CHEMBL5730966

Radioligand Binding Assay (EP) (In vitro Assay): Radioligand Binding Studies: Saturation experiments. A representative compound of formula (I) having a methyl group was tritiated. Three tritiums were incorporated in place of methyl hydrogens to generate [3H]compound. Binding of this radioligand was preformed in 5 mL borosilicate glass test tubes at room temperature. Binding was initiated by adding membranes to increasing concentrations of [3H]compound in 100 mM NaCl, 20 mM TrisHCl, pH 7.4 buffer containing 0.01% w/v bovine serum albumin (BSA) for 18 h. Non-specific binding was determined in the presence of 1 μM unlabelled compound. After 18 h, the reactants were filtered through GF/C glass fiber filters presoaked in 0.5% w/v polyethylene imine. Filters were washed with 15 mL ice cold 100 mM NaCl, 20 mM TrisHCl, pH7.4 buffer containing 0.25% BSA to separate bound from free ligand. [3H]compound bound to filters was quantified by liquid scintillation counting.Competitive binding experiments: Binding reactions were preformed its 96-well polypropylene plates at room temperature for 18 h. In 360 μL, membranes were incubated with 100 pM [3H]compound and increasing concentrations of Test Compound. Non-specific binding was defined in the presence of 1 μM unlabelled compound. Reactions were transferred and filtered through 96-well glass fiber/C filter plates presoaked with 0.5% polyethylene imine. The filtered reactions were washed 5 times with 200 μL ice cold butter containing 0.25% BSA. Bound radioactivity was determined by liquid scintillation counting.