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BioAssay: AID 2199

Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5

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(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(1)
 
 
Inactive(1)
 
 
AID: 2199
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (mglur4 PAM at rmGluR5)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-11
Modify Date: 2010-10-27

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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
743419 On Hold
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
2197Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4Confirmatory 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
Basic protocol:
1. Cells are plated at a density of 20,000 cells/20 ul well 1 day prior to assay in Assay Media (DMEM with 20mM HEPES, 10% dialyzed FBS, 1mM sodium pyruvate).
2. The following day, media is removed from the cells.
3. 20 ul/well of Fluo-4 AM dye (1 uM) in Assay Buffer (HBSS + 2.5mM probenecid) is added.
4. The plates are incubated for 45 min to one hour at RT.
5. Dye is removed from the cells.
6. 20 ul of assay buffer is added and removed. 20 ul of assay buffer is added.
8. Plates are loaded into a Hamamatsu FDSS.
9. Compounds are tested for selectivity at a 10 uM final concentration (prepared as a 2X stock, 20 ul added per well) and the agonist is added at a 5X concentration (10 ul of agonist added per well, 50 uL 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) 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). 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 max fluorescence increase beginning after the agonist addition was calculated. Curves were fitted using a four point logistical equation using Microsoft XLfit (IDBS, Bridgewater, NJ).
This compound was not active as a positive allosteric modulator of mGluR5 and was assigned an 'Outcome' of 'Inactive' and a 'Score' of '0'. Furthermore, the 3-fold decrease in glutamate EC50 in the presence of compound would suggest antagonistic modulation.
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
1GluRep1_1000_uM (1000μM**)Value for replicate 1 for glutamate-only at 1000 uM GluFloat
2GluRep2_1000_uM (1000μM**)Value for replicate 2 for glutamate-only at 1000 uM GluFloat
3GluRep3_1000_uM (1000μM**)Value for replicate 3 for glutamate-only at 1000 uM GluFloat
4GluRep4_1000_uM (1000μM**)Value for replicate 4 for glutamate-only at 1000 uM GluFloat
5AvgGlu_1000_uMAverage for glutamate-only across all replicates at 1000 uM GluFloat
6CompoundGluRep1_1000_uM (1000μM**)Value for replicate 1 for glutamate + compound at 1000 uM GluFloat
7CompoundGluRep2_1000_uM (1000μM**)Value for replicate 2 for glutamate + compound at 1000 uM GluFloat
8AvgCompoundGlu_1000_uMAverage for glutamate + compound across all replicates at 1000 uM GluFloat
9GluRep1_199_uM (199μM**)Value for replicate 1 for glutamate-only at 199 uM GluFloat
10GluRep2_199_uM (199μM**)Value for replicate 2 for glutamate-only at 199 uM GluFloat
11GluRep3_199_uM (199μM**)Value for replicate 3 for glutamate-only at 199 uM GluFloat
12GluRep4_199_uM (199μM**)Value for replicate 4 for glutamate-only at 199 uM GluFloat
13AvgGlu_199_uMAverage for glutamate-only across all replicates at 199 uM GluFloat
14CompoundGluRep1_199_uM (199μM**)Value for replicate 1 for glutamate + compound at 199 uM GluFloat
15CompoundGluRep2_199_uM (199μM**)Value for replicate 2 for glutamate + compound at 199 uM GluFloat
16AvgCompoundGlu_199_uMAverage for glutamate + compound across all replicates at 199 uM GluFloat
17GluRep1_39.8_uM (39.8μM**)Value for replicate 1 for glutamate-only at 39.8 uM GluFloat
18GluRep2_39.8_uM (39.8μM**)Value for replicate 2 for glutamate-only at 39.8 uM GluFloat
