Trace amine-associated receptor 1 (TAAR1) (Ki = 1.9 nM, 2.7 nM, 31 nM and 24 nM)

When TAAR1 is persistently expressed in HEK293 cells, RO5166017 has excellent functional activity and high affinity against mouse, tumor, cynomolgus monkey, and human TAAR1. It also demonstrates high selectivity against other targets [1].
The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and β-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT1A receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission [1].

In addition to inhibiting cocaine in WT electrodes, RO5166017 also prevents cocaine-induced hyperlocomotion, hyperburn in dopamine-stressed hyperlocomotion, and NMDA antagonist-induced hyperkinetic effects in cocaine-treated and dopamine transporter knockout RUBS [1]. RO5166017 (0.01-1 mg/kg, RO5166017 exhibits TAAR1-mediated anxiolytic-like properties at doses of 0.1-0.3 mg/kg]. RO5166017 also inhibits cocaine in WT electrodes. RO5166017 increases INS1E cells and human insulin-like peptide 1 (GLP-1) in subchronic processing of diet-induced nutrition (DIO) using RO5166017.

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Rats rarely yawned after vehicle administration. Two-way repeated measures ANOVA revealed a significant RO5263397 × quinpirole interaction (F(15, 140) = 2.68, p < 0.01) with significant main effects of RO5263397 dose (F(3, 140) = 9.10, p < 0.001) and quinpirole dose (F(5, 140) = 23.06, p < 0.001). Bonferroni’s post hoc tests revealed that 0.032 and 0.1 mg/kg quinpirole significantly increased yawning in the vehicle pretreatment group. Bonferroni’s post hoc tests also revealed significant effects from 3.2 mg/kg RO5263397 at 0.1 mg/kg quinpirole and from 5.6 and 10 mg/kg RO5263397 at 0.032 and 0.1 mg/kg quinpirole as compared to vehicle pretreatment (Fig. 1, upper left). Two-way repeated measures ANOVA also revealed a significant RO5166017 × quinpirole interaction (F(10, 105) = 2.14, p < 0.05) with significant main effects of RO5166017 dose (F(2, 105) = 5.52, p < 0.05) and quinpirole dose (F(5, 105) = 20.43, p < 0.0001). Bonferroni’s post hoc tests revealed that 0.032 and 0.1 mg/kg quinpirole significantly increased yawning in the vehicle pretreatment group. Bonferroni’s post hoc tests also revealed significant effects from 3.2 mg/kg RO5166017 at 0.1 mg/kg quinpirole and from 10 mg/kg RO5166017 at 0.032 and 0.1 mg/kg quinpirole as compared to vehicle pretreatment (Fig. 1, upper right). [2]
After vehicle administration, rats had a body temperature of 37.8 ± 0.1 °C. Two-way repeated measures ANOVA revealed a significant main effect of quinpirole dose (F(5, 140) = 336.70, p < 0.0001) but not of RO5263397 dose (F(3, 140) = 1.96, NS), with no significant RO5263397 × quinpirole interaction (F(15, 140) = 0.77, NS). Bonferroni’s post hoc tests revealed that 0.032, 0.1, and 0.32 mg/kg quinpirole significantly decreased body temperature in the vehicle pretreatment group (Fig. 1, lower left). Two-way repeated measures ANOVA also revealed a significant main effect of quinpirole dose (F(5, 105) = 313.24, p < 0.001) but not of RO5166017 dose (F(2, 105) = 0.14, NS), with no significant RO5166017 × quinpirole interaction (F(15, 105) = 1.19, NS). Bonferroni’s post hoc tests revealed that 0.032, 0.1, and 0.32 mg/kg quinpirole significantly decreased body temperature in the vehicle pretreatment group (Fig. 1, lower right).[2]

