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BioAssay: AID 625

Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Primary Screen

The focus of this screening campaign was to identify highly selective small molecules that act as allosteric agonists of the M4 muscarinic receptor. The assay utilized thallium influx through G-protein Inwardly Rectifying K+ (GIRK) channels as a measure of M4 activation. Compounds were tested at 10uM final concentration against Human Embryonic Kidney (HEK) 293 cells stably expressing cDNAs for more ..
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 Tested Compounds
 Tested Compounds
All(12364)
 
 
Active(69)
 
 
Inactive(12295)
 
 
 Tested Substances
 Tested Substances
All(12369)
 
 
Active(69)
 
 
Inactive(12300)
 
 
 Related BioAssays
 Related BioAssays
AID: 625
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (VMLSCN00000002)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-03-21
Modify Date: 2007-09-19

Data Table ( Complete ):           Active    All
BioActive Compounds: 69
Depositor Specified Assays
AIDNameTypeComment
643Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation Screenother
2616Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activitysummary
Description:
Assay Provider: Colleen Niswender
Assay Provider Affliation: Vanderbilt University
Grant Title: Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor
Grant Number: 1 X01 MH077607-1

The focus of this screening campaign was to identify highly selective small molecules that act as allosteric agonists of the M4 muscarinic receptor. The assay utilized thallium influx through G-protein Inwardly Rectifying K+ (GIRK) channels as a measure of M4 activation. Compounds were tested at 10uM final concentration against Human Embryonic Kidney (HEK) 293 cells stably expressing cDNAs for GIRK 1, GIRK 2, and the M4 muscarinic receptor. The Hamamatsu FDSS 6000 plate reader was used to collect kinetic fluorescence intensities during treatment with the test compound. The muscarinic agonist carbachol was the positive control, and DMSO, the compound vehicle, was used as the negative control.
Protocol
Assay Protocol:
M4/GIRK expressing HEK293 cells were plated at 15,000 cells per well in Dulbecco's modified medium (DMEM) containing 10% dialyzed fetal bovine serum (FBS), 100units/mL penicillin/streptomycin, and 20mM HEPES. 384-well, black walled, clear bottom, poly-D-lysine coated plates were used. After overnight incubation at 37degreesC in the presence of 5% CO2, the medium was removed and BTC-AM (Molecular Probes) dye in assay buffer (Hanks Balanced Salt Solution (HBSS)) was added. After 1hour, the dye was replaced with HBSS. Test compounds were prepared in thallium assay buffer (125mM sodium gluconate, 1mM magnesium sulfate, 1.8mM calcium gluconate, 5mM glucose, 12mM thallium sulfate, 10mM HEPES, pH 7.3). The compounds were added to the cell plate by the Hamamatsu FDSS. Each cell plate was imaged at 1Hz for 10seconds, 10uL of test compound was added, and the cell plate was imaged at 1Hz for 2minutes total sampling time.

Data Processing:
Each kinetic trace was normalized to the initial fluorescence intensity to correct for dye loading of the cells. The ratios were used to calculate the slope of the response after compound addition (time 15 to 24 seconds). Slope values used resulted from a linear fit with 30 iterations or with a chi squared value less than or equal to 0.0001. The slope for each test compound response was compared to the sample population treated as a Gaussian distribution. Compounds selected as 'Outcome' = 'Active' and 'Score' = 100 had slope values that differed from the mean sample distribution at 99.7% confidence level.
Comment
These data are from a primary screen where the number of independent measurements is one (n=1). Possible artifacts of this assay include, but are not limited to dust in or on the microtiter plate, compounds that fluoresce, compounds that interact with BTC-AM, and compounds that cause nonspecific influx of thallium.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1SlopeSlope calculated from a linear fit of normalized fluorescence values versus time for a well.Float
2PosNPosN refers to the number of data points for the positive controls from the assay plate.Integer
3PosAvgPosAvg refers to the average of the positive controls from the assay plate.Float
4PosSDPosSD refers to the standard deviation of the positive controls from the assay plate.Float
5NegNNegN refers to the number of data points for the negative controls from the assay plate.Integer
6NegAvgNegAvg refers to the average of the negative controls from the assay plate. Float
7NegSDNegSD refers to the standard deviation of the negative controls from the assay plate.Float
8zPrime zPrime refers to the Zprime value of the assay plate which was calculated as 1-(3*(PosSD + NegSD)/ ABS(PosAvg -NegAvg)).Float

Data Table (Concise)
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