extracel- lular monoamine signaling and replenishes intracellular stores The principal role of neurotransmitter transporters is to of the vesicular transmitter. The physiologic significance of retrieve previously released neurotransmitter molecules transporter-mediated uptake is clinically relevant since ligands from the extracellular space (Kristensen et al., 2011). This is that interact with monoamine transporters [serotonin trans- an economical way of regulating synaptic transmission and porter (SERT), dopamine transporter (DAT), and norepineph- rine transporter (NET)] are used as medications to treat a This work was supported by the Austrian Science Fund/FWF (Fonds zur number of psychiatric disorders that involve an underlying Förderung der Wissenschaft und Forschung) [Grant W1232] to G.F.E. and H.H.S., imbalance of monoamine signaling and homeostasis (Iversen, Austrian Science Fund/FWF (Fonds zur Förderung der Wissenschaft und Forschung) [Grant F35] to G.F.E., H.H.S., and T.Sto., and a MDPhD fellowship 2000; Rothman and Baumann, 2003). On the other hand, some by the Medical University of Vienna to P.S.H. M.B.H. and A.H.N. receive transporter ligands are used for nonmedical purposes and can be support by the National Institute on Drug Abuse Intramural Research Program [Grant DA000389]. addictive, therefore blurring the borders between therapeuti- 1W.S. and T.Sto. contributed equally to this work. cally and illicitly used drugs (Kristensen et al., 2011). dx.doi.org/10.1124/mol.115.101394. s This article has supplemental material available at molpharm. The structurally diverse array of monoamine transporter aspetjournals.org. ligands can be classified with respect to their mode of action at ABBREVIATIONS: CFP, cyan fluorescent protein; DAT, dopamine transporter; dDAT, Drosophila melanogaster dopamine transporter; 5-HT, serotonin; KHB, Krebs HEPES buffer; FRET, fluorescence resonance energy transfer; HEK, human embryonic kidney; MDA, 3,4- methylenedioxyamphetamine; MDDMA, 3,4-methylenedioxy-N,N-dimethylamphetamine; MDMA, 3,4-methylenedioxy-N-methylamphetamine; MDTMA, 3,4-methylenedioxy-N,N,N-trimethylamphetamine; MOE, Molecular Operating Environment; MPP1, 1-methyl-4-phenylpyridinium; MTSEA, 2-aminoethyl methanethiosulfonate hydrobromide; NET, norepinephrine transporter; SERT, serotonin transporter; YFP, yellow fluorescent protein. 165 166 Sandtner et al. the transporter. Inhibitors (e.g., cocaine) elevate extracellular were synthesized, purified, and characterized according to the meth- monoamine concentrations by blocking reuptake of endoge- ods described in the Supplemental Data. All other reagents, buffer nous substrate from the extracellular space. Most of these salts, and chemicals were obtained from Sigma Chemical (St. Louis, inhibitors are competitive, and their binding sites overlap MO) unless otherwise noted. Reagents used in the experiments for with the substrate-binding sites (Beuming et al., 2008; uptake and efflux assays in cells were purchased and used according to previous work (Hofmaier et al., 2014). Plasmids encoding human Penmatsa et al., 2013, 2015; Wang et al., 2015). Substrates SERT were a generous gift of Dr. Randy D. Blakely. (e.g., D-amphetamine), in contrast, increase extracellular monoamine concentrations via two mechanisms: 1) they Animals and Housing compete with the endogenous substrate for reuptake from the extracellular space; and 2) they induce release of endog- Male Sprague-Dawley rats (Charles River, Wilmington, MA) – enous substrate molecules from intracellular stores by re- weighing 250 350 g were housed in standard conditions (lights on from 7:00 a.m. to 7:00 p.m.), with food and water freely available. Rats versing the normal direction of transporter flux (Sitte and were maintained in facilities fully accredited by the Association for Freissmuth, 2015). The mechanistic difference between com- Assessment and Accreditation of Laboratory Animal Care, and petitive inhibitors and substrates raises questions about the experiments were performed in accordance with the Institutional structural determinants that distinguish both compound Care and Use Committee of the National Institute on Drug Abuse groups. It also suggests the possibility that some compounds Intramural Research Program (IRP). might have mixed substrate and inhibitory properties at Downloaded from different transporters (Blough et al., 2014; Reith et al., 2015; Uptake and Release Assay in Rat Brain Synaptosomes Saha et al., 2015). Uptake and release assays were carried out in rat brain synapto- Recently, crystal structures of the Drosophila melanogaster somes as previously described (Baumann et al., 2013). Synaptosomes DAT (dDAT) bound to different substrates and inhibitors have were prepared from rat striatum for DAT assays, whereas synapto- been reported (Penmatsa et al., 2013, 2015; Wang et al., 2015). somes were prepared from whole brain minus striatum and cerebel- These studies have advanced our understanding of how the lum for NET and SERT assays. molpharm.aspetjournals.org monoamine transporters coordinate ligand binding. Yet the For uptake inhibition assays, 5 nM [3H]dopamine, [3H]norepinephrine, structural features that separate substrates from inhibitors and [3H]5-HT were used to assess transport activity at DAT, NET, and remain ill-defined, in part because conclusions are difficult to SERT, respectively. Where necessary, the selectivity of uptake assays draw when comparing the actions of substrate drugs based on was optimized for a single transporter of interest by including unlabeled 3 dissimilar chemical scaffolds. blockers to prevent uptake of a [ H]transmitter by competing trans- porters. Specifically, NET assays were carried out in the presence of Here, we tackled this issue by synthesizing and investigat- 3 – 50 nM GBR12935 to prevent uptake of [ H]norepinephrine by DAT, ing a series of 3,4-methylenedioxy ring substituted amphet- whereas SERT assays were carried out in the presence of 50 nM amine compounds that share the same structural scaffold GBR12935 and 100 nM nomifensine to prevent uptake of [3H]5-HT by and differ only in the number of methyl substituents at the DAT and NET, respectively. No unlabeled blockers are required for at ASPET Journals on September 26, 2021 terminal amine. Specifically, 3,4-methylenedioxyamphetamine