Radiolabeled
compounds were purchased from PerkinElmer (Waltham, MA, U.S.). Opioid
ligand binding assays were performed by competitive displacement of
0.2 nM [3H]diprenorphine (250 μCi, 1.85 TBq/mmol)
by the peptidomimetic from membrane preparations containing opioid
receptors as described above. The assay mixture, containing membranes
(20 μg protein/tube) in 50 mM Tris-HCl buffer (pH 7.4), [3H]diprenorphine, and various concentrations of test peptidomimetic,
was incubated at room temperature for 1 h to allow binding to reach
equilibrium. The samples were rapidly filtered through Whatman GF/C
filters using a Brandel harvester (Brandel, Gaithersburg, MD, U.S.)
and washed five times with 50 mM Tris-HCl buffer. Bound radioactivity
on dried filters was determined by liquid scintillation counting,
after saturation with EcoLume liquid scintillation cocktail, in a
Wallac 1450 MicroBeta (PerkinElmer, Waltham, MA, U.S.). Nonspecific
binding was determined using 10 μM naloxone. The results presented
are the mean ± standard error (SE\M) from at least three separate
assays performed in duplicate. Ki (nM)
values were calculated using nonlinear regression analysis to fit
a logistic equation to the competition data using GraphPad Prism,
version 6.0c, for Mac OS X (GraphPad Software Inc., La Jolla, CA).
compounds were purchased from PerkinElmer (Waltham, MA, U.S.). Opioid
ligand binding assays were performed by competitive displacement of
0.2 nM [3H]diprenorphine (250 μCi, 1.85 TBq/mmol)
by the peptidomimetic from membrane preparations containing opioid
receptors as described above. The assay mixture, containing membranes
(20 μg protein/tube) in 50 mM Tris-HCl buffer (pH 7.4), [3H]diprenorphine, and various concentrations of test peptidomimetic,
was incubated at room temperature for 1 h to allow binding to reach
equilibrium. The samples were rapidly filtered through Whatman GF/C
filters using a Brandel harvester (Brandel, Gaithersburg, MD, U.S.)
and washed five times with 50 mM Tris-HCl buffer. Bound radioactivity
on dried filters was determined by liquid scintillation counting,
after saturation with EcoLume liquid scintillation cocktail, in a
Wallac 1450 MicroBeta (PerkinElmer, Waltham, MA, U.S.). Nonspecific
binding was determined using 10 μM naloxone. The results presented
are the mean ± standard error (SE\M) from at least three separate
assays performed in duplicate. Ki (nM)
values were calculated using nonlinear regression analysis to fit
a logistic equation to the competition data using GraphPad Prism,
version 6.0c, for Mac OS X (GraphPad Software Inc., La Jolla, CA).