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BioAssay: AID 652201

Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu1 selectivity Assay

Selectivity of mGlu5 SAMs was assessed by investigating the ability of 10uM to modulate orthosteric agonist activity at rat mGlu1 (1, 2, 3). ..more
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 Tested Compounds
 Tested Compounds
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Inactive(1)
 
 
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 Tested Substances
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 Related BioAssays
 Related BioAssays
AID: 652201
Data Source: Vanderbilt Specialized Chemistry Center (Metabotropic Glutamate Receptor 5 (mGlu5) Allosteric Modulat..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-03-27
Hold-until Date: 2014-03-27
Modify Date: 2014-03-27

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
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AIDNameTypeProbeComment
588721Optimization of novel mGluR5 positive allosteric modulators (PAM)sSummary depositor-specified cross reference
652263Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: BioAssay Summary, ML 353Summary depositor-specified cross reference
588710mGluR5_3H_methoxyPEPy_BindingConfirmatory2 same project related to Summary assay
588715Optimization of novel mGluR5 positive allosteric modulators with different mechanisms of action and modes of efficacyConfirmatory same project related to Summary assay
588728rmGluR3_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588730hmGluR6_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588731rmGluR7_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588732rmGluR8_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588733rmGluR2_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588734rmGluR4_Thallium_Fold_Shift (GIRK)Other same project related to Summary assay
588735rmGluR5_Calcium_Fold_ShiftOther same project related to Summary assay
588736rmGluR1_Calcium_Fold_ShiftOther same project related to Summary assay
588737hmGluR5_Calcium_Fold_ShiftOther same project related to Summary assay
588753ML254 and ML273 Competition in Radioligand Binding assays (Riserca)Other same project related to Summary assay
651835Identification of a glycine sulfonamide based non-MPEP site positive allosteric potentiator (PAM) of mGlu5 (calcium_assay)Confirmatory16 same project related to Summary assay
651852Identification of a glycine sulfonamide based non-MPEP site positive allosteric potentiator (PAM) of mGlu5 (binding_assay)Confirmatory same project related to Summary assay
652185Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu2 selectivity AssayConfirmatory same project related to Summary assay
652186Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu8 selectivity AssayConfirmatory same project related to Summary assay
652191Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: 3pt binding AssayConfirmatory1 same project related to Summary assay
652196Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: full Ki determinationConfirmatory1 same project related to Summary assay
652198Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: Fold-shift AssayConfirmatory1 same project related to Summary assay
652202Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu3 selectivity AssayConfirmatory same project related to Summary assay
652203Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu7 selectivity AssayConfirmatory same project related to Summary assay
652204Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu4 selectivity AssayConfirmatory same project related to Summary assay
652205Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: human mGlu6 selectivity AssayConfirmatory same project related to Summary assay
686927ML353 Eurofin Panel Assay for mGlu5 SAM Inhibitor (Probe Compound)Other same project related to Summary assay
652185Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu2 selectivity AssayConfirmatory same project related to Summary assay
652186Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu8 selectivity AssayConfirmatory same project related to Summary assay
652198Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: Fold-shift AssayConfirmatory1 same project related to Summary assay
