CHEMBL5738698

Opioid Receptor Binding Assay: The measurement of opioid receptor binding affinity was conducted using a radioligand binding assay on the membranes prepared from HEK293 cells (human embryonic kidney cell line) that were heterologously expressed for the recombinant human mu, delta or kappa opioid receptors.The assay buffers used for opioid receptor binding studies were 50 mM Tris.HCl (pH 7.4) for KOR, 50 mM Tris.HCl (pH 7.4) with 5 mM MgCl2 for MOR, and 50 mM Tris.HCl (pH 7.4) with 10 mM MgCl2 plus 1 mM EDTA for DOR. The wash buffer solution contained 50 mM Tris.HCl with pH 7.4.The opioid receptor binding affinity were compared to three known standards: Naltrindole, U-50488 (trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]phenylacetamide, see M. Doi, T. Ishida and M, Inoue; Structure of K-agonist, U-50488 Acta Cryst. (1990). C46, 676-678), and DAMGO (D-Ala2 MePhe4,Gly(ol)5]encephalin, see Allan D. Blake, George Bot, John C. Freeman, and Terry Reisine‡ Differential Opioid Agonist Regulation of the Mouse m Opioid Receptor* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 2, Issue of January 10, pp. 782-790, 1997).The radio ligands were prepared at the final concentration of 0.5 nM for [3H]DAMGO, 0.5 nM for [3H]diprenorphine, and 0.5 nM for [3H] DADLE, which were used as the competing radioligands for mu, kappa and delta receptor respectively.Cell membrane of HEK293 cells transfected with opioid receptors was prepared in the amount of 20 ug of MOR, 6.7 ug of KOR and 6.7 ug of DOR per each well respectively. These membranes containing the receptor of interest were incubated with increasing concentrations of test compound in the presence of a single concentration of radioligand. The fixed concentration of the radioligand was used and serial dilutions of the test compound were prepared.Testing started at 10 uM of testing compound to 4-fold serial dilution for 8-points detection. 1 μl of compounds/high control/low control was transferred in to the 96 well plates according to the plate map, and then 100 μl of membrane stock solution was dispensed into the plate followed by 100 μl of radio ligand solution. The well plated were incubated for 1 hour at room temperature with 300 rpm gentle agitation. Then, soaked the Unifilter-96 GF/C filter plates with 50 μl of 0.3% Poly ethyleneimine per well for at least 0.5 hour at room temperature, and filtered the reaction mixture through the plates using FilterMate™harvester, then wash each plate for four times with cold wash buffer. The filter plates are then dried for 1 hour at 50° C. After drying, the filter was sealed in polyethylene and adds 50 μl of Perkin Elmer Microscint 20 cocktail and the radioactivity counted in a Perkin Elmer MicroBeta2 counter.Specific binding is determined by subtraction of the Bound CPM values in the presence of 50-100× excess of cold ligand. Data is fitted using the saturation analysis non-linear curve fitting routines in Prism®. Calculation of the inhibition was conducted using following equation: % Inhibition=(1−(Assay well−Average_LC)/(Average_HC−Average_LC))*100%Binding data was analyzed using GraphPad Prism 5.0 and IC50 data was generated by non-linear regression from dose response curves. Use the model “log (inhibitor) vs. response—Variable slope” was used to fit the data.