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BioAssay: AID 651840

Modulation of the Metabotropic Glutamate Receptor mGluR3 (Negative Allosteric Modulators mGlu3 Galpha15 Calcium Potency)

The hippocampus is a limbic cortical structure that plays an important role in a number of normal physiological processes and is a primary site of pathology in certain neurological disorders, such as Alzheimer's disease and temporal lobe epilepsy (see (Brown and Zador 1990) for review). Because of its important role in both normal and pathological processes, a great deal of effort has been more ..
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 Tested Compounds
 Tested Compounds
All(28)
 
 
Probe(15)
 
 
Active(16)
 
 
Inactive(12)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Probe(15)
 
 
Active(16)
 
 
Inactive(12)
 
 
AID: 651840
Data Source: Vanderbilt Specialized Chemistry Center (mGluR3_Nam_Galpha15_Calcium_CRC)
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: 2013-07-18
Modify Date: 2013-07-18

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 15    Active: 16
Related Experiments
AIDNameTypeProbeComment
602451Optimization of novel mGluR3-selective allosteric modulatorsSummary depositor-specified cross reference: Optimization of novel mGluR3-selective allosteric modulators
623885Modulation of the Metabotropic Glutamate Receptor mGluR3 (mGlu3 GIRK Potency)Confirmatory2 same project related to Summary assay
623888Modulation of mGlu3 (mGlu3_GIRK_Schild)Other same project related to Summary assay
623929Modulation of the Metabotropic Glutamate Receptor mGluR3 (mGlu2 GIRK Potency)Confirmatory same project related to Summary assay
623931ML289 Competition in Radioligand Binding assays (Ricerca)Other same project related to Summary assay
624028Modulation of the Metabotropic Glutamate Receptor mGluR3 (mGlu3 Galpha15 Calcium Potency)Confirmatory1 same project related to Summary assay
651839Modulation of the Metabotropic Glutamate Receptor mGluR3 (Negative Allosteric Modulators, mGlu3 GIRK Potency)Confirmatory5 same project related to Summary assay
Description:
The hippocampus is a limbic cortical structure that plays an important role in a number of normal physiological processes and is a primary site of pathology in certain neurological disorders, such as Alzheimer's disease and temporal lobe epilepsy (see (Brown and Zador 1990) for review). Because of its important role in both normal and pathological processes, a great deal of effort has been focused on developing a detailed understanding of the cellular mechanisms involved in regulation of transmission through the hippocampal circuit. Evidence suggests that the metabotropic glutamate receptors (mGlus) play a variety of roles in modulating transmission and cell excitability at each of the major excitatory synapses in the hippocampus (Anwyl 1999; Coutinho and Knopfel 2002). In particular, the Gi/o-coupled group II mGlus (mGlu2 and mGlu3) can have significant physiological effects in the hippocampus. Activation of group II mGlus reduces transmission at perforant path-dentate gyrus synapses (Macek et al. 1996), the mossy fiber synapse (Nicholls et al. 2006), and at synapses onto certain interneuron populations (Doherty et al. 2004). However, although presynaptic group II mGlus do not directly reduce synaptic transmission through actions on glutamatergic terminals at the SC-CA1 synapse (Gereau and Conn 1995b; Winder et al. 1996; Fitzjohn et al. 1999), we and others previously reported extensive studies demonstrating that group II mGlus in this region are involved in a novel form of glial-neuronal communication. In particular, activation of group II mGlus induces a marked potentiation of cAMP responses elicited by activation of beta-adrenergic receptors (betaARs) in astrocytes, leading to release of adenosine from the astrocytes and inducing a profound depression of transmission at the SC-CA1 synapse via activation of presynaptic A1 adenosine receptors (Winder et al. 1996; Moldrich et al. 2002). This unique response provides a mechanism by which activation of group II mGlus only reduces transmission at the SC-CA1 synapses under conditions where betaARs and group II mGlus are coincidentally activated. This could provide a protective mechanism to reduce risk of excitotoxicity during periods in which there is excessive excitatory drive when noradrenergic inputs are highly active, such as during periods of intense or prolonged stress.