Membrane Preparation and Radioligand Binding. [1]
For generation of HEK-293 cells stably expressing mouse, rat, human, and cynomolgus monkey TAAR1, the coding sequences were amplified from genomic DNA using the Expand High Fidelity PCR System modified by the addition of an N-terminal influenza hemagglutinin viral leader sequence followed by a FLAG tag and a Met-Gly linker cloned into the pIRESneo2 vector and transfected into HEK293 cells (CRL-1573; ATCC) using Lipofectamine 2000. After 24 h, the culture medium was supplemented with 1 mg/mL G418, and after 10 d, clones were isolated, expanded, and tested for responsiveness to trace amines (TAs) with the Upstate cAMP immunoassay kit following the manufacturer’s instructions. Monoclonal cell lines that displayed a stable EC50 for a culture period of 15 passages were used for all subsequent studies. All cell lines were maintained at 37 °C and 5% CO2 in DMEM high-glucose medium containing FCS (10%; heat-inactivated for 30 min at 56 °C), penicillin/streptomycin (1%), and 375 μg/mL Geneticin. For membrane preparation, the cells were released from culture flasks using trypsin/EDTA, harvested, washed two times with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 × g for 5 min at 4 °C, frozen, and stored at −80 °C. Frozen pellets were suspended in buffer A [20 mL Hepes-NaOH (20 mM, pH 7.4) containing 10 mM EDTA] and homogenized with a Polytron (PT 6000; Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged (30 min at 48,000 × g) at 4 °C, the supernatant was removed and discarded, and the pellet was resuspended in buffer A using the Polytron (20 s at 14,000 rpm). This procedure was repeated, and the final pellet was resuspended in buffer A and homogenized using the Polytron. Typically, aliquots of 2-mL membrane portions were stored at −80 °C. With each new membrane batch, the dissociation constant (Kd) was determined by a saturation curve. For the competitive binding assays, the TAAR1 agonists [3 H] RO5166017 (mouse and rat TAAR1) or [3 H]RO5192022 (cynomolgus monkey and human TAAR1) were used as TAAR1 radioligands at a concentration equal to Kd values, which were usually around 0.7 nM (mouse TAAR1), 2.3 nM (rat), 16 nM (human), and 28 nM (cynomolgus monkey). Nonspecific binding was defined as the amount of radioligand bound in the presence of 10 μM RO5166017. RO5166017 was tested at a broad range of concentrations (10 pM to 10 μM) in duplicates. RO5166017 (20 μL/well) was transferred into a 96-deep well plate, and 180 μL binding buffer (20 mM Hepes-NaOH, 10 mM MgCl2, 2 mM CaCl2, pH 7.4), 300 μL radioligand, and 500 μL membranes (resuspended at 60 μg protein/mL except for human TAAR1 assay, which was resuspended at 400 μg protein/mL) were added. The plates were incubated at 4 °C for 90 min (mouse, rat, and monkey TAAR1) or 60 min (human TAAR1). For the mouse, rat, and monkey TAAR1 assays, incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument) and glass filters GF/C presoaked for 1 h in polyethylenimine (0.3%) and washed three times with 1 mL cold binding buffer. After the addition of 45 μL Microscint 40, the Unifilter-96 plate was sealed, and after 1 h, the radioactivity was counted using a TopCount Microplate Scintillation Counter. For the human TAAR1 assays, incubations were terminated by filtration with a Brandel cell harvester through glass filters GF/C presoaked for 1 h in polyethylenimine (0.3%) on glass filters GF/B and washed three times with 1 mL cold binding buffer. Each filter GF/C was put into a vial containing 10 mL UltimaGold. After 1 h, the vials were counted on a Beta counter Tricarb 2500TR.