19GluRep3_39.8_uM (39.8μM**)Value for replicate 3 for glutamate-only at 39.8 uM GluFloat
20GluRep4_39.8_uM (39.8μM**)Value for replicate 4 for glutamate-only at 39.8 uM GluFloat
21AvgGlu_39.8_uMAverage for glutamate-only across all replicates at 39.8 uM GluFloat
22CompoundGluRep1_39.8_uM (39.8μM**)Value for replicate 1 for glutamate + compound at 39.8 uM GluFloat
23CompoundGluRep2_39.8_uM (39.8μM**)Value for replicate 2 for glutamate + compound at 39.8 uM GluFloat
24AvgCompoundGlu_39.8_uMAverage for glutamate + compound across all replicates at 39.8 uM GluFloat
25GluRep1_7.94_uM (7.94μM**)Value for replicate 1 for glutamate-only at 7.94 uM GluFloat
26GluRep2_7.94_uM (7.94μM**)Value for replicate 2 for glutamate-only at 7.94 uM GluFloat
27GluRep3_7.94_uM (7.94μM**)Value for replicate 3 for glutamate-only at 7.94 uM GluFloat
28GluRep4_7.94_uM (7.94μM**)Value for replicate 4 for glutamate-only at 7.94 uM GluFloat
29AvgGlu_7.94_uMAverage for glutamate-only across all replicates at 7.94 uM GluFloat
30CompoundGluRep1_7.94_uM (7.94μM**)Value for replicate 1 for glutamate + compound at 7.94 uM GluFloat
31CompoundGluRep2_7.94_uM (7.94μM**)Value for replicate 2 for glutamate + compound at 7.94 uM GluFloat
32AvgCompoundGlu_7.94_uMAverage for glutamate + compound across all replicates at 7.94 uM GluFloat
33GluRep1_1.58_uM (1.58μM**)Value for replicate 1 for glutamate-only at 1.58 uM GluFloat
34GluRep2_1.58_uM (1.58μM**)Value for replicate 2 for glutamate-only at 1.58 uM GluFloat
35GluRep3_1.58_uM (1.58μM**)Value for replicate 3 for glutamate-only at 1.58 uM GluFloat
36GluRep4_1.58_uM (1.58μM**)Value for replicate 4 for glutamate-only at 1.58 uM GluFloat
37AvgGlu_1.58_uMAverage for glutamate-only across all replicates at 1.58 uM GluFloat
38CompoundGluRep1_1.58_uM (1.58μM**)Value for replicate 1 for glutamate + compound at 1.58 uM GluFloat
39CompoundGluRep2_1.58_uM (1.58μM**)Value for replicate 2 for glutamate + compound at 1.58 uM GluFloat
40AvgCompoundGlu_1.58_uMAverage for glutamate + compound across all replicates at 1.58 uM GluFloat
41GluRep1_0.323_uM (0.323μM**)Value for replicate 1 for glutamate-only at 0.323 uM GluFloat
42GluRep2_0.323_uM (0.323μM**)Value for replicate 2 for glutamate-only at 0.323 uM GluFloat
43GluRep3_0.323_uM (0.323μM**)Value for replicate 3 for glutamate-only at 0.323 uM GluFloat
44GluRep4_0.323_uM (0.323μM**)Value for replicate 4 for glutamate-only at 0.323 uM GluFloat
45AvgGlu_0.323_uMAverage for glutamate-only across all replicates at 0.323 uM GluFloat
46CompoundGluRep1_0.323_uM (0.323μM**)Value for replicate 1 for glutamate + compound at 0.323 uM GluFloat
47CompoundGluRep2_0.323_uM (0.323μM**)Value for replicate 2 for glutamate + compound at 0.323 uM GluFloat
48AvgCompoundGlu_0.323_uMAverage for glutamate + compound across all replicates at 0.323 uM GluFloat
49GluRep1_0.0645_uM (0.0645μM**)Value for replicate 1 for glutamate-only at 0.0645 uM GluFloat
50GluRep2_0.0645_uM (0.0645μM**)Value for replicate 2 for glutamate-only at 0.0645 uM GluFloat
51GluRep3_0.0645_uM (0.0645μM**)Value for replicate 3 for glutamate-only at 0.0645 uM GluFloat
52GluRep4_0.0645_uM (0.0645μM**)Value for replicate 4 for glutamate-only at 0.0645 uM GluFloat
53AvgGlu_0.0645_uMAverage for glutamate-only across all replicates at 0.0645 uM GluFloat
54CompoundGluRep1_0.0645_uM (0.0645μM**)Value for replicate 1 for glutamate + compound at 0.0645 uM GluFloat
55CompoundGluRep2_0.0645_uM (0.0645μM**)Value for replicate 2 for glutamate + compound at 0.0645 uM GluFloat
56AvgCompoundGlu_0.0645_uMAverage for glutamate + compound across all replicates at 0.0645 uM GluFloat
57GluRep1_0.0128_uM (0.0128μM**)Value for replicate 1 for glutamate-only at 0.0128 uM GluFloat
