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BioAssay: AID 652203

Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu7 selectivity Assay

Selectivity of mGlu5 SAMs was assessed by investigating the ability of 10uM to modulate orthosteric agonist activity at mGlu7. As glutamate has low affinity and efficacy at mGlu7, L-AP4 was used to probe modulation of receptor function (1,2,3), and Galpha15 was co-expressed to facilitate coupling to calciumm mobilization. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Inactive(1)
 
 
AID: 652203
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 ):           All
Target
Tested Compound:
Depositor Specified Assays
AIDNameTypeComment
652263Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: BioAssay Summary, ML 353summary
588721Optimization of novel mGluR5 positive allosteric modulators (PAM)ssummary
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 mGlu7. As glutamate has low affinity and efficacy at mGlu7, L-AP4 was used to probe modulation of receptor function (1,2,3), and Galpha15 was co-expressed to facilitate coupling to calciumm mobilization.

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
rmGlu7+Galpha15/TRex293 cells were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 20 mM HEPES, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, 500ug/mL G418, 5ug/mL Blasticin and 100ug/mL hygromycin.
Experiment Preparation.
rmGlu7+Galpha15/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) . 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 10 minutes at 37degrees Celcius, 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 L-AP4 ranged from 3.9uM 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 L-AP4was 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
9Cmpd Conc1 Rep1 (3.9μM**)Value for compound + L-AP4 3.9uM replicate 1Float%
10Cmpd Conc2 Rep1 (7.8μM**)Value for compound + L-AP4 7.8uM replicate 1Float%
11Cmpd Conc3 Rep1 (15.6μM**)Value for compound + L-AP4 15.6uM replicate 1Float%
12Cmpd Conc4 Rep1 (31.3μM**)Value for compound + L-AP4 31.3uM replicate 1Float%
13Cmpd Conc5 Rep1 (62.5μM**)Value for compound + L-AP4 62.5uM replicate 1Float%
14Cmpd Conc6 Rep1 (125μM**)Value for compound + L-AP4 125uM replicate 1Float%
15Cmpd Conc7 Rep1 (250μM**)Value for compound + L-AP4 250 uM replicate 1Float%
16Cmpd Conc8 Rep1 (500μM**)Value for compound +L-AP4 500 uM replicate 1Float%
17Cmpd Conc9 Rep1 (1000μM**)Value for compound +L-AP4 1mM replicate 1Float%
18EC50_uM_Rep1Calculated EC50 for replicate 1FloatμM
19Fold_shift_Rep1Calculated fold shift for replicate 1Float
20Cmpd Conc1 Rep2 (3.9μM**)Value for compound + L-AP4 3.9uM replicate 2Float%
21Cmpd Conc2 Rep2 (7.8μM**)Value for compound + L-AP4 7.8uM replicate 2Float%
22Cmpd Conc3 Rep2 (15.6μM**)Value for compound + L-AP4 15.6uM replicate 2Float%
23Cmpd Conc4 Rep2 (31.3μM**)Value for compound + L-AP4 31.3uM replicate 2Float%
24Cmpd Conc5 Rep2 (62.5μM**)Value for compound + L-AP4 62.5uM replicate 2Float%
25Cmpd Conc6 Rep2 (125μM**)Value for compound + L-AP4 125uM replicate 2Float%
26Cmpd Conc7 Rep2 (250μM**)Value for compound + L-AP4 250 uM replicate 2Float%
27Cmpd Conc8 Rep2 (500μM**)Value for compound +L-AP4 500 uM replicate 2Float%
28Cmpd Conc9 Rep2 (1000μM**)Value for compound +L-AP4 1mM replicate 2Float%
29EC50_uM_Rep2Calculated EC50 for replicate 2FloatμM
30Fold_shift_Rep2Calculate Fold shift for replicate 2Float
31Average_EC50_uM*Calculated Avg EC50 for replicatesFloatμM
32SD_EC50_uMCalculated Stand. Dev of EC50 for replicatesFloatμM
33Average_Fold_ShiftCalculated Avg Fold shift for replicatesFloat
34SD_Fold_ShiftCalculated Stand. Dev for replicatesFloat

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

Data Table (Concise)
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