Trace amine-associated receptor 1 (TAAR1)

RO5203648 showed high affinity and potency at TAAR1, high selectivity versus other targets [2].

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Researchers first selected the partial agonist RO5203648/36 (= RO5203648) for further studies. A Cerep screen11 showed high selectivity across a panel of 149 enzymes and receptors, and pharmacokinetic analysis revealed that this compound was well suited for in vivo studies in rat. To our knowledge, RO5203648/36 was the first selective partial TAAR1 agonist tested in behavioral studies, and it clearly demonstrated antipsychotic, antidepressant, and antiaddictive activities in a number of animal models. Unfortunately, additional in vitro testing of 36 revealed a very high metabolic clearance of this compound in human hepatocytes, which was not obvious in prior testing in human liver microsomes, and this led to a deselection of 36 for further development. These observations prompted us to compare in vitro microsomal clearance with in vitro hepatocyte clearance for a number of other compounds from the same series, whereby a surprisingly big discrepancy between the clearance data in the two assay systems was apparent for most of the compounds [1].

In mouse brain slices, RO5203648 increased the firing frequency of dopaminergic and serotonergic neurons in the ventral tegmental area and the dorsal raphe nucleus, respectively. In various behavioral paradigms in rodents and monkeys, RO5203648 demonstrated clear antipsychotic- and antidepressant-like activities as well as potential anxiolytic-like properties. Furthermore, it attenuated drug-taking behavior and was highly effective in promoting attention, cognitive performance, and wakefulness [2].

Bioluminescence Resonance Energy Transfer (BRET) Measurement [1]
Bioluminescence resonance energy transfer experiments were performed as described previously (Espinoza et al., 2013). RO5203648 and EPPTB powder was dissolved in DMSO at the concentration of 10 mM and then diluted in PBS to the desired concentration. For time course experiments, the plate was read immediately after the addition of RO5203648 and for approximately 20 min. In order to calculate the EC50 values, a concentration response curve was performed using different concentration of the agonist. To evaluate the antagonistic effect of EPPTB, the antagonist was added 5 min before RO5203648. All the experiments were conducted in presence of the phosphodiesterase inhibitor 3-Isobutyl-1-methylxanthine at the final concentration of 200 μM. Readings were collected using a Tecan Infinite instrument that allows the sequential integration of the signals detected in the 465 to 505 nm and 515 to 555 nm windows using filters with the appropriate band pass and by using iControl software. The acceptor/donor ratio was calculated as previously described (Espinoza et al., 2013). We used an EPAC BRET biosensor to monitor cAMP levels. With this sensor an increase in cAMP is reflected in a decrease in the BRET ratio. Curve was fitted using a non-linear regression and one site specific binding with GraphPad Prism 5. Data are representative of four independent experiments and are expressed as means ± SEM.

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Assay for determining functional activity at the human adrenergic α2A receptor: [1]
Membrane Preparation: CHL cells stably expressing the adrenergic α2A receptor were maintained at 37 °C and 5% CO2 in DMEM high glucose medium containing fetal calf serum (5%, heat inactivated for 30 min at 56 °C) and 250 µg/ml geneticin. Cells were released from culture flasks using trypsin/ EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1’000 rpm for 5 min at 4 °C, frozen and stored at -80 °C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron at 14’000 rpm for 20 s. The homogenate was centrifuged at 48’000 x g for 30 min at 4 °C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14’000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80 °C.

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Wheatgerm agglutinin SPA beads assay: [1]
The radioligand [35S] GTPγS was used at a concentration of 0.5 nM final concentration. Nonspecific binding was defined as the amount of GTPγS bound in the presence of 10 µM final concentration of GTP (unlabeled ligand). Compounds were tested at a broad range of concentrations (30 pM to 30 µM) in duplicates. Norepinephrine was used as a reference for agonistic activity and RX821002 as an antagonist reference. A mix M was prepared containing GDP at 1.5 µM final concentration, membranes at 5 µg protein/well and wheatgerm agglutinin SPA beads at 1 mg/well. The test compounds (20 µl/well) were transferred into an OptiPlate. 30 µl/well of buffer containing 50 mM Tris, 5 mM MgCl2, 100 mM NaCl, 1 mM EDTA and 1 mM DTT were added. In order to determine the total binding 20 µl of buffer was used and for the nonspecific binding 20 µl of 3 GTP at 10 µM. For agonist testing 100 µl of mix M and 50 µl of [35S] GTPγS were added. The OptiPlate was then incubated for 30 min at RT under shaking at 350 rpm/min and centrifuged for 3 min at 3000 rpm. Radioactivity was counted using a TopCount Microplate Scintillation Counter. For antagonist testing 100 µl of mix M and 50 µl of [35S] GTPγS were added. The OptiPlate was then incubated for 5 min at RT under shaking at 350 rpm/min. 30 µl norepinephrine at 20 µM was added to the wells containing the compounds. The plate was then incubated for 25 min at RT under shaking at 350 rpm/min and centrifuged for 3 min at 3000 rpm. Radioactivity was counted using a TopCount Microplate Scintillation Counter. 50 µl of the 35S-GTPγS stock were counted in 5 ml of ReadySafe scintillation cocktail to determine the total counts added to the respective assays.

