GoKawiilBinding screen
These experiments were performed by a commercial vendor (Eurofins) under contract with the National Institutes of Health (NIH). Membrane homogenates from stable cell lines or animal tissues expressing each protein were incubated with the respective radioligand in the absence or presence of FNZ or DFNZ (100 nM and 10 μM). In each experiment, the respective reference compound was tested concurrently with the test compound to assess the assay reliability. Nonspecific binding was determined in the presence of a specific agonist or antagonist at the target. Following incubation, the samples were filtered rapidly under vacuum through glass fibre filters presoaked in buffer and rinsed several times with an ice-cold buffer using a 48-sample or 96-sample cell harvester. The filters were counted for radioactivity in a scintillation counter using a scintillation cocktail.
Animals
Sprague Dawley rats (6 to 8 weeks old) were purchased from Charles River. C57BL/6 J mice (8 weeks old) were obtained from Charles River or Jackson Laboratory. Adult TH-Cre mice (B6.Cg-7630403G23RikTg(Th-cre)1Tmd/J) (10 to 12 weeks old) were bred at the National Institute on Drug Abuse (NIDA). Oprm1-knockout mice (B6.129S2-Oprm1tm1Kff/J) were obtained from Jackson Laboratory (strain 007559) (6 to 8 weeks old). PGP/BCRP-knockout mice (FVB.129P2-Abcb1atm1BorAbcb1btm1BorAbcg2tm1Ahs) (6 to 8 weeks old) were obtained from Taconic Biosciences. Animals were single or group housed in an environment with stable temperature (21–23 °C) and humidity (35–55%) and maintained on a 12 h:12 h light:dark reverse cycle with ad libitum access to food and water. Experiments and procedures complied with ethical regulations for animal testing and research, followed the NIH guidelines and were conducted according to the guidelines and approved by the Institutional Animal Care and Use Committees at the National Institute on Drug Abuse, the Chobanian and the Avedisian School of Medicine at Boston University and the University of Barcelona. Animals were randomly assigned to experimental groups and treatment conditions. Sample sizes were estimated based on experience from past work. The experimenters were blinded to the group allocation and treatment when applicable and as noted below.
Competition binding assays
Dissected rat brains (minus cerebellum) were suspended in Tris-HCl 50 mM buffer supplemented with protease inhibitor cocktail (1:1,000) and disrupted with a Polytron homogenizer (Kinematica). Homogenates were centrifuged at 48,000g (50 min, 4 °C) and washed twice in the same conditions to isolate the membrane fraction. Protein was quantified by the bicinchoninic acid method (Pierce). Membrane preparations were incubated with 50 mM Tris-HCl (pH 7.4) containing [3H]DAMGO (~5 nM, 46 Ci mmol−1, NIDA Drug Supply) and increasing concentrations (10 nM to 1 mM) of FNZ, DFNZ or DAMGO. Nonspecific binding was determined in the presence of 100 μM of unlabelled DAMGO. Free and membrane-bound radioligand were separated by rapid filtration of 500-μl aliquots in a 96-well plate harvester (PerkinElmer) and washed with 2 ml of ice-cold Tris-HCl buffer. Microscint-20 scintillation liquid (65 μl per well, PerkinElmer) was added to the filter plates, plates were incubated overnight at room temperature, and radioactivity counts were determined in a MicroBeta2 plate counter with an efficiency of 41%. One-site competition curves from three independent experiments with three replicates per experiment were fitted using GraphPad Prism 10 (GraphPad Software). K i values were calculated using the Cheng–Prusoff equation.
Kinetic binding assays
Association
To each well was added 150 µl membrane preparation from cells expressing human MOR (hMOR) (Revvity, ES-542-M400UA) and 50 µl of naloxone (Tocris) or buffer (50 mM Tris, 10 mM MgCl 2 , 0.1 mM EDTA (pH 7.4)). The plate was pre-incubated for approximately 30 min. [3H]FNZ (~0.3 nM, 43 Ci mmol−1, Novandi Chemistry) or [3H]DFNZ (~0.3 nM, 54 Ci mmol−1, Novandi Chemistry) solution (50 µl in buffer) was then added over fixed time intervals between 0–120 min.
Disassociation
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