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| | In Vitro Binding Assay (IC50) and In Vitro Agonism/Antagonism Measurements (EC50/IC50) |
| Description: | Compounds were tested for their inhibitory effects on ligand binding to the human ORL1 receptor. Bound and free radioligands are separated by filtration using a unifilter plate GF/C-96. The remaining radioactivity on the filter was counted using a TopCount-HTS (PerkinElmer). Specific binding of [125I][Tyr14]nociceptin is defined as the difference between total binding and nonspecific binding in the presence of 1 uM nociceptin. J-113397 was used as internal control across all assay plates for internal validation. IC50 values are calculated using GraphPad Prism. Agonist/antagonist activities were measured by the [35S]GTPgamma-S binding to proteins using membrane fractions of CHO cells expressing ORL1. Nonspecific binding is determined in the presence of 10 uM unlabeled GTPgammaS. Membrane-bound radioactivity is measured using a TopCount-HTS (PerkinElmer). Specific binding of [35S]GTPgammaS is determined as a difference between the control (in the presence of appropriate concentration of nociceptin), and the baseline (in the presence of DMSO instead of compound). EC50/IC50 values are calculated from % of specific binding in the presence of various concentration of the compound using GraphPad Prism 3.03. |
| Affinity data for this assay | |
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