TAAR1 (EC50 = 17~35 nM)

RO5263397 Increases cAMP Mediated BRET Signal in a Dose-Dependent Manner in HEK-293 Cells [3]
To study the pharmacology of RO5263397, we first used an in vitro approach. Since TAAR1 is a GPCR coupled to stimulatory G protein, its activation evokes the production of cAMP. To monitor in real time TAAR1 activation, we transfected, in addition of mouse and human TAAR1, a cAMP BRET biosensor that changes the emission of the light according to the cAMP fluctuation (see section “Materials and Methods”). We then tested a range of concentrations of RO5263397 both in cells transfected with mouse or human TAAR1. As shown in Figure 1, RO5263397 increases cAMP-mediated BRET signal in a concentration dependent manner (Figures 1A,B), with no desensitization over the observation period. RO5263397 did not provoke a BRET signal in cells not transfected with TAAR1 (data not shown). Comparison with a maximal concentration (10 μM) of β-phenylethylamine (PEA), a known TAAR1 full agonist indicated, RO5263397 behaves as a full agonist at the mTAAR1 (EC50: 0.12 nM and Emax: 100%) and partial agonist at the hTAAR1 (EC50: 47 nM and Emax: 82%) as shown in Figures 1C,D, respectively. The data also indicate that RO5263397 shows species related difference with 392-fold higher potency at the mTAAR1 compared to hTAAR1.

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The TAAR1 Antagonist EPPTB Inhibits RO5263397 Effect [3]
We further verified that the cAMP-mediated BRET signal seen with RO5263397 was due to activation of TAAR1 by pretreating the cells with the selective TAAR1 antagonist EPPTB. For these experiments, we used cells with mTAAR1 since EPPTB shows species selectivity mouse over human and rat. Pretreatment with EPPTB (1 μM) partially blocked the response to a maximal concentration (0.1 μM) of RO5263397 (Figure 1E). EPPTB alone did not affect BRET signal in mTAAR1 transfected cells. Furthermore, it did not affect the cAMP-BRET signal provoked by the adenylyl cyclase activator, forskolin (20μM) as shown in Figure 1F. These data indicate that mTAAR1 activation is necessary to observe the antagonist properties of EPPTB.

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RO5263397 Mediated ERK and CREB Phosphorylation in HEK293 Cells [3]
Apart from cAMP induction, TAAR1 signaling could involve other proteins, such as β-arrestin2 and AKT/GSK3 pathways. However, cAMP and also β-arrestin2 are linked to downstream effectors such as CREB and ERK, and TAAR1 seems to modulate the activity of these two proteins. In order to verify this hypothesis, we used HEK293 cells expressing mTAAR1 or hTAAR1. First, we treated the cells expressing mTAAR1 with RO5263397 and we performed a time course at a concentration of the compound that was the first to reach the efficacy of 100%, according to our previous BRET experiments (10 nM for mTAAR1 and 100 nM for hTAAR1). As shown in Figures 2A,B, RO5263397 was able to induce the phosphorylation of ERK2 and CREB, with the maximum effect at 5 min for pERK and at 15 min for pCREB. After measuring the optimal time for the induction of pERK and pCREB, we performed a concentration response curve using different concentrations of RO5263397 (Figures 2C,D). We then repeated the same time course experiment in cells expressing hTAAR1, and obtained similar results (Figure 3).

Animal/Disease Models: Adult 4-5 month old male WT littermates (C57BL/6 background) [2]
Doses: 0.1, 0.3, 1 mg/kg Mode of
Route of Administration: po; almost completely suppressed for 6 hrs (hrs (hours)) [3]. Mid-light phase dosing (ZT6)
Experimental Results: increased wake time at 0.3 and 1 mg/kg. TAAR1 agonism. All mice received p.o. RO5263397 (0.1, 0.3, and 1 mg/kg), Caf (10 mg/kg), or vehicle (0.3% Tween-80) in the mid-light phase (ZT6) in balanced order with at least 3 days between treatments. Mice were acclimated to oral dosing with vehicle (Veh) for at least 3 days before data collection.[2]

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Locomotor Activity [3]
Effect of RO5263397 on spontaneous locomotor activity of DAT-KO or WT mice was tested as described previously (Revel et al., 2011). Briefly, DAT-KO or WT mice were placed in the locomotor activity chambers for 30 min and then were treated with either saline or RO5263397 at different doses and total distance traveled was measured by analyzing infrared beam interruptions for another 90 min.

