CHEMBL195 ATROPINE BDBM86231 NSC_3661 CAS_51-55-8 US9333195, ATROPINE ATR Atropine,(-)
ATROPINE BDBM50014540
ATROPINE BDBM50403547 ATROPEN
BDBM50497835 ENDO-ATROPINE
BDBM200229 AB00694549-11 Atropine cid_174174
Atropine Methonitrate BDBM50487058 Eumydrin Methylatropine Nitrate Ekomine
3-(3-Hydroxy-2-phenyl-propionyloxy)-8,8-dimethyl-8-azonia-bicyclo[3.2.1]octane; bromide(atropine methyl bromide) CHEMBL63452 BDBM50015728
- ChEBML_138367 Contraction of guinea pig ileum by muscarinic AChR activation, which could be inhibited by application of atropine
- ChEBML_141661 Effective dose was measured on NK1 receptors of guinea pig ileum for maximal contraction in the presence of 3*10e-7 M atropine
- ChEMBL_2279670 Displacement of atropine from human muscarinic M3 receptor expressed in CHO cells incubated for 2 hrs by flow cytometric competition binding assay
- ChEMBL_2279662 Binding affinity to saturated human muscarinic M3 expressed in CHO cells assessed as dissociation constant incubated for 2 hrs in presence of atropine by flow cytometry
- ChEMBL_2279663 Binding affinity to saturated human muscarinic M1 expressed in CHO cells assessed as dissociation constant incubated for 2 hrs in presence of atropine by flow cytometry
- ChEMBL_1972547 (CHEMBL4605365) Binding affinity to human muscarinic M2 receptor expressed in CHO-K9 cells at 22 +/- 1 degree C preincubated for 180 mins followed by atropine addition by flow cytometric saturation binding study
- ChEMBL_1972548 (CHEMBL4605366) Binding affinity to human muscarinic M2 receptor expressed in CHO-K9 cells at 22 +/- 1 degree C preincubated for 120 mins followed by atropine addition by flow cytometric saturation binding study
- ChEMBL_1972549 (CHEMBL4605367) Binding affinity to human muscarinic M2 receptor expressed in CHO-K9 cells at 22 +/- 1 degree C preincubated for 110 mins followed by atropine addition by flow cytometric saturation binding study
- ChEMBL_2060793 (CHEMBL4716046) Binding affinity to human muscarinic M2 receptor expressed in CHO-K9 cells assessed as unspecific binding at Kd incubated in dark at 22 degree C for 2 hrs in presence of atropine by FACSCantoII flow cytometric saturation binding study relative to control
- ChEMBL_2060799 (CHEMBL4716052) Binding affinity to human muscarinic M2 receptor expressed in CHO-K9 cells assessed as unspecific binding at Kd incubated in dark at 22 degree C for 2 hrs in presence of atropine by FACSCalibur flow cytometric saturation binding study relative to control
- ChEMBL_2223583 (CHEMBL5136917) Antagonist activity at chick alpha7 nAChR expressed in stage VI oocytes assessed as inhibition of acetylcholine-induced current response pretreated for 30 secs followed by co-application with acetylcholine measured after 2 to 4 days in presence of atropine by voltage-clamp technique
- [3H]-NMS Binding Assay Assays were carried out at 25° C. Membrane preparations from stably transfected chinese hamster ovary-K1 cells (CHO) expressing the genes for the human muscarinic receptors Hm3 were used. For determination of IC, membrane preparations were suspended in DPBS to a final concentration of 89 μg/ml for the Hm3 subtype. The membrane suspension was incubated with the tritiated compound for 60 min. After incubation the membrane fraction was separated by filtration and the bound radioactivity determined. Non specific binding was determined by addition of 10^−4 M atropine. At least six concentrations were assayed in duplicate to generate individual displacement curves.
- Radioligand Binding Assay Radioligand binding studies were carried out with M3 receptor cell homogenates as described (Peralta et al., The EMBO Journal 6, 3923-3929, (1987)). Incubations of test ligands (or standard) with 0.2 nM [3H]4-DAMP were incubated for 120 minutes at 22 C. using human M3 receptor-expressing cell homogenates. Specific ligand binding to the receptors was defined as the difference between the total radioligand binding and the nonspecific binding determined in the presence of an excess of unlabelled ligand (10 atropine). The results were expressed as a percent of control specific binding ((measured specific binding/control specific binding)x100) obtained in the presence of various concentrations of the test compounds.
