CHEMBL5736054

Ligand Binding Assay (LBA): hNaV1.7 binding affinities were determined with a filtration binding assay using purified membranes from HEK293 cells stably expressing hNaV1.7. HEK293 cells from a 10-stack cell culture flask (approximately 1010 cells) were dissociated, frozen, and stored at −80° C. To prepare membranes, the frozen cell pellet was thawed and suspended in 6 ml hypotonic lysis buffer (50 mM HEPES, 0.1% mammalian protease inhibitor cocktail). 1 ml of resuspended cells was added to an additional 6 ml of lysis buffer and homogenized with 30 strokes of a tight pestle in a glass homogenizer. Homogenate was centrifuged at 1000×g for 10 minutes at 4° C. and the resulting supernatant was further centrifuged at 38,500×g for 60 minutes at 4° C. The resulting pellet was resuspended in binding buffer (50 mM HEPES, 130 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 5 mM glucose, pH 7.4) and needle homogenized with a 25 gauge needle. Protein concentration was determined with a BCA protein assay. Purified membranes were aliquoted, flash frozen in an ethyl alcohol dry ice bath, and stored at −80° C. To measure displacement of a radiolabeled ligand, 50 μg of purified hNaV1.7 HEK cell membranes were incubated with test compounds (eight concentrations, in duplicate) and 0.5 nM [3H] labeled radioligand in a 96 well plate for 24 hours at room temperature on a shaker. The total binding reaction volume was 250 μl, consisting of 200 μl purified hNaV1.7 HEK cell membranes, 25 μl test compound, and 25 μl radioligand. Non-specific binding was defined by 20 μM of a reference hNaV1.7 inhibitor. Binding reactions were terminated by filtration through GF/B filters presoaked in 0.5% polyethyleneamine. Filters were washed 5 times with 2 ml each of 4° C. wash buffer (50 mM Tris-HCl, pH 7.4 at 4° C.). Bound radioactivity captured on the filters was counted on a liquid scintillation counter. Specific binding, expressed as % inhibition, was fit with Graphpad Prism software to determine binding IC50 values.