ChEMBL ligand: CHEMBL4592045 (BTRX-140, Btrx-335140, BTRX-335140, BTRX335140, CYM-53093, CYM53093, Navacaprant, NMRA-140, Nmra-335140, NMRA-335140, NMRA335140)
δ receptor/Delta-type opioid receptor in Human [ChEMBL: CHEMBL236] [GtoPdb: 317] [UniProtKB: P41143] There should be some charts here, you may need to enable JavaScript!
κ receptor/Kappa-type opioid receptor in Human [ChEMBL: CHEMBL237] [GtoPdb: 318] [UniProtKB: P41145] There should be some charts here, you may need to enable JavaScript!
μ receptor/Mu-type opioid receptor in Human [ChEMBL: CHEMBL233] [GtoPdb: 319] [UniProtKB: P35372] There should be some charts here, you may need to enable JavaScript!
Kv11.1/Voltage-gated inwardly rectifying potassium channel KCNH2 in Human [ChEMBL: CHEMBL240] [GtoPdb: 572] [UniProtKB: Q12809] 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
δ receptor/Delta-type opioid receptor in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL236] [GtoPdb: 317] [UniProtKB: P41143]
ChEMBL	Antagonist activity at GAL4-VP16-fused DOR (unknown origin) expressed in human U2OS cells assessed as inhibition of DAMGO-induced beta-arrestin migration preincubated for 30 mins followed by agonist addition and measured after 3 hrs by PathHunter assay	B	5.19	pIC50	6500	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
GtoPdb	-	-	5.19	pIC50	6500	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
κ receptor/Kappa-type opioid receptor in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL237] [GtoPdb: 318] [UniProtKB: P41145]
ChEMBL	Displacement of [3H]diprenorphine from human KOR	B	8.82	pKi	1.5	nM	Ki	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
ChEMBL	KOR Antagonist Assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements: The carboxy terminus of the OPRK1 receptor has a 7-amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues and this induces recruitment of B-arrestin2-TEV protease. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluorescein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluorescein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the coumarin fluorophore from the fluorescein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio.OPRK1 TANGO U2OS cells are cultured in growth media (McCoy's 5A medium, 10% Dialyzed FBS, Non-essential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, penicillin/streptomycin). Two million cells are added to a T175 flask in 30 mL of growth medium and incubated at 37° C./5% CO2 for four days at which point they are 70-90% confluent. Growth medium is removed by aspiration, 5 mL of 0.25% Trypsin/EDTA is added to the flask and gently washed over the cells. The trypsin is then removed by aspiration. Cells are allowed to round up and are detached by tapping the flask. Cells are suspended in assay medium (DMEM high glucose with 1% charcoal dextran stripped FBS, Non-essential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, penicillin/streptomycin) triturated, counted and pelleted by centrifugation. Cells are resuspended at 1.6 million cells per mL and 10 ul added to each well of a black, clear-bottom 384-well assay plate (Greiner part number 788092). Assay plates are placed in a humidified box and incubated 16-24 hours at 37° C./5% CO2. Compounds dissolved in DMSO are serially diluted in DMSO in a 384-well polypropylene plate. 50 nl of each compound dilution is added to the wells of the assay plate using pintools. Control wells receive 50 nl DMSO. The plates are returned to the incubator for 30 minutes. After the preincubation with compound, 50 nl of 600 nM (−)-U-50,488 (agonist challenge) is added to the compound wells of the assay plate. Control wells receive 50 nl of 5.6 uM (−)-U-50,488 (100% response control), 50 nl of 600 nM (−)-U-50,488 (EC80 control) or 50 nl of DMSO (0% response control). Assay plates are returned to the incubator for 4 hours at 37° C./5% CO2. Assay plates are then removed from the incubator and 2.5 ul of LiveBlazer CCF4-AM substrate dye (Invitrogen) is added to each well. The assay plates are then placed on the benchtop for two hours at room temperature covered in foil to avoid light.The plates are then read on a fluorescence plate reader with an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 525 nm. Results are calculated using the blue/green emission ratio. Percent inhibition is calculated by the following equation, with IC50 being the concentration of compound required to achieve 50% inhibition:% ⁢ ⁢ Inhibition = 100 - ( 1 ⁢ 0 ⁢ 0 ⁢ ( Compound ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well EC ⁢ ⁢ 80 ⁢ ⁢ Control ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well ) ).	B	7	pIC50	>100	nM	IC50	US-11124504-B2. Kappa opioid receptor antagonists and products and methods related thereto (2021)
