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BioAssay: AID 2197

Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4

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(33)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(36)
 
 
Active(33)
 
 
Inactive(3)
 
 
AID: 2197
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (human mGluR4 PAM potency)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-10
Modify Date: 2010-10-27

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

Gene:GRM4     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 33
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
2185Modulation of the Metabotropic Glutamate Receptor mGluR4: Rat PAM PotencyConfirmatory 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
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. Future compounds developed should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes 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 line creation and culture of the human mGluR4/ Gqi5/CHO line. Human mGluR4 (hmGluR4)/CHO cells were stably transfected with the chimeric G protein Gqi5 (Conklin et al., 1993) in pIRESneo3 (Invitrogen, Carlsbad, CA) and single neomycin-resistant clones were isolated and screened for mGluR4-mediated calcium mobilization using the method described below. hmGluR4/CHO cells were cultured in 90 percent Dulbecco's Modified Eagle Media (DMEM), 10 percent dialyzed fetal bovine serum (FBS), 100 units/ml penicillin/streptomycin, 20 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 20 ug/ml proline, 2 mM glutamine, 400 ug/ml G418 sufate (Mediatech, Inc., Herndon, VA) and 5 nM methotrexate (Calbiochem, EMD Chemicals, Gibbstown, NJ). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.
Potency determinations. Assays were performed within Vanderbilt University's High-Throughput Screening Center. Human mGluR4/Gqi5/CHO cells (30,000 cells/20 ul/well) were plated in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in DMEM containing 10 percent dialyzed FBS, 20 mM HEPES, 100 units/ml penicillin/streptomycin, and 1 mM sodium pyruvate (Plating Medium). The cells were grown overnight at 37 degrees C in the presence of 5 percent CO2. The next day, the medium was removed
and replaced using a Thermo Fisher Combi (Thermo Fisher, Waltham, MA) with 20 uL of 1 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 (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 minutes at 37 degrees C. Dye was removed and replaced with 20 uL of Assay Buffer. Test compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer to generate a 2x stock. Ca2+ flux was measured using the Functional Drug Screening System 6000 (FDSS6000, 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. For concentration-response curve experiments, compounds were serially diluted 1:3 into 10 point concentration response curves in DMSO and were transferred to daughter plates using the Echo. 20 ul of test compounds (2X concentration) were applied and followed 2.5 minutes later by an EC20 concentration of glutamate (10 ul of a 5x final concentration). An EC80 concentration of glutamate was added 2 minutes later (12 ul of a 5x final concentration). Curves were fitted using a four point logistical equation using Microsoft 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).
Compounds with average EC50s greater than or equal to 30uM were assigned as 'Outcome' equals 'Inactive'. For compounds with average EC50s less than 30uM, 'Outcome' equals 'Active.' The 'Score' for 'Active' compounds was as follows EC50 <30 to >= 10uM equals '25', EC50 <10 to >= 1uM equals '50' and EC50 <1uM equals '100'.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
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
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