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BioAssay: AID 588728

rmGluR3_Thallium_Fold_Shift (GIRK)

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
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Inactive(2)
 
 
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 Tested Substances
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Inactive(2)
 
 
AID: 588728
Data Source: Vanderbilt Specialized Chemistry Center (mGlu5_PAM_SecondaryAssay4)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-30
Hold-until Date: 2012-10-25
Modify Date: 2012-10-27

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
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
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.
Protocol
Rat mGluR3 Thallium Fold Shift (Secondary Assay 4)

Summary. This assay is designed to determine the change in an agonist's CRC response in a functional assay induced by a test compound. These assays are used for determining selectivity of a compound across mGlu receptor subtypes. All cell lines except the mGlu7 cell line use Glutamate as the agonist. mGlu7 uses L-AP4 as the agonist for that receptor.

Description of Cell Lines. The cells lines used in the thallium flux fold shift assays include rat mGlu2, rat mGlu3, rat mGlu4, human mGlu6, rat mGlu7, and rat mGlu8. All cell lines were generated using a HEK/GIRK(M4) background and the pIRESpuro3 plasmid.

Cell Culture. All components are supplied by Invitrogen unless otherwise noted. Cell lines are grown in a medium consisting of 42% DMEM, 41.6% F12, 10% Heat-inactivated FBS, 20 mM HEPES, pH 7.4 (Sigma), 2 mM L-Glutamine (Sigma), 1X Nonessential Amino Acids, 1 mM sodium pyruvate, 0.7 mg/mL G418, 0.6 microg/mL puromycin dihydrochloride (Sigma). Cells are grown to 80% confluency then plated the day before the experiment in Greiner or Falcon optical PDK-coated 384 well plates in assay medium consisting of 87% DMEM, 20 mM HEPES, pH 7.4, 10% dialyzed FBS, and 1 mM sodium pyruvate.

Thallium Flux Assay Solutions. The day of the experiment, the following solutions are made up

1. Assay buffer: HBSS (Invitrogen), 20 mM HEPES, pH 7.4
2. Thallium buffer: 125 mM Sodium Bicarbonate (Sigma), 1 mM Magnesium Sulfate (Fisher), 1.8 mM Calcium Sulfate (Acros), 5 mM Glucose (Acros), 10 mM HEPES, pH 7.4, 12 mM Thallium Sulfate (Sigma; hazardous material, handle with care and dispose all reagent and containers appropriately)
3. 2X FluoZin2-AM (Invitrogen): Dye is supplied in 50 microg units. A 2X solution (0.33 microM) is made by dissolving 50 microg dye in 20 microL DMSO. 1.15 microL of the dye solution is added to 1.15 microL 20% pluronic acid (Invitrogen). This solution is then mixed with 10 mL assay buffer (#1 above)
4. Agonist dilutions in Thallium buffer using stock glutamate and stock L-AP4 of 100 mM (made up in 1 equivalent NaOH). The agonist CRCs are made 5X due to their dilution during the experiment. Eleven-point CRC's are run in addition to control conditions with buffer only.
5. Test compound: compound stock occurs as a 10 mM solution in DMSO, when possible. Compound plates are made 2X in test compound (20 microM resulting in a 10 microM final concentration during the experiment)

Basic protocol:

1. Media is removed from the cells and cells are washed 3X with assay buffer using an ELX405 washer. 20 microL of buffer is left in each well after this step.
2. 20 microl/well of Fluo Zn dye (0.16 microM final conc.) in Assay Buffer is added
3. The plates are incubated for 45 min at 37 degrees C/5% CO2
4. Dye is removed from the cells and washed with assay buffer using the ELX405 washer. The washer will leave 20 microl of assay buffer per well
5. Plates are incubated for 10 minutes at RT
6. Plates are loaded into a Hamamatsu FDSS.
7. Compounds are tested for selectivity at a 10 microM final concentration of test compound (prepared as a 2X stock, 20 microl added per well) and the agonist is added at a 5X concentration (10 microl of agonist added per well, 50 microL total volume). A baseline read is taken for 1.5 seconds, the first addition (compound or DMSO-matched vehicle) occurs at 1.5 seconds and the second addition (agonist in Tl+ buffer) is added at 142 seconds. The read continues for 300 seconds.

Data analysis. Data are analyzed using Microsoft Excel. Each data point in a given trace is divided by the first data point from that trace (static ratio). The slope of the fluorescence increase beginning five seconds after thallium/agonist addition and ending fifteen seconds after thallium/agonist addition is calculated. Each slope value is corrected by subtracting out the average of the slopes for the lowest agonist concentrations. These baseline corrected values are then normalized to the baseline corrected maximum agonist response. Curves are fit using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ) to determine %Emax, Emin, slope, and logEC50. The EC50 and %Emax values between curves with and without test compound are compared. Fold shift is calculated by dividing the control EC50 by the EC50 of the test compound.

Compound activity is scored as follows:

1. Fold Shift < or = 0.5, Antagonist
2. %Emax between 50 and 75, Weak NAM
3. %Emax < 50, NAM
4. Fold Shift > 2, PAM
5. %Emax > 125%, PAM

Note that some compounds will increase or decrease the baseline thallium flux. These compounds may have activity at the receptor in question, but further follow up studies are warranted to determine if this is the case.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Concentration_uM (10μM**)Glutamate agonist concentration in micromolarFloatμM
2Fold shiftFold shift for tested compound relative to vehicle controlsFloat
3EC50_uMEC50 value in micromolarFloatμM
4Ec50_ucl_uMEC50 value upper confidence limit in micromolarFloatμM
5Ec50_lcl_uMEC50 value lower confidence limit in micromolarFloatμM
6Glu_maxPercent maximum Glutamate responseFloat%
7Glu_max uclPercent maximum Glutamate response upper confidence limitFloat%
8Glu_max lclPercent maximum Glutamate response lower confidence limitFloat%
9Glu_minPercent minimum Glutamate responseFloat%
10Glu_min uclPercent minimum Glutamate response upper confidence limitFloat%
11Glu_min lclPercent minimum Glutamate response lower confidence limitFloat%
12SlopeConcentration Response Curve slope valueFloat
13Slope uclConcentration Response Curve slope value upper confidence limitFloat
14Slope lclConcentration Response Curve slope value lower confidence limitFloat
15Value_at_0.0001_uM_1 (0.0001μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
16Value_at_0.0001_uM_2 (0.0001μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
17Value_at_0.00051_uM_1 (0.00051μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
18Value_at_0.00051_uM_2 (0.00051μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
19Value_at_0.0026_uM_1 (0.0026μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
20Value_at_0.0026_uM_2 (0.0026μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
21Value_at_0.013_uM_1 (0.013μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
22Value_at_0.013_uM_2 (0.013μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
23Value_at_0.064_uM_1 (0.064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
24Value_at_0.064_uM_2 (0.064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
25Value_at_0.32_uM_1 (0.32μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
26Value_at_0.32_uM_2 (0.32μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
27Value_at_1.6_uM_1 (1.6μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
28Value_at_1.6_uM_2 (1.6μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
29Value_at_8_uM_1 (8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
30Value_at_8_uM_2 (8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
31Value_at_40_uM_1 (40μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
32Value_at_40_uM_2 (40μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
33Value_at_200_uM_1 (200μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
34Value_at_200_uM_2 (200μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
35Value_at_1000_uM_1 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
36Value_at_1000_uM_2 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float

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

Data Table (Concise)
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