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BindingDB_Patents: Radioligand Binding Assay. A bivalent ligand 1 (FIG. 14) that combines the pharmacophores of naltrexone (a MOR antagonist) and maraviroc (a CCR5 antagonist) into one molecule was designed and synthesized. Herein is reported the characterization of this novel molecular probe in for its binding affinity, Ca2- flux functional activity, and HIV-1 inhibition potency. Bivalent ligand 1 was first characterized in hMOR-expressed CHO cells in the competitive radioligand binding assay as described previously.
Assay data:3 Active, 1 Activity ≤ 1 nM, 3 Activity ≤ 1 µM, 3 Tested
SummaryCompounds, ActiveCompounds, activity ≤ 1 µMRelated BioAssays by Target
BindingDB_Patents: Flux Assay. As Ca2+ flux is associated with the activation of the MOR, the functional activity of bivalent ligand 1, monovalent ligand 2, and naltrexone was then evaluated in a Ca2+ mobilization assay in hMORCHO cells transfected with chimeric Ca2+ following a published protocol.23 No agonism was observed for any of the tested compounds (data not shown). Thus, they were further assessed for their antagonist properties as the ability to inhibit DAMGO (a MOR agonist) induced Ca2+ flux.
Assay data:3 Active, 3 Activity ≤ 1 µM, 3 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. d-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 microg membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 microl binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 microM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25 degrees C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 microl ice-cold binding buffer. Filter plates were subsequently dried at 50 degrees C. fo
Assay data:10 Active, 6 Activity ≤ 1 µM, 12 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose-displacement binding assays for -opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and plate
Assay data:20 Active, 18 Activity ≤ 1 µM, 20 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Membranes from recombinant HEK-293 cells expressing the human kippa opioid receptor (cloned in house) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Membranes were collected by centrifugation at 30,000xg for 15 min at 4 C. and pellets were resuspended in hypotonic buffer to a final concentration of 1-3 mg/mL. Protein concentrations were determined using the BioRad protein assay reagent with bovine serum albumen as standard. Aliquots of kippa receptor membranes were stored at -80 C.Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 ug membrane protein (recombinant K opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 ul binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4).
Assay data:24 Active, 12 Activity ≤ 1 nM, 23 Activity ≤ 1 µM, 24 Tested
BindingDB_Patents: Radioligand Binding Assay. Radioligand dose displacement assays used 0.4-0.8 nM [3H]-U69,593 (NEN; 40 Ci/mmole) with 10-20 microg membrane protein (recombinant kappa opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 microL binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 microM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 h at a temperature of about 25 degrees C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 200 microL ice-cold binding buffer. Filter plates were subsequently dried at 50 degrees C. for 1-2 hrs. Fifty microL/well scintillation cocktail (MicroScint20, Packard) was added and plates were cou
Assay data:34 Active, 32 Activity ≤ 1 µM, 34 Tested
BindingDB_Patents: Radioligand Binding Assay. Radioligand binding assays were conducted using freshly thawed membranes expressing human mu-receptors (Perkin Elmer, Shelton, Conn.). Radioligand dose-displacement binding assays for human mu-opioid receptors used 0.2 nM[3H]-diprenorphine (NEN, Boston, Mass.), with 5-20 mg membrane protein/well in a final volume of 500 uL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 1-2 hrs at about 25 C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard, Meriden, Conn.) presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by performing three filtration washes with 500 uL of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-
Assay data:27 Active, 5 Activity ≤ 1 µM, 31 Tested
BindingDB_Patents: Radioligand Binding Assay. Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (NEN; 33.0 Ci/mmole) with 10-20 microg membrane protein (recombinant delta opioid receptor expressed in CHO-K1 cells; Perkin Elmer) in a final volume of 500 microL binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 microM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 h at a temperature of about 25 degrees C. Binding reactions were determined by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by five filtration washes with 500 microL ice-cold binding buffer. Filter plates were subsequently dried at 50 degrees C. for 1-2 hrs. Fifty microL/well scintillation cocktail (MicroScint20, Packard) was added and plates we
Assay data:12 Active, 30 Tested
SummaryCompounds, ActiveRelated BioAssays by Target
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose-displacement binding assays for mu-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and pla
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose displacement assays used 0.4 nM [3]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 ug membrane protein (recombinant kippa opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 ul binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 uM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hr at a temperature of about 25 C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 200 ul ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 1-2 hours. Fifty ul/well scintillation cocktail (Perkin Elmer, S
Assay data:5 Active, 1 Activity ≤ 1 µM, 5 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 ug membrane protein (recombinant kippa opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 ul binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 uM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hour at a temperature of about 25 C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 200 ul ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 1-2 hours. Fifty ul/well scintillation cocktail (Perkin Elmer
Assay data:9 Active, 5 Activity ≤ 1 nM, 9 Activity ≤ 1 µM, 12 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Delta-Opioid Receptor Binding Assay Procedures: delta-Opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hour at a temperature of about 25 C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequent
Assay data:2 Active, 2 Activity ≤ 1 µM, 2 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. mu-Opioid Receptor Binding Assay Procedures: Radioligand dose-displacement binding assays for mu-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hours at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer,
Assay data:9 Active, 1 Activity ≤ 1 nM, 6 Activity ≤ 1 µM, 10 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. d-Opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.2 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 microg membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 microl binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 microM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hour at a temperature of about 25 degrees C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 microl ice-cold binding buffer. Filter plates were subsequently dried at 50 degrees C.
