Modulation of mGlu3 (mGlu3_GIRK_Schild)
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 ..
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.
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.
mGlu3 Schild Assay - The effect of multiple concentrations of test compound on a glutamate dose response
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mGlu3 GIRK Schild
Culture of the rat mGlu3 GIRK cell line
HEK/GIRK cells stably expressing the M4 muscarinic receptor and rat mGlu3 were grown in 45% Dulbecco's Modified Eagle Media (DMEM), 45% Ham's F12, 10% fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 2 mM glutamine, 600 ng/ml puromycin dihydrochloride (Sigma-Aldrich) and 700 ug/ml G418 (Mediatech, Inc., Herndon, VA). (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.
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). 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 pH 7.3) using an ELX405 uplate washer (BioTek), leaving 20 uL/well, followed by addition of with 20 uL of 330 nM FluoZin-2 AM (Invitrogen, Carlsbad, CA) prepared as a 2.85 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 1 hour at room temperature. 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. Test compounds were prepared in DMSO, and transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA), and diluted into Assay Buffer to generate a 2x stock. Glutamate concentration-responses were diluted in Thallium Buffer (125 mM sodium bicarbonate (added fresh the morning of the experiment), 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES, pH 7.3) at 5x the final concentration to be assayed. Thallium 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 of test compounds or vehicle were added using the FDSS's integrated pipettor. Approximately 2.5 minutes later 10 uL of Thallium Buffer +/- a concentration-response of glutamate was added. After the addition of glutamate, data were collected for an approximately 3 additional min.
Thallium sulfate requires special handling and disposal precautions and investigators are cautioned to contact their Environmental Health and Safety Department to ensure proper procedures are followed.
Data were analyzed using usoft 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 immediately before the agonist addition to correct for any subtle differences in the baseline traces after the compound incubation period. The slope of the fluorescence increase beginning five seconds after thallium/agonist addition and ending fifteen seconds after thallium/agonist addition was calculated. Curves were fitted using a four point logistical equation using XLfit (IDBS, Bridgewater, NJ). Subsequent confirmations of concentration-response parameters were performed using independent serial dilutions of source compounds and data from multiple days experiments were integrated and fit using a four point logistical equation in GraphPad Prism (GraphPad Software, Inc., La Jolla, CA. The log of the dose ratio will be calculated and linear regression performed to determine if responses are consistent with competitive (orthosteric) or noncompetitive (allosteric) interaction.
Compounds showing a Schild slope significantly different from unity (1.0) will be assigned 'Mechanism' = 'Allosteric'.
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