Compound (SMILES): CCc1cc2c(C)c(-c3nc(C)no3)c(N3CCC(N[C@H]4CCCOC4)CC3)nc2c(F)c1F
InChIKey: RZBYXXRYLZQFED-SFHVURJKSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: 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.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 471.55, ALogP 4.48, HBA 7, HBD 1, PSA 76.31, QED 0.59