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BioAssay: AID 2438

Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M4

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(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
AID: 2438
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M1 PAM Ca CRC M4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2010-02-26
Modify Date: 2010-03-26

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2626Discovery of novel allosteric modulators of the M1 Muscarinic receptor: PAM Activity with Acetylcholinescreening
2543Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Summarysummary
2651Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SARconfirmatory
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 rat M4/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). Compound dose-response curves with R2 > 0.5 were assigned as 'Active.' The 'Score' was assigned as '100' for compounds with EC50 < 1uM and as '50' for compounds with EC50 > 1uM. All other compounds were assigned as 'Inactive' with a 'Score' of '0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
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%
17Rep3 Conc1 (30μM**)replicate 3 at 30uM compound concentrationFloat%
18Rep3 Conc2 (10μM**)replicate 3 at 10uM compound concentrationFloat%
19Rep3 Conc3 (3μM**)replicate 3 at 3uM compound concentrationFloat%
20Rep3 Conc4 (1μM**)replicate 3 at 1uM compound concentrationFloat%
21Rep3 Conc5 (0.3μM**)replicate 3 at 0.3uM compound concentrationFloat%
22Rep3 Conc6 (0.1μM**)replicate 3 at 0.1uM compound concentrationFloat%
23Rep3 Conc7 (0.03μM**)replicate 3 at 0.03uM compound concentrationFloat%
24Rep3 Conc8 (0.01μM**)replicate 3 at 0.01uM compound concentrationFloat%
25BottomBottom fitted value for curveFloat
26TopTop fitted value for curveFloat
27LogEC50Log of EC50 value in uMFloat
28HillslopeHillslopeFloat
29EC50*Calculated EC50 value using all replicates to fit the curveFloatμM
30Std Error BottomStandard error for bottom fitted valueFloat
31Std Error TopStandard error for top fitted valueFloat
32Std Error LogEC50Standard error of LogEC50 valueFloat
33Std Error HillslopeStandard error of HillslopeFloat
3495%CI Bottom95% Confidence Interval for bottom fitted valueString
3595%CI Top95% Confidence Interval for top fitted valueString
3695%CI LogEC5095% Confidence Interval for LogEC50 valueString
3795%CI Hillslope95% Confidence Interval for HillslopeString
3895%CI EC5095% Confidence Interval for fitted EC50 valueString
39Deg FreedomDegrees of freedomFloat
40R squaredR squared fit statisticFloat
41VUIDinternal identifierInteger

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH077606

Data Table (Concise)
Classification
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