652202Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu3 selectivity AssayConfirmatory same project related to Summary assay
652203Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu7 selectivity AssayConfirmatory same project related to Summary assay
652204Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu4 selectivity AssayConfirmatory same project related to Summary assay
652205Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: human mGlu6 selectivity AssayConfirmatory same project related to Summary assay
686927ML353 Eurofin Panel Assay for mGlu5 SAM Inhibitor (Probe Compound)Other same project related to Summary assay
Description:
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University
Selectivity of mGlu5 SAMs was assessed by investigating the ability of 10uM to modulate orthosteric agonist activity at rat mGlu1 (1, 2, 3).
1. Noetzel, M.J., Rook, J.M., Vinson, P.N., Cho, H., Days, E., Zhou, Y., Rodriguez, A.L., Lavreysen, H., Stauffer, S.R., Niswender, C.M., Xiang, Z., Daniels, J.S., Lindsley, C.W., Weaver, C.D. and Conn, P.J., 2011. Functional Impact of Allosteric Agonist Activity of Selective Positive Allosteric Modulators of mGlu5 in Regulating CNS Function. Molecular Pharmacology, in press.
2. Hammond, A. S., Rodriguez, A. L., Townsend, S. D., Niswender, C. M., Gregory, K. J., Lindsley, C. W., Conn, P. J. (2010) Discovery of a Novel Chemical Class of mGlu(5) Allosteric Ligands with Distinct Modes of Pharmacology ACS Chem. Neurosci. 10:702-716
3. Mueller, R., Dawson, E. S., Meiler, J., Rodriguez, A. L., Chauder, B. A., Bates, B. S., Felts, A. S., Lamb, J. P., Menon, U. N.Jadhav, S. B., Kane, A. S., Jones, C. K., Gregory, K. J., Niswender, C. M., Conn, P. J., Olsen, C. M., Winder, D. G., Emmitte, K. A., Lindsley, C. W. (2012)Discovery of 2-(2-benzoxazoyl amino)-4-aryl-5-cyanopyrimidine as negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu(5)): from an artificial neural network virtual screen to an in vivo tool compound. ChemMedChem 7:406-414.
Protocol
rmGlu1/TRex293 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% tetracycline-free fetal bovine serum (FBS), 20 mM HEPES, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, 100ug/mL hygromycin and 5ug/mL blasticidin S.
Experiment Preparation.
rmGlu1/TRex293 cells were plated the day before the experiment at a density of 40,000 cells/well in Poly-D-Lysine coated 96-well Microplates (black walled/clear bottom, Costar) in assay medium (DMEM, 20 mM HEPES, 10% dialyzed FBS, 1 mM sodium pyruvate) and treated with 50ng of tetracyline to induce expression of mGlu1. On the day of the experiment, the media was removed and the cells washed with assay buffer (HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) using an ELX405 cell plate washer. A Ca2+-sensitive fluorescent dye (Fluo4-AM, Invitrogen Corp) was added at a final concentration of 1 uM. Note: the presence of probenecid, an anion exchange inhibitor, minimizes the dye being pumped out of the intracellular environment allowing it to be available to bind intracellular Ca+2. The cells were allowed to take up the dye during an incubation period of 60 min at 37 degrees C/5% CO2. The cells were then washed with assay buffer and, after an incubation period of 3 minutes at room temperature, the assay was started.
A Flexstation2 or Flexstation3 (Molecular Devices) was utilized to evaluate changes in fluorescence intensity of Fluo4. Test compounds were provided as 10 mM stocks in DMSO. Final concentration of DMSO in the assay was 1%.
The concentration of glutamate ranged from 10nM to 1mM (final concentrations) in a 9-point curve and were made 10X in duplicate. Two additions were made within the Flexstation. First, compound alone (made at 3X; 10uM final) was added to each well at 20 seconds to detect any agonist activity. Next, different concentrations of glutamate was added at 80 seconds to detect any modulation of the glutamate response by the test compound.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Average_%_Veh_MaxCalculated Average for Avg. Max vehicle concentrationFloat%
2SD_Average_%_Veh_MaxCalculated Std. Deviation for the Avg. Max vehicle concentrationFloat%
