TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity Florent G. Revela, Jean-Luc Moreaua, Raul R. Gainetdinovb, Amyaouch Bradaiac, Tatyana D. Sotnikovab, Roland Morya, Sean Durkina, Katrin Groebke Zbindend, Roger Norcrossd, Claas A. Meyere, Veit Metzlera, Sylvie Chaboza, Laurence Ozmena, Gerhard Trubea, Bruno Pouzeta, Bernhard Bettlerf, Marc G. Carong, Joseph G. Wettsteina, and Marius C. Hoenera,1 aNeuroscience Research, dDiscovery Chemistry, and eDiscovery Technologies, Pharmaceuticals Division, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland; bDepartment of Neuroscience and Brain Technologies, Italian Institute of Technology, 16163 Genoa, Italy; cNeuroservice, Domaine de Saint-Hilaire, 13593 Aix-en-Provence, France; fDepartment of Biomedicine, Institute of Physiology, Pharmazentrum, University of Basel, CH-4056 Basel, Switzerland; and gDepartment of Cell Biology, Duke University Medical Center, Durham, NC 27710 Edited by Richard D. Palmiter, University of Washington, Seattle, WA, and approved March 31, 2011 (received for review February 24, 2011) The trace amine-associated receptor 1 (TAAR1), activated by en- sitive to the locomotor-stimulating effect of d-amphetamine dogenous metabolites of amino acids like the trace amines and show elevated striatal release of dopamine (DA), noradren- p-tyramine and β-phenylethylamine, has proven to be an impor- aline (NA), and serotonin [5-hydroxytryptamine (5-HT)] after a d-amphetamine challenge (10, 12). Furthermore, the spontaneous tant modulator of the dopaminergic system and is considered −/− firing rate of the VTA DA neurons is augmented in Taar1 mice, a promising target for the treatment of neuropsychiatric disorders. fi To decipher the brain functions of TAAR1, a selective TAAR1 ago- and only in WT mice does pTyr decrease this ring rate (10). These fi observations suggest that TAAR1 is a negative modulator of mono- nist, RO5166017, was engineered. RO5166017 showed high af nity aminergic neurotransmission. and potent functional activity at mouse, rat, cynomolgus monkey, Apart from the TAs, TAAR1 is activated by a range of endoge- and human TAAR1 stably expressed in HEK293 cells as well as high nous molecules such as other biogenic amines (2, 5), thyroid selectivity vs. other targets. In mouse brain slices, RO5166017 PHARMACOLOGY hormone-derivative 3-iodothyronamine (T1AM) (13, 14), and inhibited the firing frequency of dopaminergic and serotonergic catechol-O-methyl transferase products (e.g., 3-methoxytyramine) neurons in regions where Taar1 is expressed (i.e., the ventral teg- (2, 15) or by synthetic substances such as amphetamine derivatives mental area and dorsal raphe nucleus, respectively). In contrast, and ergolines (2, 12). However, all these ligands have TAAR1- RO5166017 did not change the firing frequency of noradrenergic independent effects through other targets, such as the mono- neurons in the locus coeruleus, an area devoid of Taar1 expression. aminergic transporters and receptors or the σ-receptors (3, 11, 13, Furthermore, modulation of TAAR1 activity altered the desensiti- 16). The lack of selective ligands has rendered identification of zation rate and agonist potency at 5-HT receptors in the dorsal TAAR1 biological functions challenging. Recently, Bradaia et al. 1A (17) described the first selective TAAR1 antagonist, N-(3-Ethoxy- raphe, suggesting that TAAR1 modulates not only dopaminergic fl but also serotonergic neurotransmission. In WT but not Taar1−/− phenyl)-4-pyrrolidin-1-yl-3-tri uoromethyl-benzamide (EPPTB). Use of EPPTB revealed that TAAR1 tonically activates inwardly mice, RO5166017 prevented stress-induced hyperthermia and rectifying K+ channels in VTA DA neurons to reduce the basal blocked dopamine-dependent hyperlocomotion in cocaine-treated firing activity (17). Importantly, EPPTB increased agonist potency at and dopamine transporter knockout mice as well as hyperactivity DA D2 receptors while reducing their desensitization rate, strongly induced by an NMDA antagonist. These results tie TAAR1 to the suggesting a functional link between TAAR1 and D2 receptors. control of monoamine-driven behaviors and suggest anxiolytic- Further elucidation of TAAR1 physiological functions and and antipsychotic-like properties for agonists such as RO5166017, validation of its promising therapeutic potential require selective opening treatment opportunities for psychiatric disorders. agonists. The ideal characteristics of such molecules are (i) high affinity and intrinsic activity (equal or superior to TAs), (ii) high drug discovery | serotonin | depression | schizophrenia | anxiety selectivity for TAAR1 (in contrast to TAs), (iii) low metabolic turnover (in contrast to TAs), and (iv) favorable pharmacokinetic fi properties, such as a good brain penetration, to enable in vivo n 2001, identi cation of the trace amine-associated receptor fi I1 (TAAR1) provided evidence for a direct biological effect of studies. Here, we report the identi cation of such a compound, so-called trace amines (TAs) (1, 2), a subgroup of biogenic amines RO5166017, which was used for the study of TAAR1 function in previously denoted as false neurotransmitters (3). TAs such as vitro and in vivo. p-tyramine (pTyr), β-phenylethylamine (PEA), octopamine, and tryptamine are metabolites of amino acids with structural simi- larity to classical biogenic amines. Although they are only found Author contributions: F.G.R., J.