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BioAssay: AID 623878

Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu2_Selectivity)

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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AID: 623878
Data Source: Vanderbilt Specialized Chemistry Center (Rat_mGlu2_Thallium_Flux_Fold_Shift)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-03-27
Hold-until Date: 2013-03-26
Modify Date: 2013-03-26

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
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AIDNameTypeProbeComment
602453Chemical Optimization of Advanced mGlu4 Lead CandidatesSummary depositor-specified cross reference
623874Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu1_Selectivity)Other same project related to Summary assay
623875Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu4_Potency)Confirmatory1 same project related to Summary assay
623876Chemical Optimization of Advanced mGlu4 Lead Candidates (human_mGlu4_potency)Confirmatory1 same project related to Summary assay
623879Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu3_Selectivity)Other same project related to Summary assay
623889Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu4_Fold_Shift)Other1 same project related to Summary assay
623890Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu5_Selectivity)Other same project related to Summary assay
623891Chemical Optimization of Advanced mGlu4 Lead Candidates (human_mGlu6_Selectivity)Other same project related to Summary assay
623892Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu8_Selectivity)Other same project related to Summary assay
623893Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu7_Selectivity)Other same project related to Summary assay
623906Chemical Optimization of Advanced mGlu4 Lead Candidates (rat_mGlu4_Selectivity)Other1 same project related to Summary assay
Description:
Modulation of the Metabotropic Glutamate Receptor mGlu4: Selectivity at rat mGlu2
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 (mGlus), including the group III mGlus (mGlu4, -7 and -8), play important roles in regulating transmission through the BG and could serve as targets for novel PD therapeutics (3). For instance, mGlu4 activation reduces overactive GABA release at a specific inhibitory BG synapse (12,19,9) and reverses motor deficits in a variety of rodent PD models (12,19,10,8,16).

To more selectively activate mGlu4 and improve upon the pharmacokinetic liabilities of glutamate analogs, we and others have developed novel positive allosteric modulators (PAMs) which potentiate glutamate function at mGlu4 (11,12,14,15,4,21,7,6); several of these tool compounds exhibit antiparkinsonian and neuroprotective effects in multiple rodent PD models (12,1,14,7,6). Unfortunately, many available compounds have lacked the necessary pharmacokinetic properties required for study of mGlu4 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 mGlu4 biology, which will undoubtedly allow the intense study of mGlu4 activation in multiple areas of neuroscience such as psychiatric disorders (18,17), cancer (5), and addiction (2).

Potency and efficacy of compounds will be determined by performing concentration-response curves (CRCs, 10 points, ranging from approximately 30 uM-1 nM at 0.3% final DMSO concentration) at human mGlu4 using a calcium assay in which the cells express the chimeric G protein Gqi5 to couple mGlu4 to calcium mobilization (13,14,7,6). PAMs with EC50 values less than 500 nM versus human mGlu4 will next be evaluated for potency versus rat mGlu4 in a thallium flux assay measuring coupling of rat mGlu4 to G Protein-coupled Inwardly Rectifying Potassium (GIRK) Channels (13,6). Following potency evaluation at human and rat mGlu4, PAMs with EC50 values less than 500 nM at rat mGlu4 will be evaluated for their ability to left-shift the CRC of glutamate for rat mGlu4 in a thallium flux assay (13,14,7,6). PAMs with a fold-shift of the glutamate CRC of > 5 will then be evaluated for Tier 1 DMPK assays including plasma protein binding (PPB), intrinsic clearance, and inhibition of cytochrome p450 enzymes (CYP) (4,6). Next, compounds demonstrating PPB > 1% free and moderate clearance will be evaluated for CNS exposure and Plasma:Brain levels using an in vivo snapshot PK paradigm and will be examined for their selectivity for mGlu4 relative to other mGlu subtypes evaluated (4,6). Novel mGlu4 PAMs showing a CNS exposure > PAM EC50, a Brain:Plasma ratio of >0.5, and at least a 50-fold or greater selectivity versus other mGlu subtypes will next be evaluated in a preclinical model of PD: haloperidol-induced catalepsy (HIC) (20). mGlu4 PAMs demonstrating activity in HIC at a dose of < 30 mg/kg will have their ancillary pharmacology fully evaluated and those compounds that show no significant off-target activity, CaCo permeability > 10 cm/s-6, and suitable solubility will be declared an MLPCN probe. The ultimate goal of this project from the PI's perspective is to generate compounds which should exhibit sufficient potency, efficacy, and pharmacokinetic properties, including brain penetration, to make useful probes to progress mGlu4 biology.

