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BioAssay: AID 652198

Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: Fold-shift Assay

Silent allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) occupy allosteric sites but have no intrinsic modulator activity. Thus, such compounds have potential as crucial tools to dissect receptor function and efficacy of novel positive and negative allosteric modulators both in vitro and in vivo. Previous mGlu5 SAMs, VU0365396 and 5MPEP, have low affinity and are therefore more ..
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 Tested Compounds
 Tested Compounds
All(34)
 
 
Probe(1)
 
 
Active(20)
 
 
Inactive(14)
 
 
 Tested Substances
 Tested Substances
All(34)
 
 
Probe(1)
 
 
Active(20)
 
 
Inactive(14)
 
 
AID: 652198
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 ):           Active    All
Target
BioActive Compounds: Chemical Probe: 1    Active: 20
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

Silent allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) occupy allosteric sites but have no intrinsic modulator activity. Thus, such compounds have potential as crucial tools to dissect receptor function and efficacy of novel positive and negative allosteric modulators both in vitro and in vivo. Previous mGlu5 SAMs, VU0365396 and 5MPEP, have low affinity and are therefore limited in their utility as tools (1, 2). The mGlu5 acetylene PAM series is prone to "molecular switches" within the SAR and certain PAMs had previously been shown to exhibit these "molecular switches" when key residues within the the common allosteric site were mutated (3). Thus, this series was predicted to also contain hitherto unappreciated mGlu5 SAMs.
To identify and evaluate mGlu5 SAMs, we screened previously identified inactive compounds in an acetylene PAM series at 10uM, to confirm they were inactive. Confirmed "inactives" were then screened in a 3 pt binding assay to rapidly identify compounds that retained appreciable affinity for the common allosteric site and thus categorise these compounds as "SAMs". SAMs with sub-100nM affinities were then selected for determination of binding affinity, using a more rigorous 10 pt inhibition binding curve,

1. Rodriguez, A. L., Nong, Y., Sekaran, N. K., Alagille, D., Tamagnan, G. D., Conn, P. J. (2005) A close structural analog of 2-methyl-6-(phenylethynyl)-pyridine acts as a neutral allosteric site ligand on metabotropic glutamate receptor subtype 5 and blocks the effects of multiple allosteric modulators. Mol. Pharmacol. 68:1793-1802.
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. Gregory, K. J., Nguyen, E. D., Reiff, S. D., Squire, E. F., Stauffer, S. R., Lindsley, C. W., Meiler, J., Conn, P. J. (2013) Probing the Metabotropic Glutamate Receptor 5 (mGlu5) Positive Allosteric Modulator (PAM) Binding Pocket: Discovery of Point Mutations that Engender a "Molecular Switch" in PAM Pharmacology. Mol Pharmacol (in press)
4. 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.
Protocol
Creation and culture of the rat mGlu5 cell line. Monoclones of stable HEK293A cells expressing rat mGlu5 were generated, selected, and tested for functional Ca+2 response (4). The selected clone (designated as "R10A") exhibited robust Ca+2 response to addition of glutamate, demonstrated specific binding of the specific mGlu5 radioligand [3H]-mPEPy, and showed an mGlu5-postive signal in a Western blot analysis.
rmGlu5/HEK293A 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, and 500 ug/ml G418 (Mediatech, Inc., Herndon, VA). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Experiment Preparation.
rmGlu5/HEK293A 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 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
ScoreThe BioAssay activity ranking scoreInteger
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 0.01MICROMOLAR replicate 1Float%
11Cmpd Conc2 Rep1 (0.03μM**)Value for compound + glu 0.03MICROMOLAR replicate 1Float%
12Cmpd Conc3 Rep1 (0.1μM**)Value for compound +glu 0.1 MICROMOLAR replicate 1Float%
13Cmpd Conc4 Rep1 (0.2μM**)Value for compound + glu 0.2MICROMOLAR replicate 1Float%
14Cmpd Conc5 Rep1 (0.3μM**)Value for compound + glu 0.3 MICROMOLAR replicate 1Float%
15Cmpd Conc6 Rep1 (1μM**)Value for compound + glu 0.1 MICROMOLAR replicate 1Float%
16Cmpd Conc7 Rep1 (3μM**)Value for compound +glu 1MICROMOLAR replicate 1Float%
17Cmpd Conc8 Rep1 (10μM**)Value for compound +glu 10 MICROMOLAR replicate 1Float%
18Cmpd Conc9 Rep1 (100μM**)Value for compound +glu 100 MICROMOLAR replicate 1Float%
19EC50_MICROMOLAR_Rep1Calculated EC50 for replicate 1FloatμM
20Fold_Shift_Rep1Calculated Fold Shift for Replicate 1.Float
21Cmpd Conc1 Rep2 (0.01μM**)Value for compound + glu 0.01MICROMOLAR replicate 2Float%
22Cmpd Conc2 Rep2 (0.03μM**)Value for compound + glu 0.03MICROMOLAR replicate 2Float%
23Cmpd Conc3 Rep2 (0.1μM**)Value for compound +glu 0.1 MICROMOLAR replicate 2Float%
24Cmpd Conc4 Rep2 (0.2μM**)Value for compound + glu 0.2MICROMOLAR replicate 2Float%
25Cmpd Conc5 Rep2 (0.3μM**)Value for compound + glu 0.3 MICROMOLAR replicate 2Float%
26Cmpd Conc6 Rep2 (1μM**)Value for compound + glu 0.1 MICROMOLAR replicate 2Float%
27Cmpd Conc7 Rep2 (3μM**)Value for compound +glu 1MICROMOLAR replicate 2Float%
28Cmpd Conc8 Rep2 (10μM**)Value for compound +glu 10 MICROMOLAR replicate 2Float%
29Cmpd Conc9 Rep2 (100μM**)Value for compound +glu 100 MICROMOLAR replicate 2Float%
30EC50_MICROMOLAR_Rep2Calculated EC50 for replicate 2FloatμM
31Fold_Shift_Rep2Calculated Fold Shift for Replicate 2Float
32Cmpd Conc1 Rep3 (0.01μM**)Value for compound + glu 0.01MICROMOLAR replicate 3Float%
33Cmpd Conc2 Rep3 (0.03μM**)Value for compound + glu 0.03MICROMOLAR replicate 3Float%
34Cmpd Conc3 Rep3 (0.1μM**)Value for compound +glu 0.1 MICROMOLAR replicate 3Float%
35Cmpd Conc4 Rep3 (0.2μM**)Value for compound + glu 0.2MICROMOLAR replicate 3Float%
36Cmpd Conc5 Rep3 (0.3μM**)Value for compound + glu 0.3 MICROMOLAR replicate 3Float%
37Cmpd Conc6 Rep3 (1μM**)Value for compound + glu 0.1 MICROMOLAR replicate 3Float%
38Cmpd Conc7 Rep3 (3μM**)Value for compound +glu 1MICROMOLAR replicate 2Float%
39Cmpd Conc8 Rep3 (10μM**)Value for compound +glu 10 MICROMOLAR replicate 3Float%
40Cmpd Conc9 Rep3 (100μM**)Value for compound +glu 100 MICROMOLAR replicate 3Float%
41EC50_MICROMOLAR_Rep3Calculated EC50 for replicate 3FloatμM
42Fold_Shift_Rep3Calculated Fold Shift for replicate 3Float
43Average_EC50_MICROMOLAR*Calculated Avg EC50 for the first 3 replicatesFloatμM
44SD_EC50_MICROMOLARCalculated Stand. Dev of EC50 for the first 3 replicatesFloatμM
45SEM_EC50_MICROMOLARCalculated SEM for EC50 for the first 3 replicatesFloatμM
46Average_Fold_ShiftCalculated Average Fold Shift for the first 3 replicatesFloat
47SD_Fold_ShiftCalculated Stand. Dev of Fold Shifts for the first 3 replicatesFloat
48SEM_Fold_ShiftCalculated S.E.M. of Fold shifts for the first 3 replicatesFloat

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

Data Table (Concise)
Classification
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