- [3H]NMS/Carbachol Binding Assay In brief, binding reactions containing ~10 μg of membrane protein per tube were carried out for 2 h (22 °C) in 1 ml of binding buffer containing 25 mM sodium phosphate and 5 mM MgCl2 (pH 7.4). In saturation binding assays, we employed six different [3H]NMS concentrations (200 to 7,000 pM). In competition binding assays, we used a fixed concentration of [3H]NMS (500 pM) in the presence of 10 different concentrations of carbachol, the cold competitor. Nonspecific binding was defined as binding observed in the presence of 10 μM atropine. Binding reactions were terminated by rapid filtration over GF/C Brandel filters, followed by three washes (~4 ml/wash) with ice-cold distilled water. The amount of radioactivity that remained bound to the filters was determined by liquid scintillation spectrometry.
- Binding Assay Cell membrane proteins (Perkin Elmer) wherein human muscarinic M3 receptor was overexpressed, [3H]-methyl scopolamine and test compounds in various concentration were cultured in 0.2 ml of Tris-HCl buffer at 25° C. for 120 minutes. The same was filtered under suction through glass fiber filter (Whatman GF/B), and then the filter was washed 5 times with 1 ml of Tris-HCl buffer. The radioactivity of [3H]-methyl scopolamine adsorbed on the filter was measured by a liquid scintillation counter. Non-specific binding was evaluated under existence of 5 μM of atropine. Affinity of the compound of the present invention to muscarinic M3 receptor was calculated as the dissociation constant (Ki), which can be calculated from concentration (IC50) of test compounds inhibiting 50% of binding of [3H]-methyl scopolamine (i.e. labeled ligand) according to Cheng and Prusoff [Cheng and Prusoff, Biochem. Pharmacol., 22, 3099, 1973]. In following Table, compounds having stronger binding affinity to human muscarinic M3 receptor have lower dissociation constant (Ki).
- Muscarinic Receptor Binding Assay The study of binding to human muscarinic M1, M2, M3, M4 and M5 receptors was performed using commercial membranes (Perkin Elmer) prepared from CHO-K1 cells. Radioligand binding experiments were conducted in 96 polypropylene well plates in a total volume of 200 ul. All reagents were dissolved in assay binding buffer (PBS with calcium and magnesium, SIGMA), except compounds that were dissolved in DMSO 100%. Non-specific binding (NSB) was measured in the presence of 1 uM atropine. [3H]-NMS was used as the radioligand at a concentration of 1 nM for M2, M3 and M5 and 0.3 nM for M1 and M4. [3H]-NMS and antagonists were incubated with membranes that express human muscarinic receptors M1, M2, M3, M4 and M5 at concentrations of 8.1, 10, 4.9, 4.5 and 4.9 ug/well, respectively. After an incubation period of two hours with gentle shaking, 150 ul of the reaction mix were transferred to 96 GF/C filter plates (Millipore), previously treated with wash buffer.
- M3 Binding Assay Human M3 receptor membranes (15 μg/well) from Perkin Elmer are incubated with 0.52 nM Scopolamine Methyl Chloride, [N-methyl-3H] with or without test compounds, or a saturating concentration of Atropine (5 μM) for the determination of non-specific binding. The assay is carried out in 96-well polypropylene plates in a volume of 250 μl. The assay buffer used is 50 mM Tris-HCl, 154 mM NaCl (pH 7.4). The final assay concentration of DMSO is 0.5% (v/v). The plates are sealed and incubated for 2 h at room temperature on an orbital shaker (slow speed). Membranes are harvested onto 96-well unifilter GF/C filter plates pre-treated with 0.5% polyethyleneimine (v/v), using a filter manifold, washed four times with 200 μl of assay buffer. The plates are dried before addition of 50 μl of microscint-0, sealed then read in a Trilux Microbeta scintillation counter. IC50 values are determined from competition curves using a non-linear curve fitting program. Ki values are calculated from IC50 values by the Cheng and Prusoff equation.
- Radioligand Binding Assay Human M3 receptor membranes (15 μg/well) from Perkin Elmer are incubated with 0.52 nM Scopolamine Methyl Chloride, [N-methyl-3H] with or without test compounds, or a saturating concentration of Atropine (5 μM) for the determination of non-specific binding. The assay is carried out in 96-well polypropylene plates in a volume of 250 μl. The assay buffer used is 50 mM Tris-HCl, 154 mM NaCl (pH 7.4). The final assay concentration of DMSO is 0.5% (v/v). The plates are sealed and incubated for 2 h at room temperature on an orbital shaker (slow speed). Membranes are harvested onto 96-well unifilter GF/C filter plates pre-treated with 0.5% polyethyleneimine (v/v), using a filter manifold, washed four times with 2000 of assay buffer. The plates are dried before addition of 50 μl of microscint-0, sealed then read in a Trilux Microbeta scintillation counter. IC50 values are determined from competition curves using a non-linear curve fitting program. Ki values are calculated from IC50 values by the Cheng and Prusoff equation.