cAMP Assay. [1]
Recombinant HEK293 cells expressing TAAR1 were grown as above and harvested when 80–90% confluent. The culture medium was removed, and cells were washed one time with PBS; then, the cells were detached by incubation with 5 mL trypsin/EDTA for 5 min at 37 °C. Afterward, 45 mL culture medium were added, and the solution was centrifuged at 900 × g for 5 min at room temperature (RT). The cell pellet was resuspended in fresh culture medium and brought to 5.105 cells/ mL. The cells were plated in 96-well plates (BIOCOAT 6640; Becton Dickinson) (100 μL/well with 50,000 cells/well) and incubated for 20 h at 37 °C. For the stimulation of the cells, the culture medium was removed, and 100 μL PBS (AMIMED endotoxine free) were added; after 5 min under shaking at RT, PBS was removed, and 90 μL PBS containing 1 mM 3-isobutyl-1-methylxanthine (IBMX) was added. After shaking the cells for 2 min, they were incubated for 10 min at 37 °C and 5% CO2/95% air. All compounds were tested at a broad range of concentrations (100 pM to 10 μM) in duplicates. Typically, 10 μL compound solution in PBS and 1 mM IBMX, 10 μL 0.3 mM β-phenylethylamine (PEA) solution (as maximal response), or 10 μL 2% DMSO solution (as basal level) were added, and after 10 min shaking, the cells were incubated for 30 min at 37 °C. Afterward, the solutions were removed, and the cells were lysed with 150 μL lysis buffer (cAMP kit); the plates were shaken for 30 min and stored at −20 °C. For the cAMP assay, the Upstate cAMP kit was used according to the manufacturer’s instructions. In the 96-well rabbit anti-cAMP antibody-coated plates, 50 μL cAMP standards (eight standards from 1 to 0.0039 pmol/μL and one standard without cAMP) or 50 μL samples from the cell plates were added. A standard curve was performed on each plate; 25 μL diluted cAMP alkaline phosphatase-conjugated tracer were added followed by 50 μL diluted rabbit anti-cAMP antibody. After sealing, the plates were incubated for 30 min at RT under shaking followed by removal of the supernatant with an automated plate washer and washing five times using 1× wash buffer. Then, 100 μL diluted alkaline phosphatase substrate were added, and the plates were sealed and incubated for 30 min at RT under shaking. The plates were finally read for 1 s with a luminometer.

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Electrophysiological Recordings. [1]
Electrophysiology in Xenopus oocytes. Electrophysiological experiments in Xenopus oocytes expressing mouse TAAR1 and human Kir3.1/3.2 were performed as reported previously.

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Electrophysiology in brain slices. [1]
Electrophysiological experiments were performed as reported in 250-μm-thick horizontal slices prepared from adult (3–6 mo of age) WT and Taar1−/− mice. Visual whole-cell voltage-clamp or current-clamp techniques were used to measure holding currents at −50 mV or record spontaneous spiking activity, respectively. All cells used for the statistical analysis displayed a stable firing activity for at least 30 min. Data were obtained with an Axopatch 700B, filtered at 2 kHz, digitized at 10 kHz, and acquired and analyzed with pClamp10. Neurons were visualized using infrared differential interference contrast microscopy with a 60× objective. In the ventral tegmental area (VTA), dopaminergic cells were identified by their large hyperpolarization-activated current (Ih) evoked by hyperpolarizing pulses (−50 to −120 mV) and an outward current in response to quinpirole (10 μM). In the dorsal raphe nucleus (DRN), serotonergic cells were identified by their large Ih current in response to ipsapirone (100 nM). In the locus coeruleus (LC), noradrenergic neurons were identified by their response to U.K.14,304 followed by a block with yohimbine. Current voltage (I–V) relationships were determined by ramp commands from −20 to −140 mV (250 ms duration) in the presence of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) in the external solution.