Unfortunately, despite concerted efforts by multiple groups, selective reagents that differentiate between mGlu2 and mGlu3 have not been available to allow determination of the specific mGlu subtype involved in modulating betaAR-mediated cAMP responses and effects on synaptic transmission. However, within our mGlu5 program, we have discovered compounds with mGlu3 NAM activity, such as VU0092273 (Rodriguez et al. 2010), that have excellent aqueous solubility and achieve high CNS exposure. However, this compound also acts as an mGlu5 PAM. While the mGlu5 PAM activity prevents use of VU0092273 as a selective mGluR3 NAM probe, discovery of this compound still represents a major breakthrough in that it is the first compound that selectively blocks mGlu3 relative to mGlu2 and establishes the feasibility of optimizing novel molecules that act at mGlu3 without effects on the closely related mGlu2 subtype. Thus, it will now be critical to further optimize compounds based on VU0092273 that are selective for mGlu3 relative to all other mGlu subtypes. Establishing novel, highly selective mGlu3 probes represents a critical need that could directly impact our understanding of mGlu3 function and could have a major impact on the direction of current drug discovery efforts focused on discovery and development of agents that activate or inhibit group II mGlus.

This unique response provides a mechanism by which activation of group II mGlus only reduces transmission at the SC-CA1 synapses under conditions where betaARs and group II mGlus are coincidentally activated. This could provide a protective mechanism to reduce risk of excitotoxicity during periods in which there is excessive excitatory drive when noradrenergic inputs are highly active, such as during periods of intense or prolonged stress. However, it is also possible that this reduction in transmission at a key hippocampal synapse could impair hippocampal function and could contribute to the known disrupting effects of stress on hippocampal synaptic plasticity and cognitive function (Howland and Wang 2008). If so, selective antagonists of the specific group II mGlu subtype that mediates this response also have potential utility as cognition-enhancing agents. Consistent with this, while non-selective group II mGlu agonists can reduce stress responses (Swanson et al. 2005) these agents also impair hippocampal-dependent cognitive function (Higgins et al. 2004). While speculative, this hypothesis provides a critical impetus for determining the specific group II mGlu subtype responsible for this response and establishing the impact of selectively blocking this receptor on hippocampal function.

Compounds will be initially screened by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) to determine the potency and efficacy of novel compounds for mGlu3 in a thallium flux assay measuring coupling of mGlu3 to G Protein-coupled Inwardly Rectifying Potassium (GIRK) Channels (Niswender et al. 2008; Jin et al. 2010; Dhanya et al. 2011). Compounds with IC50 values less than 1 uM will next be evaluated for potency versus mGlu2 in a thallium flux CRC assay. Compounds demonstrating at least 20-fold selectivity for mGlu3 versus mGlu2 will then be evaluated for the mechanism of mGlu3 antagonism through Schild functional thallium flux assays at mGlu3 to determine if the compounds are antagonizing mGlu3 in a competitive or noncompetitive manner. For noncompetetive compounds passing the above steps, we will pursue follow-up studies to determine their selectivity for mGlu3 relative to other mGlu subtypes. Our goal for this project would be to generate an mGlu3-selective compound, with an IC50 under 1 uM, and with reasonable solubility in a solvent generally useful for in vitro experimentation (i.e., DMSO). The ultimate goal of this project from the PI's perspective is to generate compounds that would be useful proof-of-concept molecules to determine the mGlu subtype involved in glial-neuronal communication in the hippocampus.

