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BioAssay: AID 588710

mGluR5_3H_methoxyPEPy_Binding

Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 5 subtype (mGluR5) have been identified as a potential novel approach to treatment for schizophrenia and other CNS disorders that lead to impaired cognitive function (1). These compounds exist across a variety of chemical scaffolds (2) and have been determined to interact with at least two distinct sites in the transmembrane region of the receptor (3). In addition, it has been observed in functional cell-based assays that different mGluR5 PAMs exhibit different properties (4). ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Probe(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Probe(2)
 
 
Active(2)
 
 
AID: 588710
Data Source: Vanderbilt Specialized Chemistry Center (mGlu5_PAM_SecondaryAssay11)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-10-27
Hold-until Date: 2012-10-25
Modify Date: 2012-10-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 2    Active: 2
Related Experiments
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AIDNameTypeProbeComment
588721Optimization of novel mGluR5 positive allosteric modulators (PAM)sSummary depositor-specified cross reference: Optimization of novel mGluR5 positive allosteric modulators (PAM)s.
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
652201Discovery of Novel Silent Allosteric Modulators (SAM) of the Metabotropic Glutamate Receptor 5: rat mGlu1 selectivity AssayConfirmatory 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:
Positive allosteric modulators (PAMs) of the metabotropic glutamate receptor 5 subtype (mGluR5) have been identified as a potential novel approach to treatment for schizophrenia and other CNS disorders that lead to impaired cognitive function (1). These compounds exist across a variety of chemical scaffolds (2) and have been determined to interact with at least two distinct sites in the transmembrane region of the receptor (3). In addition, it has been observed in functional cell-based assays that different mGluR5 PAMs exhibit different properties (4).

Most notably, some mGluR5 PAMs exhibit allosteric agonist activity whereas others are pure mGluR5 PAMs with no intrinsic agonist activity. In order to determine the significance of these differences in a physiological setting, it is necessary to develop compounds that possess these different characteristics that are also amenable to in vivo studies (good physicochemical and pharmacokinetic properties). We have proposed a series of aims to evaluate the effects of these compounds on signaling in the CNS and to test the hypothesis that mGluR5 PAMs enhance synaptic plasticity in CNS preparations. Furthermore, we proposed studies aimed at testing the hypothesis that structurally and functionally distinct allosteric activators of mGluR5 have efficacy in rodent models that predict antipsychotic activity and enhance multiple forms of cognitive function in rodent models.

In addition to evaluating mGluR5 PAMs in general, a major goal of the proposed studies was to determine whether mGluR5 PAMs that have different in vitro profiles (ie. pure PAMs versus ago-PAMs) behave in a similar manner in these studies or whether these compounds have different effects. If the latter is the case, In addition to evaluating mGluR5 PAMs in general, a major goal of the proposed studies was to determine whether mGluR5 PAMs that have different in vitro profiles (ie. pure PAMs versus ago-PAMs) behave in a similar manner in these studies or whether these compounds have different effects. If the latter is the case, this could have major implications in guiding the optimal profile of compounds that will ultimately advance to clinical development and would directly inform studies focused on optimization of clinical development candidates.

