Radioligand Binding Assays

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 tissue 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 (SEM) from at least three separate assays performed in duplicate. K_i (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).