3SEM_Average_%_Veh_MaxCalculated S.E.M. for the Avg. Max Vehicle ConcentrationFloat%
4Average_%_Veh_MinCalculated Average for the Avg. Min Vehicle ConcentrationFloat%
5SD_Average_%_Veh_MinCalculated Stand. Dev for Avg. Min Vehicle ConcentrationFloat%
6SEM_Average_%_Veh_MinCalculated S.E.M for the Avg. Min Vehicle ConcentrationFloat%
7EC50_Average_VehCalculated Average for the Vehicle glu EC50FloatμM
8SD_EC50_VehCalculated Stand. Dev for Vehicle glu EC50FloatμM
9SEM_EC50_VehCalculated S.E.M for the Vehicle glu EC50FloatμM
10Cmpd Conc1 Rep1 (0.01μM**)Value for compound + glu 0.01uM replicate 1Float%
11Cmpd Conc2 Rep1 (0.03μM**)Value for compound + glu 0.03uM replicate 1Float%
12Cmpd Conc3 Rep1 (0.1μM**)Value for compound +glu 0.1 uM replicate 1Float%
13Cmpd Conc4 Rep1 (0.2μM**)Value for compound + glu 0.2uM replicate 1Float%
14Cmpd Conc5 Rep1 (0.3μM**)Value for compound + glu 0.3 uM replicate 1Float%
15Cmpd Conc6 Rep1 (0.1μM**)Value for compound + glu 0.1 uM replicate 1Float%
16Cmpd Conc7 Rep1 (1μM**)Value for compound +glu 1uM replicate 1Float%
17Cmpd Conc8 Rep1 (10μM**)Value for compound +glu 10 uM replicate 1Float%
18Cmpd Conc9 Rep1 (100μM**)Value for compound +glu 100 uM replicate 1Float%
19EC50_uM_Rep1Calculated EC50 for replicate 1FloatμM
20Fold_Shift_Rep1Calculated Fold Shift for Replicate 1.Float
21Cmpd Conc1 Rep2 (0.01μM**)Value for compound + glu 0.01uM replicate 2Float%
22Cmpd Conc2 Rep2 (0.03μM**)Value for compound + glu 0.03uM replicate 2Float%
23Cmpd Conc3 Rep2 (0.1μM**)Value for compound +glu 0.1 uM replicate 2Float%
24Cmpd Conc4 Rep2 (0.2μM**)Value for compound + glu 0.2uM replicate 2Float%
25Cmpd Conc5 Rep2 (0.3μM**)Value for compound + glu 0.3 uM replicate 2Float%
26Cmpd Conc6 Rep2 (1μM**)Value for compound + glu 1 uM replicate 2Float%
27Cmpd Conc7 Rep2 (3μM**)Value for compound +glu 3uM replicate 2Float%
28Cmpd Conc8 Rep2 (10μM**)Value for compound +glu 10 uM replicate 2Float%
29Cmpd Conc9 Rep2 (100μM**)Value for compound +glu 100 uM replicate 2Float%
30EC50_uM_Rep2Calculated EC50 for replicate 2FloatμM
31Fold_Shift_Rep2Calculated Fold Shift for Replicate 2Float
32Cmpd Conc1 Rep3 (0.01μM**)Value for compound + glu 0.01uM replicate 3Float%
33Cmpd Conc2 Rep3 (0.03μM**)Value for compound + glu 0.03uM replicate 3Float%
34Cmpd Conc3 Rep3 (0.1μM**)Value for compound +glu 0.1 uM replicate 3Float%
35Cmpd Conc4 Rep3 (0.2μM**)Value for compound + glu 0.2uM replicate 3Float%
36Cmpd Conc5 Rep3 (0.3μM**)Value for compound + glu 0.3 uM replicate 3Float%
37Cmpd Conc6 Rep3 (1μM**)Value for compound + glu 1 uM replicate 3Float%
38Cmpd Conc7 Rep3 (3μM**)Value for compound +glu 3uM replicate 2Float%
39Cmpd Conc8 Rep3 (10μM**)Value for compound +glu 10 uM replicate 3Float%
40Cmpd Conc9 Rep3 (100μM**)Value for compound +glu 100 uM replicate 3Float%
41EC50_uM_Rep3Calculated EC50 for replicate 3FloatμM
42Fold_Shift_Rep3Calculated Fold Shift for replicate 3Float
43Cmpd Conc1 Rep4 (0.01μM**)Value for compound + glu 0.01uM replicate 4Float%
44Cmpd Conc2 Rep4 (0.03μM**)Value for compound + glu 0.03uM replicate 4Float%
45Cmpd Conc3 Rep4 (0.1μM**)Value for compound +glu 0.1 uM replicate 4Float%
46Cmpd Conc4 Rep4 (0.2μM**)Value for compound + glu 0.2uM replicate 4Float%
47Cmpd Conc5 Rep4 (0.3μM**)Value for compound + glu 0.3 uM replicate 4Float%
48Cmpd Conc6 Rep4 (1μM**)Value for compound + glu 1 uM replicate 4Float%
49Cmpd Conc7 Rep4 (3μM**)Value for compound +glu 3uM replicate 4Float%
50Cmpd Conc8 Rep4 (10μM**)Value for compound +glu 10 uM replicate 4Float%
51Cmpd Conc9 Rep4 (100μM**)Value for compound +glu 100 uM replicate 4Float%
52EC50_uM_Rep4Calculated EC50 for replicate 4FloatμM
53Fold_Shift_Rep4Calculated Fold Shift for replicate 4Float
54Average_EC50_uM*Calculated Avg EC50 for replicatesFloatμM
55SD_EC50_uMCalculated Stand. Dev of EC50 for replicatesFloatμM
56SEM_EC50_uMCalculated SEM for EC50 for replicatesFloatμM
57Average_Fold_ShiftCalculated Average Fold Shift for replicatesFloat
58SD_Fold_ShiftCalculated Stand. Dev of Fold Shifts for replicatesFloat
59SEM_Fold_ShiftCalculated S.E.M. of Fold shifts for replicatesFloat

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R01 MH062646

Data Table (Concise)
Data Table ( Complete ):     View All Data
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