The compounds selected
for the in vitro binding assay were purchased via MolPort, SIA, Riga,
Latvia (Supporting Information S2 ). Membrane
preparations from CHO-K1 cells expressing the human CB2 (ChemiSCREEN
Membrane Preparation Recombinant Human CB2 Cannabinoid Receptor. Merck,
USA) were incubated in duplicate with 0.8 nM [3H]CP-55,940
(specific activity: 101 Ci/mmole, PerkinElmer, USA) in a 50 mM Tris–HCl,
pH = 7.4 buffer supplemented with 2.5 mM EDTA, 5 mM MgCl2, 0.5 mg/mL BSA and increasing concentrations of the compounds tested.
Compounds were dissolved in 50% DMSO and added to the reaction mixture
at 10 concentrations equally spaced on a log scale (10–10–10–4.5 M). The final DMSO concentration
was 5%. Nonspecific binding was determined with 10 μM WIN 55,212-2.
The reaction mixture (500 μL) was incubated for 1.5 h at 30
°C. Before harvesting, Brandel Whatman GF/B Filter Paper was
presoaked with 0.5% polyethylenimine buffer for 30 min and then washed
with 2 mL of 50 mM Tris–HCl buffer (pH = 7.4) containing 0.5%
BSA to minimize nonspecific binding. The reaction was terminated by
depositing the samples onto the filter paper with the Brandel M–24
Cell Harvester. Samples were then rapidly washed three times with
2 mL of wash buffer (50 mM Tris–HCl pH 7.4, 2.5 mM EDTA, 5
mM MgCl2, 0.5 mg/mL BSA) to separate the bound radioligand
from free. Filters were then air-dried for 1.5 h at 60 °C. After
drying, filter discs were placed on a flexible 24-well plate and 500
μL of EcoScint-20 scintillant (PerkinElmer, USA) was added to
each well. Plates were counted (2 min per well) in a Trilux MicroBeta
counter (PerkinElmer, USA). Data were analyzed with GraphPad Prism
5.0 software. Curves were fitted with a one-site nonlinear regression
model, and inhibitory constants (pKi ±
SEM and Ki, 95% CI) were calculated from
the Cheng–Prusoff equation.
for the in vitro binding assay were purchased via MolPort, SIA, Riga,
Latvia (
preparations from CHO-K1 cells expressing the human CB2 (ChemiSCREEN
Membrane Preparation Recombinant Human CB2 Cannabinoid Receptor. Merck,
USA) were incubated in duplicate with 0.8 nM [3H]CP-55,940
(specific activity: 101 Ci/mmole, PerkinElmer, USA) in a 50 mM Tris–HCl,
pH = 7.4 buffer supplemented with 2.5 mM EDTA, 5 mM MgCl2, 0.5 mg/mL BSA and increasing concentrations of the compounds tested.
Compounds were dissolved in 50% DMSO and added to the reaction mixture
at 10 concentrations equally spaced on a log scale (10–10–10–4.5 M). The final DMSO concentration
was 5%. Nonspecific binding was determined with 10 μM WIN 55,212-2.
The reaction mixture (500 μL) was incubated for 1.5 h at 30
°C. Before harvesting, Brandel Whatman GF/B Filter Paper was
presoaked with 0.5% polyethylenimine buffer for 30 min and then washed
with 2 mL of 50 mM Tris–HCl buffer (pH = 7.4) containing 0.5%
BSA to minimize nonspecific binding. The reaction was terminated by
depositing the samples onto the filter paper with the Brandel M–24
Cell Harvester. Samples were then rapidly washed three times with
2 mL of wash buffer (50 mM Tris–HCl pH 7.4, 2.5 mM EDTA, 5
mM MgCl2, 0.5 mg/mL BSA) to separate the bound radioligand
from free. Filters were then air-dried for 1.5 h at 60 °C. After
drying, filter discs were placed on a flexible 24-well plate and 500
μL of EcoScint-20 scintillant (PerkinElmer, USA) was added to
each well. Plates were counted (2 min per well) in a Trilux MicroBeta
counter (PerkinElmer, USA). Data were analyzed with GraphPad Prism
5.0 software. Curves were fitted with a one-site nonlinear regression
model, and inhibitory constants (pKi ±
SEM and Ki, 95% CI) were calculated from
the Cheng–Prusoff equation.
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