4.3. In Vitro Binding Assays

Reagents and reference compounds used were of analytical grade and obtained from various commercial sources. All cell culture reagents were obtained from Invitrogen (Merelbeke, Belgium). Radioligands (3H-UCB30889, 1184 GBq/mmol; 3H-UCB1418435, 925 GBq/mmol; and 3H-UCB101275-1, 1110–1480 GBq/mmol) were obtained from G.E Healthcare, Amersham, UK (now Perkin Elmer, Zaventem, Belgium) and reference compounds (levetiracetam, UCB108649-1 and UCB101275-1) were custom synthesized and stored according to manufacturer’s recommendations. Test and reference compounds were dissolved in 100% DMSO or H₂O to give 1 or 10 mM stock solution. The final DMSO concentration in assays was 0.1% unless otherwise stated.

Cell lines generated at UCB Biopharma were human embryonic kidney (HEK) 293 cells expressing human SV2A, SV2B or SV2C proteins. Cells were cultured in Dulbecco’s Modified Eagle medium. The culture medium was supplemented with foetal bovine serum (FBS, 10%), 2 mM l-glutamine, 50 to 100 U/mL penicillin, 50 to 100 µg/mL streptomycin, and 200 µg/mL hygromycin B. Cells were grown at 37 °C with 95% air. Confluent cells were detached by 10 min incubation at 37 °C in phosphate buffered saline (PBS) containing 0.02% EDTA. Culture flasks were washed with 15 mL of ice-cold PBS. The cell suspension was centrifuged at 1500× g for 10 min at 4 °C. The pellet was homogenized in 15 mM Tris-HCl buffer (pH 7.5) containing 2 mM MgCl₂, 0.3 mM EDTA, and 1 mM EGTA (buffer A) using a glass/teflon homogenizer. The crude homogenate was subjected to a freeze and thaw cycle in liquid nitrogen and DNAse (1 µL/mL) was then added. The homogenate was further incubated for 10 min at 25 °C before being centrifuged at 40,000× g for 25 min at 4 °C. The pellet was re-suspended in buffer A and washed once under the same conditions. The final crude membrane pellet was re-suspended at a protein concentration of 1–3 mg/mL in 7.5 mM Tris-HCl buffer (pH 7.5 at 25 °C) containing 250 mM sucrose and stored in liquid nitrogen until use.

Membranes were incubated in binding buffer (see Table 3) containing test compound or positive control in the presence of the radioligand. The non-specific binding (NSB) was defined as the residual binding observed in the presence of a high concentration (1000 fold its Ki) of a specific unlabeled reference compound. Membrane-bound and free radioligands were separated by rapid filtration through glass fiber filters (GF/C). Samples and filters were rinsed using at least 6 mL of washing buffer. The entire filtration procedure did not exceed 10 s per sample. The radioactivity trapped on the filters was counted by liquid scintillation in a β-counter. To determine the affinity of a compound for a given target, competition curves were performed with at least 10 concentrations of compound spanning at least 5 log units.

Details of the in vitro binding assay determination. Percentage of inhibition was calculated as follows: % INHIBITION = 100 − [((BI − NSB)/(B0 − NSB)) × 100], where B0 and BI represent the binding observed in the absence and presence of the test compound, respectively (dpm), NSB is the radioligand non-specific binding (dpm). Raw data were analyzed by non-linear regression using XLfit^TM (IDBS, London, Great Britain) according to the following generic equation: B = NSB + [(B0 − NSB)/(1 + (((10^X)/(10^−pIC50))^nH))], where B is the radioligand bound in the presence of the unlabeled compound (dpm), NSB is the radioligand non-specific binding (dpm), B0 is the radioligand bound in the absence of unlabeled compound (dpm), X is the concentration of unlabeled compound (log M), pIC₅₀ is the concentration of unlabeled compound that inhibits the radioligand specific binding by 50% (−log M), and nH is the Hill coefficient.