ChEMBL ligand: CHEMBL4594376 (Ralmitaront, RG-7906, RG7906, Ro6889450, RO-6889450, RO6889450)
DAT/Sodium-dependent dopamine transporter in Human [ChEMBL: CHEMBL238] [GtoPdb: 927] [UniProtKB: Q01959] There should be some charts here, you may need to enable JavaScript!
TA1 receptor/Trace amine-associated receptor 1 in Human [ChEMBL: CHEMBL5857] [GtoPdb: 364] [UniProtKB: Q96RJ0] There should be some charts here, you may need to enable JavaScript!

DB	Assay description	Assay Type	Standard value	Standard parameter	Original value	Original units	Original parameter	Reference
DAT/Sodium-dependent dopamine transporter in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL238] [GtoPdb: 927] [UniProtKB: Q01959]
ChEMBL	Human DAT assay: Binding to dopamine transporter (DAT) in vitro. Human embryonic kidney (HEK) 293 cells (Invitrogen, Zug, Switzerland) stably transfected with human DAT were cultured. The cells were collected and washed three times with phosphate-buffered saline (PBS). The pellets were frozen at −80° C. The pellets were then resuspended in 400 ml of 20 mM HEPES-NaOH, pH 7.4, that contained 10 mM EDTA at 4° C. After homogenization with a Polytron (Kinematica, Lucerne,Switzerland) at 10000 rotations per minute (rpm) for 15 s, the homogenates were centrifuged at 48000×g for 30 min at 4° C. Aliquots of the membrane stocks were frozen at −80° C. All assays were performed at least three times. The test compounds were diluted in 20 ml of binding buffer (252 mM NaCl, 5.4 mM KCl, 20 mM Na2HPO4, 3.52 mM KH2PO4, pH 7.4) and 10 point dilution curves were made and transferred to 96-well white polystyrene assay plates (Sigma-Aldrich, Buchs, Switzerland). [3H]-WIN35,428 (˜86 Ci /mmol; Perkin-Elmer) was the radioligand for the DAT assay and had a Kd of 12 nM. Fifty microliters of [3H]-WIN35,428 (˜40 nM concentration) was added to each well of the hDAT assay plates, targeting a final [3H]-WIN35428 concentration of 10 nM. Twenty microliters of binding buffer alone in the assay plate defined the total binding, whereas binding in the presence of 10 μM indatraline defined nonspecific binding. Frozen DAT membrane stocks were thawed and resuspended to a concentration of approximately 0.04 mg protein/ml binding buffer (1:1 diluted in H2O) using a polytron tissue homogenizer. The membrane homogenates (40 μg/ml) were then lightly mixed for 5-30 min with polyvinyl toluene (PCT) wheat germ agglutinin-coated scintillation proximity assay (WGASPA; Amersham Biosciences) beads at 7.7 mg beads/ml homogenate. One hundred thirty microliters of the membrane/bead mixture were added to each well of the assay plate that contained radioligand and test compounds (final volume in each well, 200 μl) to start the assay, which was incubated for approximately 2 h at room temperature with agitation. The assay plates were then counted in the PVT SPA counting mode of a Packard Topcount. Fifty microliters of the [3H]-WIN35428 stocks were counted in 5 ml of ReadySafe scintillation cocktail (Beckman Industries) on a Packard 1900CA liquid scintillation counter to determine the total counts added to the respective assays. Non-linear regression was used to fit the data to sigmoid curves and determine IC50 values for binding and uptake. Ki values for binding and uptake were calculated using the following Cheng-Prusoff equation: Ki=IC50/(1+[S]/Km).	B	4.56	pKi	27530	nM	Ki	US-10508107-B2. Morpholine derivative (2019)
