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BioAssay: AID 2807

Modulation of the Metabotropic Glutamate Receptor mGluR4: Calcium Assay

Screening Center Name and PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 2807
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (mglur4_Ca_CMN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-05-05

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
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2181Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR8confirmatory
2182Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR7confirmatory
2188Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR2confirmatory
2191Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR6confirmatory
2190Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR3confirmatory
2193Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR1confirmatory
2199Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR5confirmatory
2179Modulation of Metabotropic Glutamate Receptor mGluR4: Rat PAM Fold-Shiftconfirmatory
2183Modulation of the Metabotropic Glutamate Receptor mGluR4: Selectivity at mGluR4confirmatory
2185Modulation of the Metabotropic Glutamate Receptor mGluR4: Rat PAM Potencyconfirmatory
2180Modulation of Metabotropic Glutmate Receptor mGluR4: Human PAM Fold-Shiftconfirmatory
2197Modulation of the Metabotropic Glutamate Receptor mGluR4: Potency at human mGluR4confirmatory
2437Modulation of the Metabotropic Glutamate Receptor mGluR4summary1
Screening Center Name and PI: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters, C. David Weaver
Chemistry Center Name and PI: Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Craig W. Lindsley
Assay Submitter and Institution: Colleen M. Niswender, 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).

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.
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% Dulbecco's Modified Eagle Media (DMEM), 10% 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% 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% 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% (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). For compounds where an EC50 was determined, the 'Activity' was assigned as 'Active' and the 'Score' was assigned as '100'. All other compounds were considered 'Inactive' and assigned a 'Score' of '0'.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Rep1 Conc1 (0.00152μM**)replicate 1 concentration 1Float%
2Rep1 Conc2 (0.00457μM**)replicate 1 concentration 2Float%
3Rep1 Conc3 (0.01372μM**)replicate 1 concentration 3Float%
4Rep1 Conc4 (0.04115μM**)replicate 1 concentration 4Float%
5Rep1 Conc5 (0.12346μM**)replicate 1 concentration 5Float%
6Rep1 Conc6 (0.37037μM**)replicate 1 concentration 6Float%
7Rep1 Conc7 (1.11111μM**)replicate 1 concentration 7Float%
8Rep1 Conc8 (3.33333μM**)replicate 1 concentration 8Float%
9Rep1 Conc9 (10μM**)replicate 1 concentration 9Float%
10Rep1 Conc10 (30μM**)replicate 1 concentration 10Float%
11Rep2 Conc1 (0.00152μM**)replicate 2 concentration 1Float%
12Rep2 Conc2 (0.00457μM**)replicate 2 concentration 2Float%
13Rep2 Conc3 (0.01372μM**)replicate 2 concentration 3Float%
14Rep2 Conc4 (0.04115μM**)replicate 2 concentration 4Float%
15Rep2 Conc5 (0.12346μM**)replicate 2 concentration 5Float%
16Rep2 Conc6 (0.37037μM**)replicate 2 concentration 6Float%
17Rep2 Conc7 (1.11111μM**)replicate 2 concentration 7Float%
18Rep2 Conc8 (3.33333μM**)replicate 2 concentration 8Float%
19Rep2 Conc9 (10μM**)replicate 2 concentration 9Float%
20Rep2 Conc10 (30μM**)replicate 2 concentration 10Float%
21Rep3 Conc1 (0.00152μM**)replicate 3 concentration 1Float%
22Rep3 Conc2 (0.00457μM**)replicate 3 concentration 2Float%
23Rep3 Conc3 (0.01372μM**)replicate 3 concentration 3Float%
24Rep3 Conc4 (0.04115μM**)replicate 3 concentration 4Float%
25Rep3 Conc5 (0.12346μM**)replicate 3 concentration 5Float%
26Rep3 Conc6 (0.37037μM**)replicate 3 concentration 6Float%
27Rep3 Conc7 (1.11111μM**)replicate 3 concentration 7Float%
28Rep3 Conc8 (3.33333μM**)replicate 3 concentration 8Float%
29Rep3 Conc9 (10μM**)replicate 3 concentration 9Float%
30Rep3 Conc10 (30μM**)replicate 3 concentration 10Float%
31EC50*Calculated EC50 value based on best-fit of all replicatesFloatμM
32Lower CL95% Confidence Limit for Bottom of curveFloat
33Upper CL95% Confidence Limit for Top of curveFloat
34BottomBottom of calculated curveFloat
35TopTop of calculated curveFloat
36VUIDVanderbilt University Internal Registration NumberString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NS053536-01

Data Table (Concise)