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Formation of Glutathione (GSH) adducts with selected compounds [1]
Microsomal suspensions were diluted in 100 mM potassium phosphate buffer, pH7.4 to make 2 mg/ml solutions. One portion of this was heat denatured at 95 °C for 10 minutes, whilst the other remained on ice. The denatured protein solutions were then cooled before use. Each incubation was made up using 220.5 µl of pretreated protein solution (see above, final concentration 1 mg/ml) and 4.5 µl substrate (final concentration 10 µM). This was warmed to 37 °C for 10 minutes before addition of 225 µl prewarmed glutathione solution in phosphate buffer (final concentration 5 mM). Incubations were stopped after 10 or 20 minutes incubation time (at 37 °C) by addition of 150 µl quench reagent (150:100:1 mix of 10% trichloroacetic acid : acetonitrile : 30% hydrogen peroxide). Samples were chilled on ice for 1 hour before centrifugation (20,000x g, 10 minutes). The supernatant was removed and 200 µl analysed by HPLC. Mass spectrometric analysis was performed on a LCQ mass spectrometer equipped with the Xcalibur 1.2 software package.

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The TAAR1 partial agonist RO5203648 was evaluated for its binding affinity and functional activity at rodent and primate TAAR1 receptors stably expressed in HEK293 cells. [2]

Biological assay section [1]
Results were obtained from at least three independent experiments. Experiments were run at least in duplicates. EC50 values are given as a mean in nM. The Emax value for the functional activity data at TAAR1 receptor describes the degree of functional activity compared to 100% for the natural ligand and full agonist phenethylamine. Compounds with Emax<85% are regarded as partial agonists. The functional selectivity ratio of hTAAR1 vs. hα2A is determined by dividing the hα2A EC50 or IC50 value by the hTAAR1 EC50 value.

[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 partial agonism reveals novel paradigm for neuropsychiatric therapeutics. Biol Psychiatry. 2012 Dec 1;72(11):934-42.

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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Background: Trace amines, compounds structurally related to classical biogenic amines, represent endogenous ligands of the trace amine-associated receptor 1 (TAAR1). Because trace amines also influence the activity of other targets, selective ligands are needed for the elucidation of TAAR1 function. Here we report on the identification and characterization of the first selective and potent TAAR1 partial agonist. Methods: The TAAR1 partial agonist RO5203648 was evaluated for its binding affinity and functional activity at rodent and primate TAAR1 receptors stably expressed in HEK293 cells, for its physicochemical and pharmacokinetic properties, for its effects on the firing frequency of monoaminergic neurons ex vivo, and for its properties in vivo with genetic and pharmacological models of central nervous system disorders. Results: RO5203648 showed high affinity and potency at TAAR1, high selectivity versus other targets, and favorable pharmacokinetic properties. In mouse brain slices, RO5203648 increased the firing frequency of dopaminergic and serotonergic neurons in the ventral tegmental area and the dorsal raphe nucleus, respectively. In various behavioral paradigms in rodents and monkeys, RO5203648 demonstrated clear antipsychotic- and antidepressant-like activities as well as potential anxiolytic-like properties. Furthermore, it attenuated drug-taking behavior and was highly effective in promoting attention, cognitive performance, and wakefulness. Conclusions: With the first potent and selective TAAR1 partial agonist, RO5203648, we show that TAAR1 is implicated in a broad range of relevant physiological, behavioral, and cognitive neuropsychiatric dimensions. Collectively, these data uncover important neuromodulatory roles for TAAR1 and suggest that agonists at this receptor might have therapeutic potential in one or more neuropsychiatric domains.[2]

C9H10CL4N2O

304.00049829483

230.001

C, 35.56; H, 3.32; Cl, 46.65; N, 9.22; O, 5.26

1043491-54-8

1043491-54-8;

24966113

White to off-white solid powder

1.6±0.1 g/cm3

351.5±52.0 °C at 760 mmHg

166.4±30.7 °C

0.0±0.8 mmHg at 25°C

1.663

2.19

1

2

1

14

247

1

C1[C@@H](N=C(O1)N)C2=CC(=C(C=C2)Cl)Cl

HGGPGNSCBBAGJN-MRVPVSSYSA-N

InChI=1S/C9H8Cl2N2O/c10-6-2-1-5(3-7(6)11)8-4-14-9(12)13-8/h1-3,8H,4H2,(H2,12,13)/t8-/m1/s1

(4S)-4-(3,4-dichlorophenyl)-4,5-dihydro-1,3-oxazol-2-amine

1043491-54-8; (S)-4-(3,4-Dichlorophenyl)-4,5-dihydrooxazol-2-amine; RO5203648; (4S)-4-(3,4-dichlorophenyl)-4,5-dihydro-1,3-oxazol-2-amine; RO5203648 hydrochloride; RO5203648 HCl; (4S)-4-(3,4-Dichlorophenyl)-4,5-dihydro-2-oxazolamine; SCHEMBL3613795;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~432.75 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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