- Muscarinic Receptor Binding Assay The study of binding to human muscarinic M1, M2, M3, M4 and M5 receptors was performed using commercial membranes (Perkin Elmer) prepared from CHO-K1 cells. Radioligand binding experiments were conducted in 96 polypropylene well plates in a total volume of 200 μl. All reagents were dissolved in assay binding buffer (PBS with calcium and magnesium, SIGMA), except compounds that were dissolved in DMSO 100%. Non-specific binding (NSB) was measured in the presence of 1 μM atropine. [3H]-NMS was used as the radioligand at a concentration of 1 nM for M2, M3 and M5 and 0.3 nM for M1 and M4. [3H]-NMS and antagonists were incubated with membranes that express human muscarinic receptors M1, M2, M3, M4 and M5 at concentrations of 8.1, 10, 4.9, 4.5 and 4.9 μg/well, respectively. After an incubation period of two hours with gentle shaking, 150 μl of the reaction mix were transferred to 96 GF/C filter plates (Millipore), previously treated with wash buffer (Tris 50 mM; NaCl 100 mM; pH: 7.4), containing 0.05% PEI (Sigma) during one hour. Bound and free [3H]-NMS were separated by rapid vacuum filtration in a manifold from Millipore and washed four times with ice cold wash buffer. After drying 30 min, 30 μl of OPTIPHASE Supermix were added to each well and radioactivity quantified using a Microbeta microplate scintillation counter.
- Receptors Binding Assay The study of binding to human muscarinic M1, M2, M3, M4 and M5 receptors was performed using commercial membranes (Perkin Elmer) prepared from CHO-K1 cells. Radioligand binding experiments were conducted in 96 polypropylene well plates in a total volume of 200 μl. All reagents were dissolved in assay binding buffer (PBS with calcium and magnesium, SIGMA), except compounds that were dissolved in DMSO 100%. Non-specific binding (NSB) was measured in the presence of 1 μM atropine. [3H]-NMS was used as the radioligand at a concentration of 1 nM for M2, M3 and M5 and 0.3 nM for M1 and M4. [3H]-NMS and antagonists were incubated with membranes that express human muscarinic receptors M1, M2, M3, M4 and M5 at concentrations of 8.1, 10, 4.9, 4.5 and 4.9 μg/well, respectively.After an incubation period of two hours with gentle shaking, 150 μl of the reaction mix were transferred to 96 GF/C filter plates (Millipore), previously treated with wash buffer (Tris 50 mM; NaCl 100 mM; pH: 7.4), containing 0.05% PEI (Sigma) during one hour. Bound and free [3H]-NMS were separated by rapid vacuum filtration in a manifold from Millipore and washed four times with ice cold wash buffer. After drying 30 min, 30 μl of OPTIPHASE Supermix were added to each well and radioactivity quantified using a Microbeta microplate scintillation counter.
- Muscarinic 3 Receptor Binding Assay The muscarinic 3 receptor binding assay was adapted from Perkin Elmer. Briefly, assay buffer (60 μL of pH 7.4 phosphate saline buffer) was added to polypropylene round bottom 96-well microtiter plates, followed by CHO cell suspension expressing the human M3 receptor (1 mg suspension/ml; 20 ug membrane suspension per well). 3H-Scopolamine (20 μL of a 7.5 nM solution) was added to each well and plates were shaken at room temperature for 2 h. Atropine (Sigma-Aldrich, St. Louis, Mo.) was used as a positive control. Test compounds and control samples were prepared in DMSO (20 mM) and diluted to give a final concentration of 20 μM. The reaction mixtures were then added to matrix 96-well GFC filtration microtiter plates that had been previously pretreated with 0.5% polyethylimine (100 μL) for 4 h and filtered. The binding reactions were terminated by filtering through the GFC plates and washing & filtering with ice-cold phosphate saline buffer (5×100 μL). Once the filters were dry, microscint scintillation cocktail (100 μL) was added to each well, allowed to sit for 20 min and the plates analyzed using a TopCount scintillation counter. Test compounds which showed >50% reduction in 3H-scopolamine binding at a final concentration of 20 μM were subjected to further serial dilutions and evaluated at various concentrations to determine their IC50 value. Curve fitting of % inhibition versus concentration using Excell software allowed determination of IC50 values for test compounds.