ChEMBL	Human DAT assay: Binding to dopamine transporter (DAT) in vitro. Human embryonic kidney (HEK) 293 cells (Invitrogen, Zug, Switzerland) stably transfected with human DAT were cultured. The cells were collected and washed three times with phosphate-buffered saline (PBS). The pellets were frozen at −80° C. The pellets were then resuspended in 400 ml of 20 mM HEPES-NaOH, pH 7.4, that contained 10 mM EDTA at 4° C. After homogenization with a Polytron (Kinematica, Lucerne, Switzerland) at 10000 rotations per minute (rpm) for 15 s, the homogenates were centrifuged at 48000×g for 30 min at 4° C. Aliquots of the membrane stocks were frozen at −80° C. All assays were performed at least three times. The test compounds were diluted in 20 ml of binding buffer (252 mM NaCl, 5.4 mM KCl, 20 mM Na2HPO4, 3.52 mM KH2PO4, pH 7.4) and 10 point dilution curves were made and transferred to 96-well white polystyrene assay plates (Sigma-Aldrich, Buchs, Switzerland). [3H]-WIN35,428 (˜86 Ci/mmol; Perkin-Elmer) was the radioligand for the DAT assay and had a Kd of 12 nM. Fifty microliters of [3H]-WIN35,428 (˜40 nM concentration) was added to each well of the hDAT assay plates, targeting a final [3H]-WIN35428 concentration of 10 nM. Twenty microliters of binding buffer alone in the assay plate defined the total binding, whereas binding in the presence of 10 μM indatraline defined nonspecific binding. Frozen DAT membrane stocks were thawed and resuspended to a concentration of approximately 0.04 mg protein/ml binding buffer (1:1 diluted in H2O) using a polytron tissue homogenizer. The membrane homogenates (40 μg/ml) were then lightly mixed for 5-30 min with polyvinyl toluene (PCT) wheat germ agglutinin-coated scintillation proximity assay (WGASPA; Amersham Biosciences) beads at 7.7 mg beads/ml homogenate. One hundred thirty microliters of the membrane/bead mixture were added to each well of the assay plate that contained radioligand and test compounds (final volume in each well, 200 μl) to start the assay, which was incubated for approximately 2 h at room temperature with agitation. The assay plates were then counted in the PVT SPA counting mode of a Packard Topcount. Fifty microliters of the [3H]-WIN35428 stocks were counted in 5 ml of ReadySafe scintillation cocktail (Beckman Industries) on a Packard 1900CA liquid scintillation counter to determine the total counts added to the respective assays. Non-linear regression was used to fit the data to sigmoid curves and determine IC50 values for binding and uptake. Ki values for binding and uptake were calculated using the following Cheng-Prusoff equation: Ki=IC50/(1+[S]/Km).	B	4.56	pKi	27530	nM	Ki	US-11312711-B2. Morpholine derivative (2022)
TA1 receptor/Trace amine-associated receptor 1 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL5857] [GtoPdb: 364] [UniProtKB: Q96RJ0]
GtoPdb	-	-	7.23	pEC50	58.5	nM	EC50	WO2017157873A1. 5-ethyl-4-methyl-pyrazole-3-carboxamide derivative having activity as agonist of taar (2017)
ChEMBL	Human TAAR1 assay: For the construction of expression plasmids the coding sequences of human TAAR 1 were amplified from genomic DNA essentially as described by Lindemann et al.[14]. The Expand High Fidelity PCR System (Roche Diagnostics) was used with 1.5 mM Mg2+ and purified PCR products were cloned into pCR2.1-TOPO cloning vector (Invitrogen) following the instructions of the manufacturer. PCR products were subcloned into the pIRESneo2 vector (BD Clontech, Palo Alto, Calif.), and expression vectors were sequence verified before introduction in cell lines.HEK293 cells (ATCC # CRL-1573) were cultured essentially as described by Lindemann et al. (2005). For the generation of stably transfected cell lines HEK293 cells were transfected with the pIRESneo2 expression plasmids containing the TAAR coding sequences (described above) with Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer, and 24 hrs post transfection the culture medium was supplemented with 1 mg/ml G418 (Sigma, Buchs, Switzerland). After a culture period of about 10 d clones were isolated, expanded and tested for responsiveness to trace