Bookmark and Share
BioAssay: AID 588737

hmGluR5_Calcium_Fold_Shift

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
_
   
 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 588737
Data Source: Vanderbilt Specialized Chemistry Center (mGlu5_PAM_SecondaryAssay10)
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 ):           Active    All
Target
BioActive Compounds: 2
Depositor Specified Assays
AIDNameTypeComment
588721Optimization of novel mGluR5 positive allosteric modulators (PAM)ssummaryOptimization of novel mGluR5 positive allosteric modulators (PAM)s.
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.
Protocol
Human mGluR5 Calcium Fold Shift (Secondary Assay 10)

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 use Glutamate as the agonist.

Description of Cell Lines. The cells lines used in the Calcium fold shift assays include rat mGlu1, human mGlu5 and rat mGlu5. The rat mGlu1 and human mGlu5 cell lines were generated using a HEK293A background and the pcDNA3.1 plasmid. The cell line expressing rat mGlu5 was generated using a HEK293 background and the pcDNA1Neo plasmid (5).

Cell Culture. All components were supplied by Invitrogen unless otherwise noted. Cell lines were grown in a medium consisting of 85% DMEM, 10% FBS, 20 mM HEPES, pH 7.4, 2 mM Glutamax, 1X (0.1 mM) Nonessential Amino Acids, 1 mM sodium pyruvate, 0.5 mg/mL G418. Cells were 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 at a density of 15,000 cells/20 microL/well.

Calcium Mobilization Assay Solutions. The following solutions are required:

1. Assay buffer: HBSS (Invitrogen), 20 mM HEPES, pH 7.4, 2.5 mM Probenecid (Sigma) (buffer may be made up a week in advance; probenecid should be added the day of the experiment)
2. Fluo4-AM (Invitrogen): Dye is supplied in 1 mg units. A 2X solution (2 microM) is made by dissolving 1 mg dye in 456 microL DMSO. 10 microL of the dye solution is added to 5 microL 20% pluronic acid (Invitrogen). This solution is then mixed with 10 mL assay buffer (#1 above)
3. Agonist dilutions in assay buffer using stock glutamate 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.
4. 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 Fluo4-AM dye (1 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 CRC) is added at 142 seconds. The read continues for 300 seconds.

Data analysis. Data were analyzed using Microsoft Excel. Raw data were opened in Excel and each data point in a given trace was divided by the first data point from that trace (static ratio). The max fluorescence increase occurring after the agonist addition minus the minimum fluorescence at the beginning of data collection was calculated to determine the net amplitude. The value was normalized to the average of the maximum amplitudes in the control curves to determine the percent of the maximum glutamate response (%Glu max). %Glu max was plotted vs log agonist concentration. Curves were 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 were compared. Fold shift was calculated by dividing the control EC50 by the EC50 of the condition with the test compound.

Compound category was 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: Ago-PAM activity can also be detected in this assay as a response upon compound addition. This typically also results in an increase in the Emin of the agonist curve.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
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)
Classification
PageFrom: