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BioAssay: AID 652196

Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: full Ki determination

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 ..
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 652196
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
Deposit Date: 2013-03-27
Hold-until Date: 2014-03-27
Modify Date: 2014-03-27

Data Table ( Complete ):           Active    All
BioActive Compounds: Chemical Probe: 1    Active: 9
Depositor Specified Assays
588721Optimization of novel mGluR5 positive allosteric modulators (PAM)ssummary
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.
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. 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.
Membrane preparations of rmGlu5/HEK293A cells were prepared as follows. Cells were harvested by trypsinization and pelleted in assay medium (DMEM, 20 mM HEPES, 10% dialyzed FBS, 1 mM sodium pyruvate). Cell pellets were snap frozen prior to re-suspension in ice cold homogenization buffer (recipe) and disruption with a Polytron homogenizer 3x10sec bursts, separated by 30sec on ice. Homogenates were centrifuged at 1000xg for 10min to clear nuclear and mitochonrial fractions, followed by centrifugation at 30000xg for 1 hr. The resulting pellet was resuspended in assay buffer (HBSS, 20 mM HEPES, 2.5 mM probenecid, pH 7.4) and stored at -80degrees until use.
Membrane preparations were diluted to give 50microgram/well. Test compounds were provided as 10 mM stocks in DMSO; final concentration of DMSO in the assay was 1%.
Displacement of [3H]methoxyPEPy binding to rmGlu5/HEK293A membranes by a range of concentrations of test compound (0.1nM to 10uM) was assessed as previously validated (2,3,4). Membranes (50microgram/well) were incubated with test compound and 2nM [3H]methoxyPEPy for 1 hr at romm teperature with skaing in assay buffer. Bound radiligand was separated from free by rapid filtration through GF/B filter plates (Perkin Elmer) with a 96 well plate Brandel cell harvester, with three washes with ice-cold binding buffer (0.9% NaCl, 50mM Tris, pH 7.4). Filter plates were allowed to dry overnight, prior to addition of 40uL Microscint20/well and determination of radioactivity using a TopCount six-detector plate reader (Perkin Elmer).

Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_Ki_uM*Calculate Ki valueFloatμM
4Value_at_0.0001_uM_1 (0.0001μM**)Value for 0.0001uMFloat%
5Value_at_0.0003_uM_1 (0.0003μM**)Value for 0.0003uMFloat%
6Value_at_0.001_uM_1 (0.001μM**)Value for 0.001uMFloat%
7Value_at_0.003_uM_1 (0.003μM**)Value for 0.003uMFloat%
8Value_at_0.01_uM_1 (0.01μM**)Value for 0.01uMFloat%
9Value_at_0.03_uM_1 (0.03μM**)Value for 0.03uMFloat%
10Value_at_0.1_uM_1 (0.1μM**)Value for 0.1uMFloat%
11Value_at_0.3_uM_1 (0.3μM**)Value for 0.3uMFloat%
12Value_at_1_uM_1 (1μM**)Value for 1uMFloat%
13Value_at_3_uM_1 (3μM**)Value for 3uMFloat%
14Value_at_10_uM_1 (10μM**)Value for 10uMFloat%
15Value_at_0.0001_uM_2 (0.0001μM**)Value for 0.0001uMFloat%
16Value_at_0.0003_uM_2 (0.0003μM**)Value for 0.0003uMFloat%
17Value_at_0.001_uM_2 (0.001μM**)Value for 0.001uMFloat%
18Value_at_0.003_uM_2 (0.003μM**)Value for 0.003uMFloat%
19Value_at_0.01_uM_2 (0.01μM**)Value for 0.01uMFloat%
20Value_at_0.03_uM_2 (0.03μM**)Float%
21Value_at_0.1_uM_2 (0.1μM**)Value for 0.1uMFloat%
22Value_at_0.3_uM_2 (0.3μM**)Value for 0.3uMFloat%
23Value_at_1_uM_2 (1μM**)Value for 1uMFloat%
24Value_at_3_uM_2 (3μM**)Value for 3uMFloat%
25Value_at_10_uM_2 (10μM**)Value for 10uMFloat%
26Value_at_0.0001_uM_3 (0.0001μM**)Value for 0.0001uMFloat%
27Value_at_0.0003_uM_3 (0.0003μM**)Value for 0.0003uMFloat%
28Value_at_0.001_uM_3 (0.001μM**)Value for 0.001uMFloat%
29Value_at_0.003_uM_3 (0.003μM**)Value for 0.003uMFloat%
30Value_at_0.01_uM_3 (0.01μM**)Value for 0.01uMFloat%
31Value_at_0.03_uM_3 (0.03μM**)Float%
32Value_at_0.1_uM_3 (0.1μM**)Value for 0.1uMFloat%
33Value_at_0.3_uM_3 (0.3μM**)Value for 0.3uMFloat%
34Value_at_1_uM_3 (1μM**)Value for 1uMFloat%
35Value_at_3_uM_3 (3μM**)Value for 3uMFloat%
36Value_at_10_uM_3 (10μM**)Value for 10uMFloat%
37Average_Value_at_0.0001_uMValue for 0.0001uMFloat%
38Average_Value_at_0.0003_uMValue for 0.0003uMFloat%
39Average_Value_at_0.001_uMValue for 0.001uMFloat%
40Average_Value_at_0.003_uMValue for 0.003uMFloat%
41Average_Value_at_0.01_uMValue for 0.01uMFloat%
42Average_Value_at_0.03_uMValue for 0.03uMFloat%
43Average_Value_at_0.1_uMValue for 0.1uMFloat%
44Average_Value_at_0.3_uMValue for 0.3uMFloat%
45Average_Value_at_1_uMValue for 1uMFloat%
46Average_Value_at_3_uMValue for 3uMFloat%
47Average_Value_at_10_uMValue for 10uMFloat%

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

Data Table (Concise)