REFERERENCES
1. Anwyl, R. (1999). "Metabotropic glutamate receptors: electrophysiological properties and role in plasticity." Brain Res Brain Res Rev 29(1): 83-120.
2. Brown, T. A. and A. M. Zador (1990). Hippocampus. The Synaptic Organization of the Brain. S. GM. New York, Oxford UP: 346-388.
3. Coutinho, V. and T. Knopfel (2002). "Metabotropic glutamate receptors: electrical and chemical signaling properties." Neuroscientist 8(6): 551-61.
4. Dhanya, R. P., S. Sidique, et al. (2011). "Design and synthesis of an orally active metabotropic glutamate receptor subtype-2 (mGluR2) positive allosteric modulator (PAM) that decreases cocaine self-administration in rats." J Med Chem 54(1): 342-53.
5. Doherty, J. J., S. Alagarsamy, et al. (2004). "Metabotropic glutamate receptors modulate feedback inhibition in a developmentally regulated manner in rat dentate gyrus." J Physiol 561(Pt 2): 395-401.
6. Fitzjohn, S. M., A. E. Kingston, et al. (1999). "DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists." Neuropharmacology 38(10): 1577-83.
7. Gereau, R. W. t. and P. J. Conn (1995b). "Roles of specific metabotropic glutamate receptor subtypes in regulation of hippocampal CA1 pyramidal cell excitability." J Neurophysiol 74(1): 122-9.
8. Jin, X., S. Semenova, et al. (2010). "The mGluR2 positive allosteric modulator BINA decreases cocaine self-administration and cue-induced cocaine-seeking and counteracts cocaine-induced enhancement of brain reward function in rats." Neuropsychopharmacology 35(10): 2021-36.
9. Macek, T. A., D. G. Winder, et al. (1996). "Differential involvement of group II and group III mGluRs as autoreceptors at lateral and medial perforant path synapses." J Neurophysiol 76(6): 3798-806.
10. Moldrich, R. X., K. Aprico, et al. (2002). "Astrocyte mGlu(2/3)-mediated cAMP potentiation is calcium sensitive: studies in murine neuronal and astrocyte cultures." Neuropharmacology 43(2): 189-203.
11. Nicholls, R. E., X. L. Zhang, et al. (2006). "mGluR2 acts through inhibitory Galpha subunits to regulate transmission and long-term plasticity at hippocampal mossy fiber-CA3 synapses." Proc Natl Acad Sci U S A 103(16): 6380-5.
12. Niswender, C. M., K. A. Johnson, et al. (2008). "A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors." Mol Pharmacol 73(4): 1213-24.
13. Rodriguez, A. L., M. D. Grier, et al. (2010). "Discovery of novel allosteric modulators of metabotropic glutamate receptor subtype 5 reveals chemical and functional diversity and in vivo activity in rat behavioral models of anxiolytic and antipsychotic activity." Mol Pharmacol 78(6): 1105-23.
14. Winder, D. G., P. S. Ritch, et al. (1996). "Novel glial-neuronal signalling by coactivation of metabotropic glutamate and beta-adrenergic receptors in rat hippocampus." J Physiol 494 ( Pt 3): 743-55.
Protocol
mGlu3 Galpha15 Calcium Potency
Culture of the mGlu3 TREx cell line
TREx293 cells stably expressing mGlu3 and the promiscuous G protein Galpha15 were grown in Dulbecco's Modified Eagle Media (DMEM), 10% Tet-tested fetal bovine serum (Atlanta Biologicals), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 2 mM glutamine, 1x MEM Non-Essential Amino Acids Solution, 500 ug/ml G418 (Mediatech, Inc., Herndon, VA), 100 ug/mL hygromycin, and 5 ug/mL blasticidin S (Growth Media). Cells for experiments were generally maintained for approximately 15-20 passages. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Potency Determinations
Cells were plated into 384 well, black-walled, clear-bottom poly-D-lysine coated plates (Greiner) at a density of 15,000 cells/20 uL/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media) supplemented with 25 ng/mL tetracycline. Plated cells were incubated overnight at 37 degrees C in the presence of 5% CO2. The following day, plated cells had their medium exchanged to Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES and 2.5 mM probenacid, pH 7.3) using an ELX405 microplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 4.5 uM Fluo 4 AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10 percent (w/v) pluronic acid F-127 and diluted in Assay Buffer for 45 min at 37 degrees C. The dye was then exchanged to Assay Buffer using an ELX405, leaving 20 uL/well and the plates were incubated at room temperature for 10 min prior to assay. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA), and diluted into Assay Buffer to generate a 2x stock. Calcium flux was measured using the Functional Drug Screening System 6000 or 7000 (FDSS6000 or FDSS7000, Hamamatsu, Japan). Baseline readings were taken (10 images at 1 Hz, excitation, 470+/-20 nm, emission, 540+/-30 nm) and then 20 uL/well test compounds were added using the FDSS's integrated pipettor. Approximately 2.5 minutes later an EC20 concentration of glutamate (10 uL of a 5x final concentration) was added followed approximately 2.0 minutes later by an EC80 concentration of glutamate (12 uL of a 5x final concentration).