Measurement of Locomotor Activity and Stereotypies. [1]
For psychostimulant studies, recordings were as reported (10). LMA was measured as the number of horizontal beam breaks (horizontal activity) cumulated over 30 min. Stereotypy time was assessed as the total time that stereotypic behaviors (repetitive beam breaks with intervals less than 1 s) were monitored over 30 min. For cocaine studies, C57BL/6 mice (n = 8 per group) were treated per os (p.o.) with vehicle (H2O + 0.3% tween 80) or RO5166017 (0.03–3 mg/kg in vehicle), placed into the activity monitor chamber for 30 min (habituation period), injected i.p. with saline (0.9% NaCl + 0.3% tween 80) or cocaine (15 mg/kg in saline), and returned to the recording chamber for immediate monitoring of behavior (recording period). The same paradigm was used in Taar1−/− mice (n = 10 per group) with RO5166017 (0.3–1 mg/kg p.o.) and cocaine (20 mg/kg i.p.) using a repeated measures design with at least 10 d between two sessions. For L-687,414 studies, NMRI mice (n = 8 per group) were dosed p.o. with vehicle or RO5166017 (0.01–1 mg/kg in vehicle) 15 min before receiving saline or L-687,414 (50 mg/kg in saline) s.c. The habituation period was 15 min. For Taar1−/− mice, animals (n = 8 per group) were dosed with RO5166017 (0.3 mg/kg p.o.), were placed into the recording chamber for 45 min, received saline or L-687,414 (75 mg/kg) s.c., and were returned to the recording chamber for 15 min (thus, 60-min habituation) before LMA was recorded.
For DAT−/−, WT, and double mutant (DAT−/−/Taar1−/−) mice, LMA was measured as reported (26). DAT−/− mice were placed into activity monitor chambers for 30 min to fully manifest their novelty-driven hyperactivity; then, they were treated with saline or RO5166017 (0.2–1 mg/kg i.p.), and horizontal activity was monitored for 90 min. Nonhabituated WT mice were treated before placement into the monitoring chambers, and LMA was recorded for 90 min.

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Forty-eight adult male Sprague-Dawley rats, at least 10 weeks old and weighing at least 300 g were housed under conditions described previously (Siemian et al., 2016). Water and standard rodent chow were always available except during testing. Rats were handled and habituated to the rectal temperature measurements for two days before testing.
Yawning behavior was defined as a prolonged (~1 s) opening of the mouth followed by rapid closure (Collins et al., 2005). On the test day, rats (n = 8 per treatment group) were placed into an empty rodent housing cage and allowed to habituate for 30 min. TAAR1 agonist or vehicle was administered 30 min prior to quinpirole (0.0032–0.32 mg/kg), which was administered using a cumulative dosing procedure. Observations of yawning began 20 min after each injection, and the total number of yawns was recorded for a 10 min period thereafter. At the end of the 10 min observation, body temperature was measured by inserting a lubricated thermal probe approximately 5 cm into the rectum prior to giving the next injection.
Quinpirole dihydrochloride was dissolved in normal saline. RO5263397 and RO5166017 were administered in a vehicle of 1 part 190 proof ethanol, 1 part Alkamuls EL-620 and 18 parts normal saline. All drugs were administered intraperitoneally in a volume of 1–2 ml/kg.[2]

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Pharmacokinetics Experiments. [1]
Male C57BL/6 mice were dosed with RO5166017 either i.v. through a jugular vein or orally [per os (p.o.)]. Plasma and brain samples were collected after euthanasia of two animals per group at 0.08, 0.25, 0.5, 1, 2, 4, and 8 h (i.v.) or 0.25, 0.5, 1, and 2 h (p.o.) after dosing. Concentrations of RO5166017 were determined using liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pharmacokinetic parameters were calculated by noncompartmental analysis of plasma concentration time curves using WinNonlin 4.1. Stress-Induced Hyperthermia. [1]
SIH test was performed in NMRI mice as described. Body temperature was measured two times in each mouse: one time at t =0(T1) and one time at t = +15 min (T2). T1 served as the handling stressor. The difference in temperature (T2 − T1) was considered to reflect SIH. Mice received RO5166017 (0.01–1 mg/kg p.o.) or vehicle (0.9% NaCl + 0.3% tween 80) 45 min before measurement of T1.

[1]. TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity. Proc Natl Acad Sci U S A.

[2]. Trace amine-associated receptor 1 agonists RO5263397 and RO5166017 attenuate quinpirole-induced yawning but not hypothermia in rats. Behav Pharmacol. 2017 Oct;28(7):590-593.

