| Assay Method Information | |
| | Characterization of Agonism of Human D2L Receptors Using Cyclic Adenosine Monophosphate Detection |
| Description: | Agonist activity at human D2L receptors was assayed by measuring cAMP levels in CHO-K1 cells expressing human D2L receptors (Eurofins DiscoverX, Fremont, CA, USA) by homogenous time-resolved fluorescence (HTRF®) using the cAMP Gi kit (Cisbio/PerkinElmer). CHO-K1 cells expressing human D2L receptors were cultured in Ham's F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin antimycotic solution, and 800 μg/mL G418 (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37° C. in a humidified atmosphere containing 5.0% CO2. For the cAMP measurements, cryopreserved cells were thawed, and seeded in white-walled, half area 96-well plates at 10,000 cells/well in PathHunter® AssayComplete™ Cell Plating 2 (CP2) reagent (Eurofins DiscoverX) and incubated overnight at 37° C. in a humidified atmosphere with 5.0% CO2. Prior to the cAMP measurement, the CP2 reagent was removed from the cells and replaced with 20 μL compound or vehicle containing assay buffer (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl ]ethane-1-sulfonic acid (HEPES), 10 mM glucose, pH 7.4) supplemented with 100 μM IBMX and incubated at ambient temperature for 20 minutes. After an additional 30-minute incubation step at ambient temperature with 0.5 μM forskolin (Eurofins DiscoverX), cell stimulation was stopped by adding the detection reagents (20 μL cAMP-d2 and 20 μL anti-cAMP cryptate, Cisbio/PerkinElmer) diluted in lysis buffer. The time-resolved fluorescence signal was quantified with a PHERAstar FS multimode reader (BMG Labtech, Ortenberg, Germany) using standard HTRF® settings with laser excitation at 337 nm after 60 minutes of incubation at ambient temperature. Results were calculated from the ratio of acceptor fluorescence signal (A665 nm) and donor fluorescence signal (A620 nm)×104 and expressed as ΔF % values using the following formula: 100×(Ratio Sample−Ratio Negative Control)/Ratio Negative Control. In experiments, all treatments were measured in multiple wells in parallel, and the mean ΔF % values were used for further analysis. Agonist activity values were calculated as percentage of inhibition of forskolin-stimulated cAMP accumulation normalized to the response evoked by a maximally effective concentration of dopamine tested in the same experiment. All calculations were done using Microsoft Excel® (Microsoft, Redmond, WA, USA). The pEC50 values shown in Table 5 (the negative logarithm of the concentration, expressed in mol/liter, of the agonist that produces 50% inhibition of forskolin-stimulated cAMP accumulation) were obtained by fitting 4-parameter sigmoidal curves to the concentration-effect data with the lower asymptote constrained to zero using GraphPad Prism (GraphPad, San Diego, CA, USA). The results indicate that the examples of the present disclosure are potent agonists of the G-protein-coupled signaling pathway of human recombinant D2L receptors. |
| Affinity data for this assay | |
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