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BioAssay: AID 488969

Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Primary Screening

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by disabling motor impairment due to the loss of substantia nigra dopaminergic neurons involved in modulating function of the basal ganglia (BG). The treatment of PD has traditionally relied on strategies for replacing lost dopamine; Levodopa (L-DOPA), the immediate precursor of dopamine, remains the most effective PD more ..
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 Tested Compounds
 Tested Compounds
All(105152)
 
 
Active(2166)
 
 
Unspecified(102986)
 
 
 Tested Substances
 Tested Substances
All(105158)
 
 
Active(2166)
 
 
Unspecified(102992)
 
 
 Related BioAssays
 Related BioAssays
AID: 488969
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (mGluR8 Primary)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-10-29
Modify Date: 2012-03-24

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 2166
Depositor Specified Assays
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AIDNameTypeProbeComment
623952Activators of the GIRK family of Potassium Channels (minus_mGlu8_GIRK_Confirmatory_CRC)confirmatory
623977Activators of the GIRK family of Potassium Channels (GIRK_1_2_Confirmatory_EP)confirmatory1
623984Activators of the GIRK family of Potassium Channels (hKir2.1_ThalliumFlux_CRC)confirmatory
624042Activators of the GIRK family of Potassium Channels (GIRK1/2_PTX_Treated_CRC)confirmatory
623909Activators of the GIRK family of Potassium Channels (GIRK_Confirmatory_CRC)confirmatory
623911Activators of the GIRK family of Potassium Channels (GIRK1/2_Confirmatory)other
623976Activators of the GIRK family of Potassium Channels (GIRK_1_4_Confirmatory_EP)confirmatory1
623868Development of Subtype-specific Activators of the GIRK family of Potassium Channels (mGlu8_nonGIRK_Counterscreen)other
623974Activators of the GIRK family of Potassium Channels (GIRK2/3_Confirmatory_EP)confirmatory
623983Activators of the GIRK family of Potassium Channels (GIRK2/3_ThalliumFlux_CRC)confirmatory
504480Assay for HTS of Gi/Go-linked GPCRs using mGluR8: Summarysummary
623975Activators of the GIRK family of Potassium Channels (GIRK1/4_ThalliumFlux_CRC)confirmatory
623869Development of Subtype-specific Activators of the GIRK family of Potassium Channels (rmGlu8_Gqi9_Counterscreen)other
623988Activators of the GIRK family of Potassium Channels (hERG_ThalliumFlux_CRC)confirmatory
623932ML297 Competition in Radioligand Binding assays (Ricerca)other
623986Activators of the GIRK family of Potassium Channels (hKV7.4_ThalliumFlux_CRC)confirmatory
743384 On Hold
743385 On Hold
743386 On Hold
743393 On Hold
743419 On Hold
743421 On Hold
743423 On Hold
743425 On Hold
743427 On Hold
743429 On Hold
743431 On Hold
743433 On Hold
743447 On Hold
743462 On Hold
743466 On Hold
Description:
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by disabling motor impairment due to the loss of substantia nigra dopaminergic neurons involved in modulating function of the basal ganglia (BG). The treatment of PD has traditionally relied on strategies for replacing lost dopamine; Levodopa (L-DOPA), the immediate precursor of dopamine, remains the most effective PD drug (1). As the disease progresses, however, L-DOPA efficacy becomes unpredictable and many patients experience debilitating dyskinesias and behavioral disturbances. Thus, novel approaches for symptomatic treatment of PD are desperately needed. New therapeutic agents that bypass the dopamine system have the potential of providing more sustained efficacy and fewer side effects or could allow for co-administration with lower doses of L-DOPA to delay the development of adverse effects.

