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BioAssay: AID 2430

Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-response with M2

Selective M1 activation is an attractive therapeutic approach for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia and a number of other CNS disorders. Until recently, no highly selective M1 activators existed, and those that claimed to be highly M1 selective were either not centrally penetrant or possessed significant ancillary pharmacology which prohibited their use as more ..
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Inactive(4)
 
 
AID: 2430
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M1 Pam Ca CRC M2)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-02-25
Modify Date: 2010-03-24

Data Table ( Complete ):           View All Data
Target
Sequence: muscarinic acetylcholine receptor M2 [Homo sapiens]
Description ..   
Protein Family: S100P-binding protein

Gene:CHRM2     Related Protein 3D Structures     More BioActivity Data..
Tested Compounds:
Related Experiments
AIDNameTypeProbeComment
2543Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM SummarySummary2 depositor-specified cross reference
2626Discovery of novel allosteric modulators of the M1 Muscarinic receptor: PAM Activity with AcetylcholineScreening depositor-specified cross reference
2651Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SARConfirmatory depositor-specified cross reference
2425Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M1Confirmatory same project related to Summary assay
2428Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M3Confirmatory same project related to Summary assay
2433Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M5Other same project related to Summary assay
2434Discovery of novel allosteric modulators of the M1 muscarinic receptor: Acetylcholine Fold-shift Activity of PAMSConfirmatory same project related to Summary assay
2438Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M4Confirmatory same project related to Summary assay
Description:
Selective M1 activation is an attractive therapeutic approach for the treatment of cognitive impairment, Alzheimer's disease, schizophrenia and a number of other CNS disorders. Until recently, no highly selective M1 activators existed, and those that claimed to be highly M1 selective were either not centrally penetrant or possessed significant ancillary pharmacology which prohibited their use as probes to study M1 receptor function. We have identified that different M1 PAM chemotypes display different modes of activity on downstream receptor signaling. Thus, all allosteric M1 activation is not equivalent, and additional tool compounds representing diverse chemotypes are required to truly dissect and study M1 function in the CNS.
Protocol
Assay Info: CHO-K1 cells stably transfected with human M2/Gqi5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37C. Dye was removed and replaced with assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were serially diluted in assay buffer for a final 2X stock in 0.6 percent DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3 percent. Acetylcholine (ACh) submax concentration (ca. EC20) was prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25C using a FLEXstation II (Molecular Devices, Sunnyvale, CA) according to the following protocol. Cells were preincubated with test compound (or vehicle) for 1.5 min prior to the addition of the agonist, acetylcholine. Cells were then stimulated for 50 sec with a submaximal ACh concentration (ca. EC20). The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. EC50 values for each compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope). None of the compounds were active against human M2/Gqi5 in the calcium flux assay at a compound concentration of 30uM. All compounds were assigned and 'Outcome' of 'Inactive' and a 'Score' of '0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Rep1 Conc1 (30μM**)replicate 1 at 30uM compound concentrationFloat%
2Rep1 Conc2 (10μM**)replicate 1 at 10uM compound concentrationFloat%
3Rep1 Conc3 (3μM**)replicate 1 at 3uM compound concentrationFloat%
4Rep1 Conc4 (1μM**)replicate 1 at 1uM compound concentrationFloat%
5Rep1 Conc5 (0.3μM**)replicate 1 at 0.3uM compound concentrationFloat%
6Rep1 Conc6 (0.1μM**)replicate 1 at 0.1uM compound concentrationFloat%
7Rep1 Conc7 (0.03μM**)replicate 1 at 0.03uM compound concentrationFloat%
8Rep1 Conc8 (0.01μM**)replicate 1 at 0.01uM compound concentrationFloat%
9Rep2 Conc1 (30μM**)replicate 2 at 30uM compound concentrationFloat%
10Rep2 Conc2 (10μM**)replicate 2 at 10uM compound concentrationFloat%
11Rep2 Conc3 (3μM**)replicate 2 at 3uM compound concentrationFloat%
12Rep2 Conc4 (1μM**)replicate 2 at 1uM compound concentrationFloat%
13Rep2 Conc5 (0.3μM**)replicate 2 at 0.3uM compound concentrationFloat%
14Rep2 Conc6 (0.1μM**)replicate 2 at 0.1uM compound concentrationFloat%
15Rep2 Conc7 (0.03μM**)replicate 2 at 0.03uM compound concentrationFloat%
16Rep2 Conc8 (0.01μM**)replicate 2 at 0.01uM compound concentrationFloat%
17VUIDinternal identifierInteger

** Test Concentration.
Additional Information
Grant Number: MH077606

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