M 24 cell harvester

The saturation assays were performed by mixing an appropriate concentration of
¹²⁵I-labelled flavonoid derivatives
(0.15–8.75 kBq, 6–350 nM) and
rMoPrP aggregates (100 nM) in NaCl/HEPES buffer (50 mM
HEPES/KOH, 300 mM NaCl, pH 7.5) containing 20% (v/v) dimethyl
sulfoxide (DMSO). After incubation for 2 h at room temperature, the
mixture was then filtered through Whatman GF/B filters using a Brandel M-24 cell
harvester. Each assay tube before filtration and the filters containing the
bound ¹²⁵I ligand were measured by an automatic gamma counter and
the bound/free ratio of [¹²⁵I]ligand was calculated. The
dissociation constant (K_d) and binding capacity
(B_max) of compounds were estimated by Scatchard analysis
using PRISM4 (GraphPad Software Inc., CA, USA). For competitive binding assays,
the mixture contained [¹²⁵I]SC-NMe₂(0.02 nM), test compound
(8.0 pM–12.5 μM), and rMoPrP
aggregates (100 nM) in NaCl/HEPES buffer (pH 7.5) containing 20%
(v/v) DMSO. After incubation for 2 h at room temperature, the
mixture was filtered and the filters were measured using the gamma counter.
Nonspecific binding was defined in the presence of 10 μM
for nonradioactive SC-NMe₂. Values for the half maximal inhibitory
concentration (IC₅₀) were determined from displacement curves of
three independent experiments using PRISM4, and those for the inhibition
constant (K_i) were calculated using the
Cheng–Prusoff equation.

A solid form of Aβ(1–40) was purchased from the Peptide Institute (Osaka, Japan). Aggregation was carried out by gently dissolving the peptide (0.50 mg/mL) in phosphate-buffered saline (PBS) (pH 7.4). The solution was incubated at 37 °C for 42 h with gentle and constant shaking. A mixture containing 50 μL Aβ(1–40) aggregates (final conc., 1.25 μg/mL), 50 μL ^99mTc-Ham complex (final conc., 8.3 kBq/mL), 50 μL PIB (final conc., 64 pM–125 μM in 30% EtOH), and 850 μL of 30% EtOH was incubated at room temperature for 3 h. The mixture was filtered through Whatman GF/B filters (Whatman, Kent, U.K.) using a Brandel M-24 cell harvester (Brandel, Maryland, USA), and the radioactivity of the filters containing the bound ^99mTc-Ham complex was measured using a γ counter (Wallac 1470 Wizard; PerkinElmer, Massachusetts, USA). Values for the half-maximal inhibitory concentration (IC₅₀) were determined from displacement curves using GraphPad Prism 5.0 (GraphPad Software, Inc., California, USA).

The binding affinity of peptidomimetics 1–9 and reference compoundsTyr-d-Ala-Gly-Phe-NH₂ for MOR and DOR was determined in competitive radioligand binding assays according to the method previously described [40 (link),41 (link),42 (link)]. The specific radioligands were [³H]DAMGO (DAMGO (a specific MOR ligand) and [³H][Ile^5,6]DELT II]) (a specific DOR ligand). Membrane fractions of rat brain homogenate were incubated at 25 °C for 60 min in the presence of radioligands (0.5 nM) specific for each receptor and the increasing concentrations of the tested compounds. For measuring non-specific binding, 10 μM naloxone was used as the competitor for opioid receptors. The reactions were carried out in assay buffer containing Tris-HCl (pH 7.4) with an addition of bovine serum albumin (BSA) and protease inhibitors (bacitracin, bestatin, captopril). After the incubation, the binding reactions were terminated by rapid filtration with M-24 Cell Harvester (Brandel/USA) through GF/B Whatman glass fiber strips (pre-soaked with 0.5% PEI in order to minimize non-specific binding). Radioactivity retained on the filters was measured in MicroBeta LS, Trilux scintillation counter (PerkinElmer, USA). The experiments were repeated at least three times in duplicate. The IC₅₀ value for each compound was determined using GraphPad Prism [43 ].

Membrane preparations
from Chem-1 cells expressing the human CB1 receptors (ChemiSCREEN
CB1 Cannabinoid Receptor Membrane Preparation. Merck, USA) were incubated
in duplicate with 1 nM [³H]CP-55,940 (specific activity:
108.5 Ci/mmole, PerkinElmer, USA) in a 50 mM Tris–HCl, pH =
7.4 buffer supplemented with 1 mM CaCl₂, 5 mM MgCl₂, 0.2% 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, 500 mM NaCl, and
5 mM MgCl₂) to separate the bound radioligand from the
free one. The rest of the procedure was the same as for CB2.