1. Conn, P.J., Lindsley, C.W. and Jones, C.K., 2009. Activation of metabotropic glutamate receptors as a novel approach for the treatment of schizophrenia. Trends in Pharmacological Sciences 30: 25-31.
2. Stauffer, S.R., 2011. Progress toward Positive Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 5 (mGlu5). ACS Chemical Neuroscience 2: 450-470.
3. Chen, Y., Goudet, C., Pin, J.-P. and Conn, P.J., 2008. N-{4-Chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) Acts through a Novel Site as a Positive Allosteric Modulator of Group 1 Metabotropic Glutamate Receptors. Molecular Pharmacology 73: 909-918.
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.
5. Romano, C., Yang, W.L. and O'Malley, K.L., 1996. Metabotropic Glutamate Receptor 5 Is a Disulfide-linked Dimer. Journal of Biological Chemistry 271: 28612-28616.
6. Cosford, N.D.P., Roppe, J., Tehrani, L., Schweiger, E.J., Seiders, T.J., Chaudary, A., Rao, S. and Varney, M.A., 2003. [3H]-Methoxymethyl-MTEP and [3H]-Methoxy-PEPy: potent and selective radioligands for the metabotropic glutamate subtype 5 (mGlu5) receptor. Bioorganic & Medicinal Chemistry Letters 13: 351-354.
Protocol
Rat mGluR5 Competition Binding Assay (Secondary Assay 11)
Creation and culture of the rat mGluR5 cell line.
HEK293 cells expressing rat mGluR5 were provided by Dr Carmelo Romano (5). They were cultured in 85% Dulbecco's Modified Eagle Media (DMEM), 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 0.25 microg amphotericin, 20 mM HEPES, pH 7.3, 1 mM sodium pyruvate, 0.1 mM NEAA, 2 mM L-glutamine, and 500 mug/ml G418 (Mediatech, Inc., Herndon, VA). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Membrane preparation protocol.
Cells expressing the rat mGlu5 receptor were grown in multiple (5 - 10) 150 mm x 25 mm dishes until approximately 80% confluent. The growth medium was removed from each and cells were washed with 5 mL ice cold phosphate buffered saline (PBS). Fifteen mL ice cold Tris buffer (50 mM Tris-HCL containing 0.9% NaCl, pH 7.4) was added and the cells were scraped from the dish using a rubber scraper. The cells were transferred to a clean beaker placed on ice and the dish was washed with 5 mL Tris buffer and the remaining cells transferred to the beaker. Cells were centrifuged at 300 x g for 3 min. The supernatant was disposed and the pellet resuspended in 10 mL Tris buffer. The cell suspension was homogenized with a Tekmar sonic disrupter using three 10 second bursts separated by 30 seconds with the cells on ice. The solution was centrifuged at 1000 x g for 10 min at 4 degrees C. The supernatant was kept and the pellet discarded. The supernatant was centrifuged at 30k x g for 30 min at 4 degrees C. The supernatant from this spin was discarded and the pellet resuspended in 2 mL Tris buffer. The resulting membrane solution was given a 2 sec burst on the homogenizer. The protein concentration was determined using a Bio-Rad BCA assay kit. The membrane preparation was stored at -80 degrees
Radioligand binding.
After thawing, membranes were homogenized using a manual homogenizer and diluted to a concentration of 10 microg protein/150 microL with Hanks Balanced Salt Solution (HBSS) buffered with 20 mM HEPES, pH 7.4. MPEP was added at a concentration of 8.34 microM to a portion of the membrane suspension for use as the non-specific binding condition (final concentration to be 5 microM MPEP). Mixtures of 150 microL (10 microg protein) membrane suspension (with or without MPEP), 50 microL of approximately 10 nM [3H]-mPEPy (nominal 2 nM final concentration, actual determined after the experiment with scintillation counting) (6), and 50 microL of 5X test compound were combined in wells of a 96-well deep well plate. Total binding was determined using membrane suspension and radioligand only in a DMSO- and volume-matched condition. The final compound concentrations in the assay were (microM) 0.000507, 0.00152, 0.00457, 0.0137, 0.0411, 0.123, 0.369, 1.11, 3.33, 10, and 30. Each condition was run in duplicate.
The mixtures were shaken at RT for 1 hour then rapidly harvested by filtration through glass-fiber 96-well filter plate (Unifilter-96 GF/B, Perkin Elmer) using a Brandel cell harvester. To ensure full transfer, the wells of the assay plate were washed three times with cold assay buffer and transferred to the filter plate. The filter plate was allowed to dry overnight. Scintillation fluid (40 microL) was added to each well. The bound radioligand was determined by scintillation counting using a PerkinElmer TopCount.
The average of the nonspecific binding counts (4 per plate) was subtracted from each well's counts. This corrected value was then expressed as a total of the average of the corrected total binding counts (12 per plate). Percent total binding for each concentration was plotted vs log[compound]. The average of the total binding results was plotted at a concentration one log unit below the lowest concentration used in the CRC. The data were analyzed using nonlinear regression and were fit to a four-parameter equation using the software Prism v5 (Graphpad, La Jolla, CA). IC50 values were determined from the curve fit. Percent inhibition of binding was determined by comparing the highest degree of inhibition by the compound to the range of the bottom and top of the curve fit (top - bottom = 100%).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_IC50_uM*Average of IC50 values from replicates 1-3FloatμM
2SD_IC50Standard deviation for the calculated IC50 valueFloatμM
3NNumber of replicates measuredInteger
4Average_Percent_InhibitionAverage percent Inhibition response from replicates 1-3Float%
5SD_Percent_InhibitionStandard deviation for the measured percent inhibition response valuesFloat%
6Value_at_30_uM_1 (30μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
7Value_at_10_uM_1 (10μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
8Value_at_3.33_uM_1 (3.33μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
9Value_at_1.11_uM_1 (1.11μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
10Value_at_0.369_uM_1 (0.369μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
11Value_at_0.123_uM_1 (0.123μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
12Value_at_0.0411_uM_1 (0.0411μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
13Value_at_0.0137_uM_1 (0.0137μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
14Value_at_0.00457_uM_1 (0.00457μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
15Value_at_0.00152_uM_1 (0.00152μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
16Value_at_0.000158_uM_1 (0.000158μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
17IC50_uM_1IC50 value in micromolar (replicate 1)FloatμM
18Percent_Inhibition_1Percent total binding corrected for nonspecific binding (replicate 1)Float%
19Value_at_30_uM_2 (30μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
20Value_at_10_uM_2 (10μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
21Value_at_3.33_uM_2 (3.33μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
22Value_at_1.11_uM_2 (1.11μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
23Value_at_0.369_uM_2 (0.369μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
24Value_at_0.123_uM_2 (0.123μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
25Value_at_0.0411_uM_2 (0.0411μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
26Value_at_0.0137_uM_2 (0.0137μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
27Value_at_0.00457_uM_2 (0.00457μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
28Value_at_0.00152_uM_2 (0.00152μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
29Value_at_0.000158_uM_2 (0.000158μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
30IC50_uM_2IC50 value in micromolar (replicate 2)FloatμM
31Percent_Inhibition_2Percent Inhibition Response (replicate 2)Float%
32Value_at_30_uM_3 (30μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
33Value_at_10_uM_3 (10μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
34Value_at_3.33_uM_3 (3.33μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
35Value_at_1.11_uM_3 (1.11μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
36Value_at_0.369_uM_3 (0.369μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
37Value_at_0.123_uM_3 (0.123μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
38Value_at_0.0411_uM_3 (0.0411μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
39Value_at_0.0137_uM_3 (0.0137μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
40Value_at_0.00457_uM_3 (0.00457μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
41Value_at_0.00152_uM_3 (0.00152μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
42Value_at_0.000158_uM_3 (0.000158μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
43Value_at_0.0005_uM_3 (0.0005μM**)Percent total binding corrected for nonspecific binding (See protocol)Float
44IC50_uM_3IC50 value in micromolar (replicate 3)FloatμM
45Percent_Inhibition_3Percent Inhibition Response (replicate 3)Float%

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

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