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BioAssay: AID 2185

Modulation of the Metabotropic Glutamate Receptor mGluR4: Rat PAM Potency

The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are more ..
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 Tested Compounds
 Tested Compounds
All(36)
 
 
Active(32)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(36)
 
 
Active(32)
 
 
Inactive(4)
 
 
AID: 2185
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rat mGluR4 PAM potency)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-08
Modify Date: 2010-10-27

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: glutamate receptor, metabotropic 4 precursor [Rattus norvegicus]
Description ..   
Protein Family: 7 transmembrane sweet-taste receptor of 3 GCPR

Gene:GRM4     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 32
Related Experiments
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AIDNameTypeProbeComment
2437Modulation of the Metabotropic Glutamate Receptor mGluR4Summary1 depositor-specified cross reference
2807Modulation of the Metabotropic Glutamate Receptor mGluR4: Calcium AssayConfirmatory depositor-specified cross reference
2179Modulation of Metabotropic Glutamate Receptor mGluR4: Rat PAM Fold-ShiftConfirmatory same project related to Summary assay
2180Modulation of Metabotropic Glutmate Receptor mGluR4: Human PAM Fold-ShiftConfirmatory same project related to Summary assay
2181Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR8Confirmatory same project related to Summary assay
2182Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR7Confirmatory same project related to Summary assay
2183Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR4Confirmatory same project related to Summary assay
2188Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR2Confirmatory same project related to Summary assay
2190Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR3Confirmatory same project related to Summary assay
2191Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR6Confirmatory same project related to Summary assay
2193Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR1Confirmatory same project related to Summary assay
2197Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4Confirmatory same project related to Summary assay
2199Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5Confirmatory same project related to Summary assay
488971Rat PAM Fold-shift Assay with mGluR4 GIRKConfirmatory same project related to Summary assay
Description:
Assay Provider: Colleen Niswender
Assay Provider Affiliation: Vanderbilt University

The primary pathophysiological change giving rise to the symptoms of Parkinson's disease (PD) is a loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) that are involved in modulating the function of basal ganglia (BG) nuclei. Unfortunately, traditional therapies for treatment of PD based on dopamine replacement strategies eventually fail in most patients and are associated with numerous side effects. A great deal of effort has been focused on developing a detailed understanding of the circuitry and function of the BG to develop novel, nondopaminergic, approaches for restoring normal BG function in PD patients. Exciting advances suggest that metabotropic glutamate receptors (mGluRs), including the group III mGluRs (mGluR4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (Conn et al., 2005). For instance, mGluR4 activation reduces overactive GABA release at a specific inhibitory BG synapse (Macinnes and Duty, 2008; Marino et al., 2003; Valenti et al., 2003) and reverses motor deficits in a variety of rodent PD models (Konieczny et al., 2007; MacInnes et al., 2004; Marino et al., 2003; Ossowska et al., 2007; Valenti et al., 2003).

To more selectively activate mGluR4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGluR4 (Engers et al., 2009; Maj et al., 2003; Marino et al., 2003; Niswender et al., 2008a; Niswender et al., 2008b; Williams et al., 2008); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (Battaglia et al., 2006; Marino et al., 2003; Niswender et al., 2008a). Unfortunately, many available compounds have lacked pharmacokinetic properties to make them useful tools for study of mGluR4 function via systemic routes of administration. The probe compound developed here exhibits sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make it a useful compound to progress mGluR4 biology, which will undoubtedly allow the intense study of mGluR4 activation in multiple areas of neuroscience such as psychiatric disorders (Stachowicz et al., 2006; Stachowicz et al., 2004), cancer (Iacovelli et al., 2006), and addiction (Blednov et al., 2004).