amines (all compounds purchased from Sigma) with the cAMP Biotrak Enzyme immunoassay (EIA) System (Amersham) following the non-acetylation EIA procedure provided by the manufacturer. Monoclonal cell lines which displayed a stable EC50 for a culture period of 15 passages were used for all subsequent studies.cAMP measurements were performed as described previously (Revel et al., Proc. Natl. Acad. Sci. USA 2011, 108, 8485-8490). In brief, cells that expressed human TAAR1 were plated on 96-well plates (BIOCOAT 6640; Becton Dickinson, Allschwil, Switzerland) and incubated for 20 h at 37° C. Prior to stimulation of the cells with a broad concentration range of agonists for 30 min at 37° C., the cells were washed with PBS and preincubated with PBS that contained 1 mM 3-isobutyl-1-methylxanthine for 10 min at 37° C. and 5% CO2. Stimulation with 0.2% DMSO was set as the basal level, and the effect of 30 μM β-PEA was set as the maximal response. Subsequently, the cells were lysed, and cAMP assays were performed according to the manufacturer's instructions (cAMP kit; Upstate/Millipore, Schaffhausen, Switzerland). Finally, the plates were read with a luminometer (1420 Multilabel counter; PerkinElmer, Schwerzenbach, Switzerland), and the amount of cAMP was calculated. The results were obtained from at least three independent experiments. Experiments were run in duplicate or triplicate. EC50 values are presented as mean ±standard deviation (in μM).	B	7.23	pEC50	58.5	nM	EC50	US-10508107-B2. Morpholine derivative (2019)
ChEMBL	Human TAAR1 assay: For the construction of expression plasmids the coding sequences of human TAAR1 were amplified from genomic DNA essentially as described by Lindemann et al. [14]. The Expand High Fidelity PCR System (Roche Diagnostics) was used with 1.5 mM Mg2+ and purified PCR products were cloned into pCR2.1-TOPO cloning vector (Invitrogen) following the instructions of the manufacturer. PCR products were subcloned into the pIRESneo2 vector (BD Clontech, Palo Alto, Calif.), and expression vectors were sequence verified before introduction in cell lines.HEK293 cells (ATCC #CRL-1573) were cultured essentially as described by Lindemann et al. (2005). For the generation of stably transfected cell lines HEK293 cells were transfected with the pIRESneo2 expression plasmids containing the TAAR coding sequences (described above) with Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer, and 24 hrs post transfection the culture medium was supplemented with 1 mg/ml G418 (Sigma, Buchs, Switzerland). After a culture period of about 10 d clones were isolated, expanded and tested for responsiveness to trace amines (all compounds purchased from Sigma) with the cAMP Biotrak Enzyme immunoassay (EIA) System (Amersham) following the non-acetylation EIA procedure provided by the manufacturer. Monoclonal cell lines which displayed a stable EC50 for a culture period of 15 passages were used for all subsequent studies.	B	7.23	pEC50	58.5	nM	EC50	US-11312711-B2. Morpholine derivative (2022)

ChEMBL data shown on this page come from version 36:

Zdrazil B, Felix E, Hunter F, Manners EJ, Blackshaw J, Corbett S, de Veij M, Ioannidis H, Lopez DM, Mosquera JF, Magarinos MP, Bosc N, Arcila R, Kizilören T, Gaulton A, Bento AP, Adasme MF, Monecke P, Landrum GA, Leach AR. (2024). The ChEMBL Database in 2023: a drug discovery platform spanning multiple bioactivity data types and time periods. Nucleic Acids Res., 52(D1). DOI: 10.1093/nar/gkad1004. [EPMCID:10767899] [PMID:37933841]
Davies M, Nowotka M, Papadatos G, Dedman N, Gaulton A, Atkinson F, Bellis L, Overington JP. (2015) 'ChEMBL web services: streamlining access to drug discovery data and utilities.' Nucleic Acids Res., 43(W1). DOI: 10.1093/nar/gkv352. [EPMCID:25883136]