ChEMBL	KOR Antagonist Assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements: The carboxy terminus of the OPRK1 receptor has a 7-amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues and this induces recruitment of B-arrestin2-TEV protease. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluorescein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluorescein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the coumarin fluorophore from the fluorescein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio.OPRK1 TANGO U2OS cells are cultured in growth media (McCoy's 5 A medium, 10% Dialyzed FBS, Non-essential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, penicillin/streptomycin). Two million cells are added to a T175 flask in 30 mL of growth medium and incubated at 37° C./5% CO2 for four days at which point they are 70-90% confluent. Growth medium is removed by aspiration, 5 mL of 0.25% Trypsin/EDTA is added to the flask and gently washed over the cells. The trypsin is then removed by aspiration. Cells are allowed to round up and are detached by tapping the flask. Cells are suspended in assay medium (DMEM high glucose with 1% charcoal dextran stripped FBS, Non-essential amino acids, 25 mM HEPES, 1 mM sodium pyruvate, penicillin/streptomycin) triturated, counted and pelleted by centrifugation. Cells are resuspended at 1.6 million cells per mL and 10 ul added to each well of a black, clear-bottom 384-well assay plate (Greiner part number 788092). Assay plates are placed in a humidified box and incubated 16-24 hours at 37° C./5% CO2. Compounds dissolved in DMSO are serially diluted in DMSO in a 384-well polypropylene plate. 50 nl of each compound dilution is added to the wells of the assay plate using pintools. Control wells receive 50 nl DMSO. The plates are returned to the incubator for 30 minutes. After the preincubation with compound, 50 nl of 600 nM (−)-U-50,488 (agonist challenge) is added to the compound wells of the assay plate. Control wells receive 50 nl of 5.6 uM (−)-U-50,488 (100% response control), 50 nl of 600 nM (−)-U-50,488 (EC80 control) or 50 nl of DMSO (0% response control). Assay plates are returned to the incubator for 4 hours at 37° C./5% CO2. Assay plates are then removed from the incubator and 2.5 ul of LiveBlazer CCF4-AM substrate dye (Invitrogen) is added to each well. The assay plates are then placed on the benchtop for two hours at room temperature covered in foil to avoid light.The plates are then read on a fluorescence plate reader with an excitation wavelength of 405 nm and emission wavelengths of 460 nm and 525 nm. Results are calculated using the blue/green emission ratio. Percent inhibition is calculated by the following equation, with IC50 being the concentration of compound required to achieve 50% inhibition.	B	9	pIC50	<1	nM	IC50	US-10676469-B2. Kappa opioid receptor antagonists and products and methods related thereto (2020)
ChEMBL	Antagonist activity at GAL4-VP16-fused KOR (unknown origin) expressed in human U2OS cells co-expressing Tango-OPRK1-BLA assessed as inhibition of U-50488-induced beta-arrestin migration after 4 hrs by FRET assay	B	9.1	pIC50	0.8	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
GtoPdb	-	-	9.1	pIC50	0.8	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
ChEMBL	Agonist activity at KOR (unknown origin)	F	6.39	pEC50	410	nM	EC50	Eur J Med Chem (2022) 243: 114785-114785 [PMID:36179400]
μ receptor/Mu-type opioid receptor in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL233] [GtoPdb: 319] [UniProtKB: P35372]
ChEMBL	Binding affinity to human MOR	B	6.35	pKi	450	nM	Ki	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
ChEMBL	OPR Mu Antagonist Assay: The purpose of this assay is to confirm the potency and selectivity of compounds synthesized to be OPRK1 Antagonists. This assay monitors the OPRMu1 activation, in membrane recruitment of β-arrestin. The assay monitors GPCR-β-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). It employs U20S cells which express OPRMu1 fused to the complementary beta-gal fragment (enzyme acceptor). As designed, compounds that act as antagonists will prevent receptor activation resulting in reduce well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.The Discover X OPRMu1-U20S cell line was routinely cultured in T175 flasks at 37° C., 5% CO2 and 95% relative humidity (RH). The growth media consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, Non-essential amino acids, 1 mM Sodium Pyruvate, 1× antibiotic mix (penicillin streptomycin) plus 500 ug/mL Geneticin and 300 ug/mL Hygromycin (selection antibiotics).On Day 1 of the assay, 5000 cells in 20 ul of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384 Corning 3570 standard white plate, and incubated 16-24 hours at 37° C., 5% CO2 and 95% (RH). On Day 2, 100 nl of test compound in DMSO was added to