References:
1. Battaglia G, Busceti CL, Molinaro G, Biagioni F, Traficante A, Nicoletti F and Bruno V (2006) Pharmacological activation of mGlu4 metabotropic glutamate receptors reduces nigrostriatal degeneration in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. J Neurosci 26(27):7222-7229.
2. Blednov YA, Walker D, Osterndorf-Kahanek E and Harris RA (2004) Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol. Alcohol 34(2-3):251-259.
3. Conn PJ, Battaglia G, Marino MJ and Nicoletti F (2005) Metabotropic glutamate receptors in the basal ganglia motor circuit. Nat Rev Neurosci 6(10):787-798.
4. Engers DW, Niswender CM, Weaver CD, Jadhav S, Menon UN, Zamorano R, Conn PJ, Lindsley CW and Hopkins CR (2009) Synthesis and evaluation of a series of heterobiarylamides that are centrally penetrant metabotropic glutamate receptor 4 (mGluR4) positive allosteric modulators (PAMs). J Med Chem 52(14):4115-4118.
5. Iacovelli L, Arcella A, Battaglia G, Pazzaglia S, Aronica E, Spinsanti P, Caruso A, De Smaele E, Saran A, Gulino A, D'Onofrio M, Giangaspero F and Nicoletti F (2006) Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas. J Neurosci 26(32):8388-8397.
6. Konieczny J, Wardas J, Kuter K, Pilc A and Ossowska K (2007) The influence of group III metabotropic glutamate receptor stimulation by (1S,3R,4S)-1-aminocyclo-pentane-1,3,4-tricarboxylic acid on the parkinsonian-like akinesia and striatal proenkephalin and prodynorphin mRNA expression in rats. Neuroscience 145(2):611-620.
7. Macinnes N and Duty S (2008) Group III metabotropic glutamate receptors act as hetero-receptors modulating evoked GABA release in the globus pallidus in vivo. Eur J Pharmacol 580(1-2):95-99.
8. MacInnes N, Messenger MJ and Duty S (2004) Activation of group III metabotropic glutamate receptors in selected regions of the basal ganglia alleviates akinesia in the reserpine-treated rat. Br J Pharmacol 141(1):15-22.
9. Maj M, Bruno V, Dragic Z, Yamamoto R, Battaglia G, Inderbitzin W, Stoehr N, Stein T, Gasparini F, Vranesic I, Kuhn R, Nicoletti F and Flor PJ (2003) (-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection. Neuropharmacology 45(7):895-906.
10. Marino MJ, Williams DL, Jr., O'Brien JA, Valenti O, McDonald TP, Clements MK, Wang R, DiLella AG, Hess JF, Kinney GG and Conn PJ (2003) Allosteric modulation of group III metabotropic glutamate receptor 4: a potential approach to Parkinson's disease treatment. Proc Natl Acad Sci U S A 100(23):13668-13673.
11. Niswender CM, Johnson KA, Weaver CD, Jones CK, Xiang Z, Luo Q, Rodriguez AL, Marlo JE, de Paulis T, Thompson AD, Days EL, Nalywajko T, Austin CA, Williams MB, Ayala JE, Williams R, Lindsley CW and Conn PJ (2008a) Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol Pharmacol 74(5):1345-1358.
12. Niswender CM, Lebois EP, Luo Q, Kim K, Muchalski H, Yin H, Conn PJ and Lindsley CW (2008b) Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4): Part I. Discovery of pyrazolo[3,4-d]pyrimidines as novel mGluR4 positive allosteric modulators. Bioorg Med Chem Lett 18(20):5626-5630.
13. Ossowska K, Konieczny J, Wardas J, Pietraszek M, Kuter K, Wolfarth S and Pilc A (2007) An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats. Amino Acids 32(2):179-188.
14. Stachowicz K, Chojnacka-Wojcik E, Klak K and Pilc A (2006) Anxiolytic-like effects of group III mGlu receptor ligands in the hippocampus involve GABAA signaling. Pharmacol Rep 58(6):820-826.
15. Stachowicz K, Klak K, Klodzinska A, Chojnacka-Wojcik E and Pilc A (2004) Anxiolytic-like effects of PHCCC, an allosteric modulator of mGlu4 receptors, in rats. Eur J Pharmacol 498(1-3):153-156.
16. Valenti O, Marino MJ, Wittmann M, Lis E, DiLella AG, Kinney GG and Conn PJ (2003) Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse. J Neurosci 23(18):7218-7226.
17. Williams R, Niswender CM, Luo Q, Le U, Conn PJ and Lindsley CW (2008) Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead. Bioorg Med Chem Lett.
Protocol
Cell culture, plating, and dye loading. HEK/GIRK cells stably expressing the M4 muscarinic receptor 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, and 700 ug/ml G418. The rat mGluR4 cell line was prepared by PCR amplification of the entire coding sequence of each receptor and cloning into pIRES puro 3 (Invitrogen). Cloning sites were BamHI/Not I. HEK/GIRK/M4 cells were transfected with 24 ug of DNA and stable transfectants were selected with puromycin. And a monoclonal cell lines was established. Cells 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, and 2 mM glutamine (Growth Media. mGluR/GIRK lines were supplemented with 600 ng/ml puromycin dihydrochloride (Sigma-Aldrich) and 700 ug/ml G418 (Mediatech, Inc., Herndon, VA). Cells for experiments were generally maintained for approximately 15-20 passages; this was particularly important for experiments examining the endogenous alpha2C receptor.

Assays were performed within Vanderbilt University's High-Throughput Screening Center. 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 oC in the presence of 5% CO2. The following day, the medium was removed from the cells and 20 uL/well of 1.7 uM concentration of the indicator dye BTC-AM (Invitrogen; prepared as a stock in DMSO and mixed in a 1:1 ratio with pluronic acid F-127) in Assay Buffer (Hanks Balanced Salt Solution (Invitrogen) containing 20 mM HEPES pH 7.3) was added to the plated cells. Cells were incubated for one hour at room temperature and the dye was replaced with the 20 ul of Assay Buffer.

Test compound preparation. Agonists 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. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO, transferred to daughter plates using the Echo, and diluted in Assay Buffer to a 2X final concentration. Cell plates and compound plates were loaded onto a Hamamatsu FDSS 6000 kinetic imaging plate reader. Appropriate baseline readings were taken (10 images at 1 Hz, excitation, 470+/-20 nm emission, 540+/-30 nm) and test compounds were added. Compounds were added in a 20 ul volume and incubated for approximately 2.5 minutes prior to the addition of 10 ul of Thallium Buffer +/- agonist. After the addition of agonist, data were collected for an additional 2 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 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 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).

