To establish an electrochemiluminescent (ECL) binding assay for NMUR2 receptor, the peptide Neuromedin U (NMU-23) is labeled at the N terminus with Ru(bpy) 2+ 3 and allowed to bind to the human NMUR2 receptor in the cell membranes immobilized on the electrode on the bottom of each assay plate. Upon application of an electric current, the receptor-bound Ru(bpy) 2 + 3 -ligand undergoes an oxidation-reduction cycle in the presence of a coreactant tripropylamine and emits light. Signal is only generated when the Ru(bpy) 2+ 3 label is in proximity to the electrode, thus discriminating the bound label from the unbound and enabling a no wash, homogenous assay format. The ECL-based NMUR2 binding assay is used to screen our corporate compound collection . Approximately 670,000 compounds with diverse structures are tested at 10 μM for their ability to displace the binding of 0.5 nM Ru(bpy) 2+ 3 -NMU-23 to human NMUR2 receptors. From binding experiments, the K i values for NMU-23 is determined to be 4.7 nM.