In recent years, G-protein-linked glutamate receptors, termed metabotropic glutamate receptors (mGluRs) have emerged as exciting new targets for PD treatment (2), particularly mGluR8. mGluR8 does not regulate transmission through the BG in normal animals and the mGluR8 agonist (S)-3,4-DCPG (DCPG) does not have antiparkinsonian activity in acute models of dopamine depletion or dopamine receptor blockade. However, DCPG has robust antiparkinsonian effects in rodent models in which dopamine is depleted or receptors are blocked for more prolonged periods as well as in animals with chronic 6-OHDA-induced lesions of dopamine neurons. Interestingly, DCPG has more robust antiparkinsonian activity than do mGluR4 PAMs or mGluR5 antagonists. Furthermore, mGluR8 is not widely expressed in normal animals (3) and the fact that mGluR8 does not regulate BG or motor function except in animals with chronic parkinsonian motor impairments could provide an ideal profile for a novel antiparkinsonian agent that may be selectively active in PD patients. We and others have found that activities of other mGluR subtypes in the basal ganglia are dramatically regulated by loss of dopamine (4-7).


1. Poewe, W. and Granata, R., In: Movement Disorders: Neurological principles and practice (Watts RL, ed.), 201 (1997).
2. Conn, P. J., Battaglia, G., Marino, M. J., and Nicoletti, F., Nat Rev Neurosci 6 (10), 787 (2005). PMID: 16276355
3. Corti, C. et al., Eur J Neurosci 10 (12), 3629 (1998); Saugstad, J. A. et al., Mol Pharmacol 51 (1), 119 (1997).
4. Poisik, O. V. et al., J Neurosci 23 (1), 122 (2003).
5. Marino, M. J., Awad-Granko, H., Ciombor, K. J., and Conn, P. J., Neuropharmacology 43 (2), 147 (2002).
6. Wittmann, M., Marino, M. J., and Conn, P. J., J Pharmacol Exp Ther 302 (2), 433 (2002).
7. Picconi, B. et al., Brain 125 (Pt 12), 2635 (2002).
Protocol
Using Human Embryonic Kidney Cells (HEKs) containing rat mGluR8, Poly-D-Lysine coated black/clear bottom Greiner 384 well plates, were seeded at 15,000 cells/well and grown overnight at 37 degrees C in 5 percent CO2. The next day, FluoZin2 was added to each well and the plate incubated for 50 minutes at RT. The cells were washed with HBSS + 20mM HEPES, pH 7.4 leaving 20uL residual volume. The cell plate was loaded in to the Hamamatsu FDSS and 5 frames of background were collected before addition of compound at 10uM final concentration. Data collection continued at 2 sec intervals until the addition of EC20 glutamate/thallium stimulus, then the sampling interval changed to 1 sec intervals for a total collection time of 4 min. EC20 and ECMax of glutamate controls were used to calculate the Z prime value for each window as a measure of assay robustness.

The data were processed using an automated data analysis protocol generated with Pipeline Pilot (Accelrys, San Diego, CA) and R statistics package (www.r-project.org). Data were normalized by dividing each value in each trace by the initial value for that trace. For each well, 3 windows were calculated as follows: 1)window 1 was the ratio value 5 seconds post-addition of compound; 2)window 2 was the ratio value 5 seconds pre-addition of EC20/thallium and 3)window 3 was the slope value between 165 and 175 seconds. Thallium flux modulators or "hits" were compounds where Z score > 3 or Z < -3 in Window 3. Windows 1 and 2 were used to evaluate fluorescent and environmentally sensitive compounds.

Compounds denoted as 'hits' were given the score of '100' and outcome of 'active'. All other compounds were denoted as 'inactive' and given a score of '0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Window1ratio value 5 seconds post addition of compoundFloat
2Window2ratio value 5 seconds before addition of EC20 glutamate/thallium stimulusFloat
3Window3slope value between 165 and 175 secondsFloat
4zprime_EC20_ECMAX_Window1Z prime value calculated for EC20 and Ecmax populations using the kinetic readings from window 1Float
5zprime_EC20_ECMAX_Window2Z prime value calculated for EC20 and Ecmax populations using the kinetic readings from window 2Float
6zprime_EC20_ECMAX_Window3Z prime value calculated for EC20 and Ecmax populations using the kinetic readings from window 3Float
7Z_Window1Z score for compound well using window 1Float
8Z_Window2Z score for compound well using window 2Float
9Z_Window3Z score for compound well using window 3Float
10Thallium flux modulatorDesignated 'thallium flux modulator' if the compound's Z score from window 3 is greater than 3String
11VUIDInternal Vanderbilt identifierString
Additional Information
Grant Number: 3R21NS053536-01S1

Data Table (Concise)
Classification
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