Triluxmicrobeta2 counter

Ten micromolar
concentrations of each compound were incubated in triplicate with
membrane preparations from CHO-K1 cells expressing the human CB2 receptor
(0.5 μg per well) (PerkinElmer, Cat. No. ES-111-M400UA) in an
assay buffer containing 50 mM Tris–HCl, pH = 7.4, 0.2 mM EGTA,
3 mM MgCl₂, 100 mM NaCl, 30 μM GDP and 1 mg/mL BSA)
in the presence of 0.08 nM [³⁵S] guanosine 5′-[γ-thio]triphosphate
([³⁵S]GTPγS) (specific activity: 1250 Ci/mmole, PerkinElmer).
Nonspecific binding was determined with 100 μM of unlabeled
GTPγS. CP-55,940 (100 nM) was used as stimulating ligand. The
final DMSO concentration in the assay was 5%. The reaction mixture
was incubated for 60 min at 30 °C. Next, the samples were deposited
under vacuum with the FilterMate Harvester (PerkinElmer, USA) onto
Unifilter GF/B Plates (PerkinElmer, USA) presoaked with wash buffer
(50 mM Tris–HCl, pH = 7.4). The samples were then rapidly washed
with 2 mL of wash buffer. Filter plates were dried for 30 min at 50
°C and 40 μL of MicroScint PS (PerkinElmer, USA) scintillation
fluid was added to each well. Radioactivity was counted in a Trilux
MicroBeta² counter (PerkinElmer, USA). Data were analyzed
with GraphPad Prism 5.0 software. Results were expressed as percent
of basal [³⁵S]GTPγS binding in the presence of CP-55,940
from three separate experiments. Basal binding was set to 100%.

Ten compound concentrations equally spaced on a log
scale (10^–3 to 10^–9 M) were incubated
in triplicate
with membrane preparations from CHO-K1 cells expressing the human
CB1 receptor (5 μg per well) (Perkin Elmer, cat. no. ES-110-M400UA)
in an assay buffer containing 50 mM Tris-HCl, pH = 7.4, 1 mM EGTA,
3 mM MgCl₂, 100 mM NaCl and 30 μM GDP) in the presence
of 0.08 nM [³⁵S]GTPγS (specific activity: 1250 Ci/mmole,
Perkin Elmer). Non-specific binding was determined with 100 μM
of unlabeled GTPγS. WIN 55,212-2 (3 μM) was used as stimulating
ligand. The final DMSO concentration in the assay was 5%. The reaction
mixture was incubated for 90 min. at 30°C . Next, the samples
were deposited under vacuum with the FilterMate Harvester® (Perkin
Elmer, USA) onto Unifilter® GF/B Plates (Perkin Elmer, USA) presoaked
with wash buffer (50 mM Tris-HCl, pH = 7.4). The samples were then
rapidly washed with 2 ml of wash buffer. Filter plates were dried
for 30 min. at 50 °C and 40 μL of MicroScint PS (Perkin
Elmer, USA) scintillation fluid was added to each well. Radioactivity
was counted in a Trilux MicroBeta² counter (Perkin Elmer,
USA). Data were analyzed with GraphPad Prism 5.0 software. Curves
were fitted with three-parameter non-linear regression model. Inhibitory
potency (IC₅₀) was calculated and expressed as means from
three separate experiments ± 95% confidence intervals (95% CI).