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose-displacement binding assays for mu-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hours at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/well), and
Assay data:15 Active, 5 Activity ≤ 1 nM, 12 Activity ≤ 1 µM, 16 Tested
BindingDB_Patents: Radioligand Dose-Displacement Binding Assay. Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 microg membrane protein (recombinant inverted question mark opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 microl binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 microM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hour at a temperature of about 25 degrees C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 200 microl ice-cold binding buffer. Filter plates were subsequently dried at 50 degrees C. for 1-2 hours
Assay data:25 Active, 8 Activity ≤ 1 nM, 22 Activity ≤ 1 µM, 25 Tested
BindingDB_Patents: Binding Assay. Kippa-Opioid Receptor Binding Assay Procedures: Membranes from recombinant HEK-293 cells expressing the human kippa opioid receptor (kippa) (cloned in house) were prepared by lysing cells in ice cold hypotonic buffer (2.5 mM MgCl2, 50 mM HEPES, pH 7.4) (10 mL/10 cm dish) followed by homogenization with a tissue grinder/Teflon pestle. Membranes were collected by centrifugation at 30,000xg for 15 mM at 4 C. and pellets were resuspended in hypotonic buffer to a final concentration of 1-3 mg/mL. Protein concentrations were determined using the BioRad protein assay reagent with bovine serum albumen as standard. Aliquots of kippa receptor membranes were stored at -80 C.Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 ug membrane protein (recombinant lc opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 ul binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4).
Assay data:38 Active, 31 Activity ≤ 1 nM, 38 Activity ≤ 1 µM, 38 Tested
BindingDB_Patents: Binding Assay. Delta-Opioid Receptor Binding Assay Procedures: delta-opioid Receptor Binding Assay Procedures were conducted as follows. Radioligand dose-displacement assays used 0.3 nM [3H]-Naltrindole (Perkin Elmer, Shelton, Conn.; 33.0 Ci/mmole) with 5 ug membrane protein (Perkin Elmer, Shelton, Conn.) in a final volume of 500 ul binding buffer (5 mM MgCl2, 5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 25 uM unlabeled naloxone. All reactions were performed in 96-deep well polypropylene plates for 1 hr at a temperature of about 25 C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 500 ul ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 1-2 hours.
Assay data:6 Active, 6 Activity ≤ 1 µM, 6 Tested
BindingDB_Patents: Binding Assay. u-Opioid Receptor Binding Assay Procedures: Radioligand dose-displacement binding assays for u-opioid receptors used 0.3 nM [3H]-diprenorphine (Perkin Elmer, Shelton, Conn.), with 5 mg membrane protein/well in a final volume of 500 ul binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 2 hr at room temperature. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.), presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by performing three filtration washes with 500 ul of ice-cold binding buffer. Filter plates were subsequently dried at 50 C. for 2-3 hours. BetaScint scintillation cocktail (Perkin Elmer, Shelton, Conn.) was added (50 ul/w
Assay data:9 Active, 3 Activity ≤ 1 nM, 9 Activity ≤ 1 µM, 9 Tested
BindingDB_Patents: Competitive Displacement Assay. Receptor Binding (in vitro Assay) The Ki (binding affinity) for mu-, delta-, and kippa-receptors was determined with a previously described method using a competitive displacement assay (Neumeyer, 2003). Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25 C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples th
Assay data:10 Active, 1 Activity ≤ 1 nM, 11 Activity ≤ 1 µM, 11 Tested
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