-L.M., R.R.G., M.G.C., and M.C.H. designed research; F.G.R., at low concentrations in the brain, TAs have been implicated in J.-L.M., R.R.G., A.B., T.D.S., R.M., S.D., K.G.Z., R.N., C.A.M., V.M., S.C., L.O., G.T., and M.C.H. a wide range of neuropathological disorders, including schizo- performed research; K.G.Z. and R.N. contributed new reagents/analytic tools; F.G.R., ’ J.-L.M., R.R.G., A.B., T.D.S., R.M., S.D., C.A.M., V.M., S.C., G.T., B.P., B.B., M.G.C., J.G.W., phrenia, major depression, anxiety states, Parkinson s disease, and M.C.H. analyzed data; and F.G.R., J.-L.M., R.R.G., G.T., and M.C.H. wrote the paper. and attention deficit hyperactivity disorder (3, 4). Conflict of interest statement: F.G.R., J.-L.M., R.M., S.D., K.G.Z., R.N., C.A.M., V.M., S.C., TAAR1, a member of the TAAR family (5, 6), is a G protein- L.O., G.T., B.P., J.G.W., and M.C.H. are F. Hoffmann-La Roche employees. R.R.G. is sup- coupled receptor (GPCR) that signals through Gs to elevate ported in part by research grants from F. Hoffmann-La Roche Ltd. (Basel, Switzerland) and intracellular cAMP levels in response to TAs (6, 7). In vitro Compagnia di San Paolo Fondazione (Turin, Italy). A.B. is employed by Neuroservice. M.G.C. studies have shown a reciprocal regulation between TAAR1 and has received funds for Sponsored Research Agreements unrelated to this work from monoaminergic transporters, particularly the dopamine trans- Forest Laboratories, NeuroSearch, and Lundbeck USA as well as consulting fees from porter (DAT) (8, 9). In the mouse brain, Taar1 is expressed Merck and F. Hoffmann-La Roche Ltd. An unrestricted gift to Duke University was pro- throughout the limbic and monoaminergic systems, including the vided by Lundbeck USA to support Neuroscience research in the laboratory of M.G.C. ventral tegmental area (VTA) and dorsal raphe nucleus (DRN) This article is a PNAS Direct Submission. −/− (10). Mice lacking Taar1 (Taar1 mice) have no overt phenotype 1To whom correspondence should be addressed. E-mail: [email protected]. and appear similar to WT littermates in most neurological and −/− This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. behavioral tests (10–12). However, Taar1 mice are hypersen- 1073/pnas.1103029108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1103029108 PNAS | May 17, 2011 | vol. 108 | no. 20 | 8485–8490 Downloaded by guest on October 1, 2021 Results Identification of RO5166017 and in Vitro Pharmacological Charac- terization. There seems to be considerable overlap between the pharmacophore space occupied by TAAR1 ligands and ligands of other biogenic amine receptors (2). Therefore, a medicinal chemistry program seeking to create TAAR1 ligands starting from Fig. 1. Chemical structure of the selective TAAR1 agonist RO5166017. adrenergic ligands as a source of lead compounds was started. One of the starting compounds was the amino-oxazoline S18616 (18), α an 2A-adrenergic receptor agonist, and an iterative series of Taar1 fi Because is also expressed in the DRN (10), we asked structural modi cations within this chemical class led to the dis- whether RO5166017 and pTyr also affect the firing frequency covery of RO5166017 [(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5- of 5-HT neurons. Both compounds decreased the spike rate of dihydro-oxazol-2-ylamine] (Fig. 1). DRN 5-HT neurons in WT mice (Fig. 2 C and D and Fig. S3), with fi RO5166017 exhibited high potency and ef cacy at mouse, rat, an IC50 value of 2.99 nM for RO5166017 (Table 1). As in the cynomolgus monkey, and human TAAR1 stably expressed in VTA, the inhibitory effects of pTyr and RO5166017 were blocked HEK293 cells (Table 1). Maximal cAMP levels reached by by EPPTB, which increased the firing frequency over the basal RO5166017 stimulation (maximal efficacy) were in a range similar level. The outward current generated by RO5166017 in the DRN to that achieved by PEA stimulation (set as 100%) at human, rat, was also K+-mediated (Fig. S2 C and D). Finally, neither and monkey TAAR1 (81–95%) and somewhat lower at mouse C D fi RO5166017 (Fig. 2 and ) nor−/− pTyr (Fig. S3) affected the ring TAAR1 (65%). Electrophysiological recordings in Xenopus oocytes rate of 5-HT neurons in Taar1 mice, supporting the specificity as well as VTA and DRN slices resulted in EC50 and IC50 values for of their effects in WT mice. Interestingly, the spontaneous spike mouse TAAR1 (1.7–8 nM) comparable with those obtained by the rate in DRN slices from mutant mice was significantly increased cAMP assay in HEK293 cells (3.3 nM).