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BioAssay: AID 651835

Identification of a glycine sulfonamide based non-MPEP site positive allosteric potentiator (PAM) of mGlu5 (calcium_assay)

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(32)
 
 
Probe(16)
 
 
Active(16)
 
 
Inactive(6)
 
 
Inconclusive(10)
 
 
 Tested Substances
 Tested Substances
All(32)
 
 
Probe(16)
 
 
Active(16)
 
 
Inactive(6)
 
 
Inconclusive(10)
 
 
AID: 651835
Data Source: Vanderbilt Specialized Chemistry Center (mGluR5_PAM_nonMPEP_probe_calcium_assay_20121203)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-03
Hold-until Date: 2013-07-26
Modify Date: 2013-07-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 16    Active: 16
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.
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
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.
Protocol
Rat mGluR5 Ca+2 Mobilization Potency Assay (Secondary Assay 1)
Creation and culture of the rat mGluR5 cell line. Monoclones of stable HEK293A cells expressing rat mGluR5 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 mGluR5 radioligand [3H]-mPEPy, and showed an mGluR5-postive signal in a Western blot analysis.
rmGluR5/HEK cells were cultured in 85% Dulbecco's Modified Eagle Media (DMEM), 10% fetal bovine serum (FBS), 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.
Experiment Preparation.
rmGluR5/HEK cells were plated the day before the experiment at a density of 15,000 cells/well in a 20 microL volume in Poly-D-Lysine 384-well Microplates (black/clear, BD # 356936) in assay medium (87% DMEM, 20 mM HEPES, pH 7.4, 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 microM. 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 45 min at 37 degrees C/5% CO2. The cells were then washed with assay buffer and, after an incubation period of 10 minutes at room temperature, the assay was started.
All data were collected at a frequency of 1 frame/sec for 350 secs monitoring fluorescence of the Ca2+-bound dye at an excitation wavelength of 480 nm and emission at 540 nm using a Functional Drug Screening System (FDSS, Hamamatsu Corp). Test compounds were provided as 10 mM stocks in DMSO. Final concentration of DMSO in the assay was 0.3%.
Concentration response curve (CRC) Ca+2 assays.
For CRC generation, the concentrations of test compound ranged from 0.37 nM to 30 microM (final concentrations) in an 11-point curve and were made 2X in triplicate on each compound addition plate using a Labcyte Echo liquid transfer system employed with Dose Response Software. Three additions were made within the FDSS. First, compound alone was added to each well at 3 seconds to detect any agonist activity. Next, a sub-optimal concentration (nominally 20% of the maximum response, EC20) of glutamate was added at 143 seconds to detect any potentiation of the glutamate response by the test compound. Lastly, a sub-maximal concentration of 3 microM (nominally 80% of the maximum response, EC80) was added at 240 seconds to detect any antagonism of the glutamate response by the test compound. The concentration of glutamate added for the EC20 concentration was determined on test day and typically fell between 200-300 nM.
For reference, the following conditions were included in each plate assayed:
1. wells receiving vehicle control in the first addition, an EC20 concentration of glutamate in the second addition, and an EC80 concentration of glutamate in the third addition. Only plates for which the average of the reference EC20 response fell between 10% and 30% of the ECmax response were accepted.
2. wells receiving only an ECmax concentration of glutamate (60 microM) in the third addition (vehicle for the first and second additions). These were used to normalize the test responses to express as %ECmax.
Data analysis for CRC Ca+2 assays. Data files containing raw fluorescence values from the FDSS were inserted into a Microsoft Excel analysis template using Excel formulas for data reduction. Each point was normalized to the initial value for that well (static ratio). In each data window of interest (starting at 143 seconds for PAM activity), the peak static ratio response was determined and corrected by subtraction of the minimum response at the beginning of the data collection. This correction was also performed for each reference condition described above. Each corrected response was expressed as a percent of the average of the ECmax. This %ECmax response was plotted versus the log of the molar concentration of the test compound and fit to a 4 parameter logistic equation in Prism v5 (Graphpad) to determine the minimum response, maximum response, the log concentration giving the half-maximal response, and the slope factor of the curve. The curve fit was applied to the mean of the triplicates for each day. The determined parameter values for all test days were averaged to achieve a mean value and standard deviation for each.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_EC50_uM*Average EC50 across 3 replicatesFloatμM
2SD_EC50_uMStd. Dev of EC50 across 3 replicatesFloatμM
3SEM_EC50_uMSEM of EC50 across 3 replicatedFloatμM
4Average_%_Glu_MaxAverage Glu Max across 3 replicatesFloat%
5SD_Average_%_Glu_MaxSD Glu Max across 3 replicatesFloat%
6SEM_Average_%_Glu_MaxSEM Glu Max across 3 replicatesFloat%
7CategoryHow the compound is classified as, PAM, AGO-PAM, WEAK PAM, INACTIVEString
8Value_at_0.001_uM_1 (0.001μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
9Value_at_0.005_uM_1 (0.005μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
10Value_at_0.01_uM_1 (0.01μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
11Value_at_0.04_uM_1 (0.04μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
12Value_at_0.1_uM_1 (0.1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
13Value_at_0.4_uM_1 (0.4μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
14Value_at_1_uM_1 (1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
15Value_at_3_uM_1 (3μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
16Value_at_10_uM_1 (10μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
17Value_at_30_uM_1 (30μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
18EC50_uM_1EC50 value in micromolar (replicate 1)FloatμM
19Fit_%_Glu_Max_1Percent Maximum Glutamate Response (replicate 1)Float%
20Value_at_0.001_uM_2 (0.001μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
21Value_at_0.005_uM_2 (0.005μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
22Value_at_0.01_uM_2 (0.01μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
23Value_at_0.04_uM_2 (0.04μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
24Value_at_0.1_uM_2 (0.1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
25Value_at_0.4_uM_2 (0.4μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
26Value_at_1_uM_2 (1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
27Value_at_3_uM_2 (3μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
28Value_at_10_uM_2 (10μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
29Value_at_30_uM_2 (30μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
30EC50_uM_2EC50 value in micromolar (replicate 2)FloatμM
31Fit_%_Glu_Max_2Percent Maximum Glutamate Response (replicate 2)Float%
32Value_at_0.001_uM_3 (0.001μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
33Value_at_0.005_uM_3 (0.005μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
34Value_at_0.01_uM_3 (0.01μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
35Value_at_0.04_uM_3 (0.04μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
36Value_at_0.1_uM_3 (0.1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
37Value_at_0.4_uM_3 (0.4μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
38Value_at_1_uM_3 (1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
39Value_at_3_uM_3 (3μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
40Value_at_10_uM_3 (10μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
41Value_at_30_uM_3 (30μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
42EC50_uM_3EC50 value in micromolar (replicate 3)FloatμM
43Fit_%_Glu_Max_3Percent Maximum Glutamate 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
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