the appropriate wells, 100 nl of DMSO added to control wells and plates were incubated for 30 min at 37° C., 5% CO2 and 95% (RH). Next 100 nl of DAMGO OPRMu1 or DMSO in assay media (EC80 Challenge consists of 100 nl of 50 uM DAMGO, final assay concentration=250 nM, 100% Response wells receive 100 nl 200 uM DAMGO). After incubation for 3 hours at 37° C., 5% CO2 and 95% (RH), 10 ul of Path Hunter Detection Mix is added to each well, plate placed on a plate rotator/mixer for ˜10 minutes and then incubated at room temperature, in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.The Percent Inhibition was calculated from the median ratio as follows:% ⁢ ⁢ Inhibition = 100 - ( 1 ⁢ 0 ⁢ 0 ⁢ ( Compound ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well EC ⁢ ⁢ 80 ⁢ ⁢ Control ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well ) )where: COMPOUND WELL is defined as the well containing test compound; EC80 CONTROL WELL is defined as wells containing DAMGO challenge (250 nM final)=0% inhibition; and 0% RESPONSE WELL is defined as wells containing DMSO=100% inhibition.IC50 is defined as the concentration of compound required to achieve 50% inhibition.	B	6.26	pIC50	550	nM	IC50	US-11124504-B2. Kappa opioid receptor antagonists and products and methods related thereto (2021)
ChEMBL	Antagonist activity at GAL4-VP16-fused MOR (unknown origin) expressed in human U2OS cells assessed as inhibition of DAMGO-induced beta-arrestin migration preincubated for 30 mins followed by agonist addition and measured after 3 hrs by PathHunter assay	B	6.96	pIC50	110	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
GtoPdb	-	-	6.96	pIC50	110	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]
ChEMBL	OPR Mu Antagonist Assay: The purpose of this assay is to confirm the potency and selectivity of compounds synthesized to be OPRK1 Antagonists. This assay monitors the OPRMu1 activation, in membrane recruitment of β-arrestin. The assay monitors GPCR-β-arrestin proximity using low affinity fragment complementation of beta-galactosidase (beta-gal). It employs U20S cells which express OPRMu1 fused to the complementary beta-gal fragment (enzyme acceptor). As designed, compounds that act as antagonists will prevent receptor activation resulting in reduce well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 10 micromolar.The Discover X OPRMu1-U20S cell line was routinely cultured in T175 flasks at 37° C., 5% CO2 and 95% relative humidity (RH). The growth media consisted of DMEM/F12 1:1 Media supplemented with 10% v/v heat inactivated fetal bovine serum, 25 mM HEPES, Non-essential amino acids, 1 mM Sodium Pyruvate, 1× antibiotic mix (penicillin streptomycin) plus 500 ug/mL Geneticin and 300 ug/mL Hygromycin (selection antibiotics).On Day 1 of the assay, 5000 cells in 20 ul of assay buffer (Discover X's Cell Plating Reagent 5) were seeded into each well of a 384 Corning 3570 standard white plate, and incubated 16-24 hours at 37° C., 5% CO2 and 95% (RH). On Day 2, 100 nl of test compound in DMSO was added to the appropriate wells, 100 nl of DMSO added to control wells and plates were incubated for 30 min at 37° C., 5% CO2 and 95% (RH). Next 100 nl of DAMGO OPRMu1 or DMSO in assay media (EC80 Challenge consists of 100 nl of 50 uM DAMGO, final assay concentration=250 nM, 100% Response wells receive 100 nl 200 uM DAMGO). After incubation for 3 hours at 37° C., 5% CO2 and 95% (RH), 10 ul of Path Hunter Detection Mix is added to each well, plate placed on a plate rotator/mixer for 10 minutes and then incubated at room temperature, in the dark for 1 hour. Well luminescence was measured on Perkin Elmer's Envision.The Percent Inhibition was calculated from the median ratio as follows:% ⁢ ⁢ Inhibition = 100 - ( 100 ⁢ ( Compound ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well EC ⁢ ⁢ 80 ⁢ ⁢ Control ⁢ ⁢ Well - 0 ⁢ % ⁢ ⁢ Response ⁢ ⁢ Well ) )where: COMPOUND WELL is defined as the well containing test compound; EC80 CONTROL WELL is defined as wells containing DAMGO challenge (250 nM final)=0% inhibition; and 0% RESPONSE WELL is defined as wells containing DMSO=100% inhibition. IC50 is defined as the concentration of compound required to achieve 50% inhibition.	B	7	pIC50	>100	nM	IC50	US-10676469-B2. Kappa opioid receptor antagonists and products and methods related thereto (2020)
ChEMBL	Agonist activity at MOR (unknown origin)	B	5.34	pEC50	4590	nM	EC50	Eur J Med Chem (2022) 243: 114785-114785 [PMID:36179400]
Kv11.1/Voltage-gated inwardly rectifying potassium channel KCNH2 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL240] [GtoPdb: 572] [UniProtKB: Q12809]
ChEMBL	Inhibition of human ERG expressed in HEK293 cells at 10 uM by Qpatch clamp assay	B	9	pIC50	1	nM	IC50	J Med Chem (2019) 62: 1761-1780 [PMID:30707578]

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]