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BioAssay: AID 643

Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation 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(48)
 
 
Active(25)
 
 
Inactive(23)
 
 
 Tested Substances
 Tested Substances
All(48)
 
 
Active(25)
 
 
Inactive(23)
 
 
AID: 643
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (VMLSCN00000012)
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-04-02
Modify Date: 2007-11-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 25
Related Experiments
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AIDNameTypeProbeComment
625Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Primary ScreenScreening depositor-specified cross reference
1921Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator: Ancillary ActivityOther depositor-specified cross reference
1923Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog PotencyConfirmatory depositor-specified cross reference
1928Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5Confirmatory depositor-specified cross reference
1929Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Foldshift Selectivity with hM3Confirmatory depositor-specified cross reference
1930Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Fold-shift Selectivity with hM2Confirmatory depositor-specified cross reference
1932Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1Confirmatory depositor-specified cross reference
1938Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): Analog Dose Response with rM4Confirmatory depositor-specified cross reference
1939Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition at rM4Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
449769Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4Confirmatory depositor-specified cross reference
449770Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine with human M4Confirmatory depositor-specified cross reference
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening same project related to Summary assay
449767Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of AcetylcholineOther same project related to Summary assay
588743Chemical optimization of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) SeriesConfirmatory same project related to Summary assay
588744Human M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588745Human M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588746Human M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588747Human M5 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588748Rat M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588749Rat M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588750Rat M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588751Rat M4 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588752Rat M5 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588755Human M4 PAM Extended Characterization Fold ShiftOther same project related to Summary assay
588758ML253 Competition in Radioligand Binding assays (Riserca)Other same project related to Summary assay
623924Chemical 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 Activity (hM1 CounterScreen)Confirmatory same project related to Summary assay
623925Chemical 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 Activity (hM2 CounterScreen)Confirmatory same project related to Summary assay
623926Chemical 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 Activity (hM3 CounterScreen)Confirmatory same project related to Summary assay
623938Chemical 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 Activity (hM4 Calcium Potency)Confirmatory1 same project related to Summary assay
623939Chemical 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 Activity (hM5 CounterScreen)Confirmatory same project related to Summary assay
623940Chemical 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 Activity (rM1 CounterScreen)Confirmatory same project related to Summary assay
623941Chemical 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 Activity (rM2 CounterScreen)Confirmatory same project related to Summary assay
623943Chemical 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 Activity (rM3 CounterScreen)Confirmatory same project related to Summary assay
623945Chemical 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 Activity (rM4 CounterScreen)Confirmatory1 same project related to Summary assay
623946Chemical 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 Activity (rM5 CounterScreen)Confirmatory same project related to Summary assay
623948Chemical 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 Activity (hM4 Fold Shift)Other1 same project related to Summary assay
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 vehicle control sample, DMSO. Compounds selected as 'Outcome' = 'Active' and 'Score' = '100' had slope values greater than the 'Cutoff_Value' calculated as the mean of the vehicle control (DMSO) plus three times the standard deviations.
Comment
These data are from experiments 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.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HEK293
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1SlopeSlope was calculated from a linear fit of normalized fluorescence values versus time for each compound.Float
2R_squaredR_squared reflects the goodness of fit for 'Slope' which resulted from 30 interations or from a chi squared value less than or equal to 0.0001.Float
3NN refers to the number of data points for the negative control (DMSO) on a per plate basis.Integer
4MeanMean refers to the mean of data points for the negative control (DMSO) on a per plate basis.Float
5StdDevStdDev refers to the standard deviation of data points for the negative control (DMSO) on a per plate basis.Float
6Cutoff_ValueCutoff_Value was calculated as 'Mean' plus three times 'StdDev.' 'Slope' greater than 'Cutoff_Value' were selected as 'Active.' Float

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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