Increasing evidence suggests that trace amine-associated receptor 1 (TAAR1) is an important modulator of the dopaminergic system. Existing molecular evidence indicates that TAAR1 regulates dopamine levels through interactions with dopamine transporters and D2 receptors. However, investigations to date have not been exhaustive and other pathways may be involved. In this study, we used a well-described set of behaviors, quinpirole-induced yawning and hypothermia, to explore the potential interaction of TAAR1 and D3 receptors, which are members of the 'D2-like' dopamine receptor subfamily. Previous studies have shown that for D2/D3 receptor agonists, the induction of yawning is a D3 receptor-mediated effect, whereas the inhibition of yawning and induction of hypothermia are D2 receptor-mediated effects. Quinpirole produced an inverted U-shaped dose-effect curve for yawning, which was shifted downward dose-dependently by each of the TAAR1 agonists RO5263397 and RO5166017. Quinpirole also produced dose-dependent hypothermia, which was not affected by either TAAR1 agonist. These results suggest that TAAR1 agonists may interact with D3 receptors and/or its downstream pathways, as opposed to D2 receptors. These findings may shed light on a previously unexplored possibility for the mechanism of TAAR1-mediated effects.[2]

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In conclusion, these results indicate that the TAAR1 agonists RO5263397 and RO5166017 reduce a D3 receptor-mediated behavior but do not affect D2 receptor-mediated behaviors induced by quinpirole in rats. Based on these in vivo data, future molecular investigations regarding the mechanism of TAAR1 should consider the potential involvement of D3 receptors. [2]

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TAAR1 is a promising therapeutic target for the treatment of neuropsychiatric disorders, but the diversity and polypharmacology of the agonists available have rendered dissection of its physiological functions challenging. Here, we report on RO5166017, a synthetic selective and in vivo active TAAR1 agonist. In vitro, RO5166017 inhibited the firing frequency of VTA DA and DRN 5-HT neurons. Furthermore, modulation of TAAR1 activity altered the pharmacology of DRN 5-HT1A receptors, demonstrating that TAAR1 modulates not only DA but also 5-HT neurotransmission. In vivo, although silent by itself, RO5166017 prevented psychostimulant-induced hyperlocomotion, inhibited novelty-driven hyperactivity of DAT−/− mice, and prevented SIH in WT but not Taar1−/− mice. These results link TAAR1 to the control of monoaminergic-driven behaviors and underline the antipsychotic and anxiolytic potential of TAAR1 agonists.[1]

219.137

C, 65.73; H, 7.81; N, 19.16; O, 7.30

1048346-74-2

25016538

White to light yellow solid powder

1.2±0.1 g/cm3

314.7±34.0 °C at 760 mmHg

144.2±25.7 °C

0.0±0.7 mmHg at 25°C

1.589

1.32

1

3

4

16

249

1

O1C(N)=N[C@H](C1)CN(C1C=CC=CC=1)CC

PPONHQQJLWPUPH-JTQLQIEISA-N

InChI=1S/C12H17N3O/c1-2-15(11-6-4-3-5-7-11)8-10-9-16-12(13)14-10/h3-7,10H,2,8-9H2,1H3,(H2,13,14)/t10-/m0/s1

(4S)-4-[(N-ethylanilino)methyl]-4,5-dihydro-1,3-oxazol-2-amine

RO5166017; RO 5166017; 1048346-74-2; RO5166017; (4S)-4-[(N-ethylanilino)methyl]-4,5-dihydro-1,3-oxazol-2-amine; RO 5166017; RO-5166017; YK98JFQ52U; (S)-4-((Ethyl(phenyl)amino)methyl)-4,5-dihydrooxazol-2-amine; CHEMBL3779993; RO-5166017

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ~25 mg/mL (~114.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.40 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 25.0 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: 2.5 mg/mL (11.40 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (11.40 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 25.0 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.)