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). For experiments in which antagonists/potentiators were added, data were again normalized by dividing each point by the fluorescence value at the time point immediately before the agonist addition to correct for any subtle differences in the baseline traces after the compound incubation period. The maximum fluorescence increase beginning after the agonist addition was calculated. Curves were fitted using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ).
Comment
Compounds with average IC50s greater than or equal to 30uM were assigned as 'Outcome' equals 'Inactive'. For compounds with average IC50s less than 30uM, 'Outcome' equals 'Active.' / 'Antagonist' The 'Score' for 'Active' compounds was as follows: IC50 <30 to >= 5uM equals '50' and IC50 <5uM equals '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_IC50_uMStandard deviation for the average IC50 valueFloatμM
3SEM_IC50_uMStandard error mean for the average IC50 valueFloatμM
4Average_%_Glu_MaxAverage percent maximum glutamate response from replicates 1-3Float%
5SD_Average_%_Glu_MaxStandard deviation for the average percent maximum glutamate responseFloat%
6SEM_Average_%_Glu_MaxStandard error mean for the average percent maximum glutamate responseFloat%
7CategoryWhether the compound is categorized as inactive, a potentiator, or an antagonistString
8Value at_0.00152_uM_1 (0.00152μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
9Value at_0.00457_uM_1 (0.00457μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
10Value at_0.0137_uM_1 (0.0137μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
11Value at_0.0412_uM_1 (0.0412μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
12Value at_0.123_uM_1 (0.123μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
13Value at_0.37_uM_1 (0.37μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
14Value at_1.11_uM_1 (1.11μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
15Value at_3.33_uM_1 (3.33μM**)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
18IC50_uM_1IC50 value in micromolar (replicate 1)FloatμM
19%_Glu_Max_1Percent Maximum Glutamate Response (replicate 1)Float%
20Value at_0.00152_uM_2 (0.00152μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
21Value at_0.00457_uM_2 (0.00457μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
22Value at_0.0137_uM_2 (0.0137μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
23Value at_0.0412_uM_2 (0.0412μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
24Value at_0.123_uM_2 (0.123μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
25Value at_0.37_uM_2 (0.37μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
26Value at_1.11_uM_2 (1.11μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
27Value at_3.33_uM_2 (3.33μM**)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
30IC50_uM_2IC50 value in micromolar (replicate 2)FloatμM
31%_Glu_Max_2Percent Maximum Glutamate Response (replicate 2)Float%
32Value at_0.00152_uM_3 (0.00152μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
33Value at_0.00457_uM_3 (0.00457μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
34Value at_0.0137_uM_3 (0.0137μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
35Value at_0.0412_uM_3 (0.0412μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
36Value at_0.123_uM_3 (0.123μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
37Value at_0.37_uM_3 (0.37μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
38Value at_1.11_uM_3 (1.11μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
39Value at_3.33_uM_3 (3.33μM**)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
42IC50_uM_3IC50 value in micromolar (replicate 3)FloatμM
43%_Glu_Max_3Percent Maximum Glutamate Response (replicate 3)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R01 NS031373

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