REFERENCES
1. Battaglia, G., C. L. Busceti, et al. (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): 7222-9.
2. Blednov, Y. A., D. Walker, et al. (2004). "Mice lacking metabotropic glutamate receptor 4 do not show the motor stimulatory effect of ethanol." Alcohol 34(2-3): 251-9.
3. Conn, P. J., G. Battaglia, et al. (2005). "Metabotropic glutamate receptors in the basal ganglia motor circuit." Nat Rev Neurosci 6(10): 787-98.
4. Engers, D. W., C. M. Niswender, et al. (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): 4115-8.
5. Iacovelli, L., A. Arcella, et al. (2006). "Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas." J Neurosci 26(32): 8388-97.
6. Jones, C. K., M. Bubser, et al. (2012). "The metabotropic glutamate receptor 4-positive allosteric modulator VU0364770 produces efficacy alone and in combination with L-DOPA or an adenosine 2A antagonist in preclinical rodent models of Parkinson's disease." J Pharmacol Exp Ther 340(2): 404-21.
7. Jones, C. K., D. W. Engers, et al. (2011). "Discovery, synthesis, and structure-activity relationship development of a series of N-4-(2,5-dioxopyrrolidin-1-yl)phenylpicolinamides (VU0400195, ML182): characterization of a novel positive allosteric modulator of the metabotropic glutamate receptor 4 (mGlu(4)) with oral efficacy in an antiparkinsonian animal model." J Med Chem 54(21): 7639-47.
8. Konieczny, J., J. Wardas, et al. (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): 611-20.
9. Macinnes, N. and S. Duty (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): 95-9.
10. MacInnes, N., M. J. Messenger, et al. (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.
11. Maj, M., V. Bruno, et al. (2003). "(-)-PHCCC, a positive allosteric modulator of mGluR4: characterization, mechanism of action, and neuroprotection." Neuropharmacology 45(7): 895-906.
12. Marino, M., O. Valenti, et al. (2003). "Glutamate receptors and Parkinson's disease : opportunities for intervention." Drugs Aging 20(5): 377-97.
13. Niswender, C. M., K. A. Johnson, et al. (2008a). "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.
14. Niswender, C. M., K. A. Johnson, et al. (2008b). "Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4." Mol Pharmacol 74(5): 1345-58.
15. Niswender, C. M., E. P. Lebois, et al. (2008c). "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): 5626-30.
16. Ossowska, K., J. Konieczny, et al. (2007). "An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats." Amino Acids 32(2): 179-88.
17. Stachowicz, K., E. Chojnacka-Wojcik, et al. (2006). "Anxiolytic-like effects of group III mGlu receptor ligands in the hippocampus involve GABAA signaling." Pharmacol Rep 58(6): 820-6.
18. Stachowicz, K., K. Klak, et al. (2004). "Anxiolytic-like effects of PHCCC, an allosteric modulator of mGlu4 receptors, in rats." Eur J Pharmacol 498(1-3): 153-6.
19. Valenti, O., M. J. Marino, et al. (2003). "Group III metabotropic glutamate receptor-mediated modulation of the striatopallidal synapse." J Neurosci 23(18): 7218-26.
20. Varty, G. B., R. A. Hodgson, et al. (2008). "The effects of adenosine A2A receptor antagonists on haloperidol-induced movement disorders in primates." Psychopharmacology (Berl) 200(3): 393-401.
21. Williams, R., C. M. Niswender, et al. (2009). "Positive allosteric modulators of the metabotropic glutamate receptor subtype 4 (mGluR4). Part II: Challenges in hit-to-lead." Bioorg Med Chem Lett 19(3): 962-6.
Protocol
mGlu2 Selectivity
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 microplate 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. Glutamate was 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 selectivity experiments, compounds are tested at a 10 uM final concentration and prepared as 2x stocks in Assay Buffer. 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 test compounds were added using the FDSS's integrated pipettor. Approximately 2.5 minutes later 10 uL of Thallium Buffer +/- agonist was added. After the addition of agonist, 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 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 Microsoft XLfit (IDBS, Bridgewater, NJ).
Comment
Compounds that gave a foldshift of >0.5 and <2.0 were assigned as 'Inactive' and a 'Score' of '0'. Compounds with foldshift >/= 2.0 were considered positive allosteric modulators and assigned an 'Outcome' of 'Active' and a 'Score' of '50'. Compounds with foldshift >/= 5.0 were assigned a 'Score' of '100'. Compounds with foldshift
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Fold_ShiftThe EC50 of the glutamate response in the presence of compound divided by the EC50 of the glutamate response in the presence of vehicleFloat
2EC50_uMEC50 value in micromolar FloatμM
3%_Glu_MaxPercent Maximum Glutamate ResponseFloat%
4%_Glu_MinPercent Maximum Glutamate Response Float%
5SlopeMeasured Concentration Response Curve slope value (replicate 1)Float
6Compound_Concentration_uM_1Concentration of compound tested in assay in micromolarFloatμM
7CategoryWhether the compound is categorized as inactive, a potentiator, or an antagonistString
8Value_at_1000_uM_1 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
9Value_at_200_uM_1 (200μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
10Value_at_40_uM_1 (40μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
11Value_at_8_uM_1 (8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
12Value_at_1.6_uM_1 (1.6μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
13Value_at_0.32_uM_1 (0.32μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
14Value_at_0.064_uM_1 (0.064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
15Value_at_0.0128_uM_1 (0.0128μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
16Value_at_0.00256_uM_1 (0.00256μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
17Value_at_0.000512_uM_1 (0.000512μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
18Value_at_0.0001024_uM_1 (0.0001024μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
19Value_at_1000_uM_2 (1000μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
20Value_at_200_uM_2 (200μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
21Value_at_40_uM_2 (40μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
22Value_at_8_uM_2 (8μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
23Value_at_1.6_uM_2 (1.6μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
24Value_at_0.32_uM_2 (0.32μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
25Value_at_0.064_uM_2 (0.064μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
26Value_at_0.0128_uM_2 (0.0128μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
27Value_at_0.00256_uM_2 (0.00256μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
28Value_at_0.000512_uM_2 (0.000512μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float
29Value_at_0.0001024_uM_2 (0.0001024μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured ECmax value (see Protocol).Float

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

Data Table (Concise)
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