For compounds with an average EC50 equal to or greater than 100uM, 'Outcome' was assigned as 'Inactive' and 'Score' as '1'. For compounds with an average EC50 less than 100uM, 'Outcome' was assigned as 'Active.' For compounds with average EC50's less than or equal to 10uM, 'Score' was asssigned as '50'. For compounds with average EC50's less than or equal to 1uM, 'Score was assigned as '100'.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Value_at_0.00151_uM_1 (0.00151μM**)Value for first replicate at 0.00151 uM concentrationFloat
2Value_at_0.00457_uM_1 (0.00457μM**)Value for first replicate at 0.00457 uM concentrationFloat
3Value_at_0.0138_uM_1 (0.0138μM**)Value for first replicate at 0.0138 uM concentrationFloat
4Value_at_0.0407_uM_1 (0.0407μM**)Value for first replicate at 0.0407 uM concentrationFloat
5Value_at_0.123_uM_1 (0.123μM**)Value for first replicate at 0.123 uM concentrationFloat
6Value_at_0.371_uM_1 (0.371μM**)Value for first replicate at 0.371 uM concentrationFloat
7Value_at_1.12_uM_1 (1.12μM**)Value for first replicate at 1.12 uM concentrationFloat
8Value_at_3.31_uM_1 (3.31μM**)Value for first replicate at 3.31 uM concentrationFloat
9Value_at_10.0_uM_1 (10μM**)Value for first replicate at 10.0 uM concentrationFloat
10Value_at_30.1_uM_1 (30.1μM**)Value for first replicate at 30.1 uM concentrationFloat
11EC50_1Calculated EC50 for first replicateFloatμM
12% Glu max_1Calculated percent max for first replicateFloat
13Value_at_0.00151_uM_2 (0.00151μM**)Value for second replicate at 0.00151 uM concentrationFloat
14Value_at_0.00457_uM_2 (0.00457μM**)Value for second replicate at 0.00457 uM concentrationFloat
15Value_at_0.0138_uM_2 (0.0138μM**)Value for second replicate at 0.0138 uM concentrationFloat
16Value_at_0.0407_uM_2 (0.0407μM**)Value for second replicate at 0.0407 uM concentrationFloat
17Value_at_0.123_uM_2 (0.123μM**)Value for second replicate at 0.123 uM concentrationFloat
18Value_at_0.371_uM_2 (0.371μM**)Value for second replicate at 0.371 uM concentrationFloat
19Value_at_1.12_uM_2 (1.12μM**)Value for second replicate at 1.12 uM concentrationFloat
20Value_at_3.31_uM_2 (3.31μM**)Value for second replicate at 3.31 uM concentrationFloat
21Value_at_10.0_uM_2 (10μM**)Value for second replicate at 10.0 uM concentrationFloat
22Value_at_30.1_uM_2 (30.1μM**)Value for second replicate at 30.1 uM concentrationFloat
23EC50_2Calculated EC50 for second replicateFloatμM
24% Glu max_2Calculated percent max for second replicateFloat
25Value_at_0.00151_uM_3 (0.00151μM**)Value for third replicate at 0.00151 uM concentrationFloat
26Value_at_0.00457_uM_3 (0.00457μM**)Value for third replicate at 0.00457 uM concentrationFloat
27Value_at_0.0138_uM_3 (0.0138μM**)Value for third replicate at 0.0138 uM concentrationFloat
28Value_at_0.0407_uM_3 (0.0407μM**)Value for third replicate at 0.0407 uM concentrationFloat
29Value_at_0.123_uM_3 (0.123μM**)Value for third replicate at 0.123 uM concentrationFloat
30Value_at_0.371_uM_3 (0.371μM**)Value for third replicate at 0.371 uM concentrationFloat
31Value_at_1.12_uM_3 (1.12μM**)Value for third replicate at 1.12 uM concentrationFloat
32Value_at_3.31_uM_3 (3.31μM**)Value for third replicate at 3.31 uM concentrationFloat
33Value_at_10.0_uM_3 (10μM**)Value for third replicate at 10.0 uM concentrationFloat
34Value_at_30.1_uM_3 (30.1μM**)Value for third replicate at 30.1 uM concentrationFloat
35EC50_3Calculated EC50 for third replicateFloatμM
36% Glu max_3Calculated percent max for third replicateFloat
37average EC50*Mean EC50 for three replicatesFloatμM
38SD EC50Standard deviation of the EC50 for three replicatesFloatμM
39SEM EC50SEM of the EC50 for three replicatesFloatμM
40Average Glu MaxAverage glu max for three replicatesFloat
41SD Glu MaxStandard deviation of the glu max for three replicatesFloat
42SEM Glu MaxSEM of the glu max for three replicatesFloat
43CategoryAssigned category for the compoundString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NS053536

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