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Forced Swim Test [3]
Male Spraque-Dawley rats (230–270 g) were used in the FST. The test was performed in a glass cylinder (46 cm × 20 cm), with tap water 25 cm deep and +24–25°C. Water was refreshed after each test and it was performed over two consecutive days. On the first day, the rats were forced to swim for a 15-min period and immediately after that the first dose of test compound or vehicle was administered (5 ml/kg p.o.). The second drug or vehicle administration was on the next day, 23 h after the first dosing. One hour after the second administration and 24 h after the first swimming session, the rats were subjected to a second 5-min swimming session and immobility, climbing and swimming time were registered. Rats were considered to show immobility when animals were inactive and displaying minor movements with one limb only; climbing behavior was registered when the rats were actively climbing at the walls of the cylinder; finally, time spent swimming (horizontal activity) was measured as the remaining time after immobility and climbing have been subtracted from total test time. After each FST session, the rats were dried with towel and allow rest under heating lamp. RO5263397 (0.1, 1.0, and 10 mg/kg) formulations were prepared in 5% Tween 80 in sterile H2O. Each formulation was freshly prepared each day of dosing. In experiments examining the effects of dopamine D1 (D1) receptor antagonist SCH23390 (0.1 mg/kg, s.c.), glutamate AMPA receptor antagonist NBQX (10 mg/kg, s.c.), and serotonin 5-HT1A antagonist WAY100635 (1 mg/kg, s.c.) the antagonists were administered 10 min prior to RO5263397. The dose selection for SCH23390, WAY100635, and NBQX was based on previous work with these agents (Jordan et al., 2005; Snigdha et al., 2011). Statistical analysis was done by using one-way ANOVA followed by Dunnett’s post hoc test.

Pharmacokinetic analyses in rat, mouse, and cynomolgous monkey revealed very favorable in vivo properties, which have already been reported elsewhere. [Mol. Psychiatry 2013, 18, 543–556]

[1]. Discovery and Characterization of 2-Aminooxazolines as Highly Potent, Selective, and Orally Active TAAR1 Agonists.ACS Med Chem Lett. 2015 Dec 30;7(2):192-7.

[2]. Trace Amine-Associated Receptor 1 Regulates Wakefulness and EEG Spectral Composition.Neuropsychopharmacology. 2017 May;42(6):1305-1314.

[3]. Biochemical and Functional Characterization of the Trace Amine-Associated Receptor 1 (TAAR1) Agonist RO5263397.Front Pharmacol. 2018 Jun 21;9:645.

2-Aminooxazolines were discovered as a novel structural class of TAAR1 ligands. Starting from a known adrenergic compound 1, structural modifications were made to obtain highly potent and selective TAAR1 ligands such as 12 (RO5166017), 18 (RO5256390), 36 (RO5203648), and 48 (RO5263397). These compounds exhibit drug-like physicochemical properties, have good oral bioavailability, and display in vivo activity in a variety of animal models relevant for psychiatric diseases and addiction. [1]

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Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor, which signals through elevating intracellular cAMP levels, and expressed in most vertebrates, including rodents and humans. In recent years, several lines of evidence indicated the role of TAAR1 in the regulation of dopaminergic system and its importance in physiological processes such as locomotion, control of emotional states and cognition. In our study, we used RO5263397, a selective TAAR1 agonist, as a tool and characterized its pharmacology in vitro in HEK293 cells and its effects in vivo in tests assessing potential antidepressant and antipsychotic actions. We found that RO5263397 not only increases cAMP levels at very low concentrations but also can induce the phosphorylation of ERK and CREB in a concentration- and time-dependent manner. Like other TAAR1 agonists, RO5263397 potently suppressed high dopamine-dependent hyperactivity in mice lacking the dopamine transporter. Moreover, RO5263397 produced a strong antidepressant-like effect in the forced swim test comparable to fluoxetine. Furthermore, the antidepressant-like activity was blocked by pretreatment with SCH23390 (dopamine D1 receptor antagonist) or NBQX (glutamate AMPA receptor antagonist) but only in part by WAY100635 (serotonin 5HT1A receptor antagonist). In conclusion, our study confirms some previous in vitro and in vivo findings in relation to the pharmacological effects of RO5263397 but more importantly provides new insight on intracellular signaling pathway and other neurotransmitter receptors modulated by TAAR1 receptor activation.[3]
Taken together, in this study we demonstrated that TAAR1 activation not only induce the increase of cAMP levels inside the cells, but also can induce the phosphorylation of two important signaling proteins, the MAP Kinase ERK and the transcription factor CREB. RO5263397 is highly effective in vivo in mice and rats demonstrating potential antipsychotic and antidepressant activity. Moreover, the antidepressant-like effect of the TAAR1 agonist RO5263397 relies on the engagement of other neurotransmitter systems, particularly involving the D1 receptor and the AMPA glutamate receptor.[3]

C10H11FN2O

194.21

194.085

C, 44.96; H, 4.91; Cl, 26.54; F, 7.11; N, 10.49; O, 5.99

1357266-05-7;1357266-80-8

White to off-white solid powder

1.4

1

3

1

14

244

1

IOHOUWIYOVWGHV-SECBINFHSA-N

InChI=1S/C10H11FN2O/c1-6-7(3-2-4-8(6)11)9-5-14-10(12)13-9/h2-4,9H,5H2,1H3,(H2,12,13)/t9-/m1/s1

(4R/S)-4-(3-fluoro-2-methylphenyl)-4,5-dihydro-1,3-oxazol-2-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~260 mg/mL (~1338.76 mM)

Solubility in Formulation 1: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 6.5 mg/mL (33.47 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 65.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)