58GluRep2_0.0128_uM (0.0128μM**)Value for replicate 2 for glutamate-only at 0.0128 uM GluFloat
59GluRep3_0.0128_uM (0.0128μM**)Value for replicate 3 for glutamate-only at 0.0128 uM GluFloat
60GluRep4_0.0128_uM (0.0128μM**)Value for replicate 4 for glutamate-only at 0.0128 uM GluFloat
61AvgGlu_0.0128_uMAverage for glutamate-only across all replicates at 0.0128 uM GluFloat
62CompoundGluRep1_0.0128_uM (0.0128μM**)Value for replicate 1 for glutamate + compound at 0.0128 uM GluFloat
63CompoundGluRep2_0.0128_uM (0.0128μM**)Value for replicate 2 for glutamate + compound at 0.0128 uM GluFloat
64AvgCompoundGlu_0.0128_uMAverage for glutamate + compound across all replicates at 0.0128 uM GluFloat
65GluRep1_0.00257_uM (0.00257μM**)Value for replicate 1 for glutamate-only at 0.00257 uM GluFloat
66GluRep2_0.00257_uM (0.00257μM**)Value for replicate 2 for glutamate-only at 0.00257 uM GluFloat
67GluRep3_0.00257_uM (0.00257μM**)Value for replicate 3 for glutamate-only at 0.00257 uM GluFloat
68GluRep4_0.00257_uM (0.00257μM**)Value for replicate 4 for glutamate-only at 0.00257 uM GluFloat
69AvgGlu_0.00257_uMAverage for glutamate-only across all replicates at 0.00257 uM GluFloat
70CompoundGluRep1_0.00257_uM (0.00257μM**)Value for replicate 1 for glutamate + compound at 0.00257 uM GluFloat
71CompoundGluRep2_0.00257_uM (0.00257μM**)Value for replicate 2 for glutamate + compound at 0.00257 uM GluFloat
72AvgCompoundGlu_0.00257_uMAverage for glutamate + compound across all replicates at 0.00257 uM GluFloat
73GluRep1_0.000512_uM (0.000512μM**)Value for replicate 1 for glutamate-only at 0.000512 uM GluFloat
74GluRep2_0.000512_uM (0.000512μM**)Value for replicate 2 for glutamate-only at 0.000512 uM GluFloat
75GluRep3_0.000512_uM (0.000512μM**)Value for replicate 3 for glutamate-only at 0.000512 uM GluFloat
76GluRep4_0.000512_uM (0.000512μM**)Value for replicate 4 for glutamate-only at 0.000512 uM GluFloat
77AvgGlu_0.000512_uMAverage for glutamate-only across all replicates at 0.000512 uM GluFloat
78CompoundGluRep1_0.000512_uM (0.000512μM**)Value for replicate 1 for glutamate + compound at 0.000512 uM GluFloat
79CompoundGluRep2_0.000512_uM (0.000512μM**)Value for replicate 2 for glutamate + compound at 0.000512 uM GluFloat
80AvgCompoundGlu_0.000512_uMAverage for glutamate + compound across all replicates at 0.000512 uM GluFloat
81GluRep1_0.000102_uM (0.000102μM**)Value for replicate 1 for glutamate-only at 0.000102 uM GluFloat
82GluRep2_0.000102_uM (0.000102μM**)Value for replicate 2 for glutamate-only at 0.000102 uM GluFloat
83GluRep3_0.000102_uM (0.000102μM**)Value for replicate 3 for glutamate-only at 0.000102 uM GluFloat
84GluRep4_0.000102_uM (0.000102μM**)Value for replicate 4 for glutamate-only at 0.000102 uM GluFloat
85AvgGlu_0.000102_uMAverage for glutamate-only across all replicates at 0.000102 uM GluFloat
86CompoundGluRep1_0.000102_uM (0.000102μM**)Value for replicate 1 for glutamate + compound at 0.000102 uM GluFloat
87CompoundGluRep2_0.000102_uM (0.000102μM**)Value for replicate 2 for glutamate + compound at 0.000102 uM GluFloat
88AvgCompoundGlu_0.000102_uMAverage for glutamate + compound across all replicates at 0.000102 uM GluFloat
89GluEC50Calculated Glutamate-only EC50FloatμM
90GluEmaxCalculated Glutamate-only EmaxFloat
91GluEminCalculated Glutamate-only EminFloat
92foldshiftCalculated EC50 fold-shift between glutamate-only experiment and glutamate+compound experimentFloat
93CompoundGluEC50*Calculated Glutamate+compound EC50FloatμM
94CompoundGluEmaxCalculated Glutamate+compound EmaxFloat
95CompoundGluEminCalculated Glutamate+compound EminFloat

* 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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