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BioAssay: AID 651776

Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift Assay

Muscarinic acetylcholine receptors are family A GPCRs comprised of five distinct mammalian subtypes (mAChR1-5 or M1-M5), which are expressed differentially throughout the body and play an important role in a variety of physiological processes. Among the mAChRs, M1 and M4 have been historically considered attractive targets for small molecule treatments of numerous CNS disorders such as more ..
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
AID: 651776
Data Source: Vanderbilt Specialized Chemistry Center (Muscarinic Receptor 5 (M5) Positive Allosteric Modulator ACh..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-14
Hold-until Date: 2013-07-12
Modify Date: 2013-07-12

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: muscarinic acetylcholine receptor M5 [Homo sapiens]
Description ..   
Protein Family: Serpentine type 7TM GPCR chemoreceptor Srx

Gene:CHRM5     Related Protein 3D Structures     More BioActivity Data..
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5Summarydepositor-specified cross reference
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreeningsame project related to Summary assay
2186Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Fold-shift AssayConfirmatorysame project related to Summary assay
2192Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine CompetitionConfirmatorysame project related to Summary assay
2194Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine Competition with AcetylcholineConfirmatorysame project related to Summary assay
2198Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: PAM SAR with Muscarinic M5Othersame project related to Summary assay
2204Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Calcium Flux AssayConfirmatorysame project related to Summary assay
2206Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with Muscarinic M1Othersame project related to Summary assay
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with AcetylcholineScreeningsame project related to Summary assay
Description:
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University

Muscarinic acetylcholine receptors are family A GPCRs comprised of five distinct mammalian subtypes (mAChR1-5 or M1-M5), which are expressed differentially throughout the body and play an important role in a variety of physiological processes. Among the mAChRs, M1 and M4 have been historically considered attractive targets for small molecule treatments of numerous CNS disorders such as Alzheimer's disease and schizophrenia due to their respective localization and involvement in regulation of certain aspects of learning, memory, sleep, motor control, reward, and pain, among others. However, discovery of subtype-selective small molecules has proven highly difficult due to the conservation of the orthosteric binding-site across the mAChRs. This has contributed to the failure of muscarinic agonists in clinical trials and has also hampered pharmacological investigation into the role(s) of each mAChR in basic neurobiology.

Among the mAChRs, M5 has remained perhaps the most challenging to investigate pharmacologically due in part to its extremely low expression level and a complete lack of M5-selective ligands. Interestingly, studies using M5-KO mice suggest that M5 is the sole mediator of acetylcholine-induced cerebrovasodilation, which has led to the hypothesis that an M5 activator would have therapeutic efficacy in treatment of cerebrovascular dementias and ischemic stroke. Furthermore, M5-KO mice show dramatically reduced reward responses to drugs of abuse, consistent with its putative localization on midbrain dopaminergic neurons of the nigrostriatal and mesolimbic pathways. This suggests that M5 antagonism or negative modulation may have utility in treatment of illicit drug addiction and withdrawal. Despite these and other related findings from M5-KO mice, there remains a strong need for small molecule tools to probe M5 function and test M5-related hypotheses in order to advance the state of the mAChR research field and provide critical proof-of-concept studies for drug discovery aims.
Protocol
Assay Info: CHO-K1 cells stably expressing human M5 were loaded with calcium indicator dye (2mM Fluo-4 AM) for 45-60 min at 37 degrees C. Dye was removed and replaced with the appropriate volume of assay buffer, pH 7.4 (1X HBSS (Hanks' Balanced Salt Solution), supplemented with 20 mM HEPES and 2.5 mM probenecid). All compounds were diluted in assay buffer for a 60 uM 2X stock concentration (30 uM final concentration) in 0.6% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine CRC serial dilutions were prepared at a 10X stock solution in assay buffer prior to addition to assay plates. Calcium mobilization was measured at 25 degrees C 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 one of eight concentrations of the Acetylcholine CRC. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. EC50 values for the Acetylcholine CRC alone (i.e. plus Vehicle) and in the presence of a fixed high concentration (30 uM final concentration) of each test compound were determined using GraphPad Prism (4.0c), which fit curves using standard non-linear regression (variable slope).
This compound shifted the EC50 of acetylcholine in the calcium flux assay by greater than 13 fold. The compound was assigned 'Outcome' as 'Active' and 'Score' as '100'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_%_Veh_MaxCalculated Average for Avg. Max vehicle concentrationFloat%
2SD_Average_%_Veh_MaxCalculated Std. Deviation for the Avg. Max vehicle concentrationFloat%
3SEM_Average_%_Veh_MaxCalculated S.E.M. for the Avg. Max Vehicle ConcentrationFloat%
4Average_%_Veh_MinCalculated Average for the Avg. Min Vehicle ConcentrationFloat%
5SD_Average_%_Veh_MinCalculated Stand. Dev for Avg. Min Vehicle ConcentrationFloat%
6SEM_Average_%_Veh_MinCalculated S.E.M for the Avg. Min Vehicle ConcentrationFloat%
7Cmpd Conc1 Rep1 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 1FloatμM
8Cmpd Conc2 Rep1 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 1FloatμM
9Cmpd Conc3 Rep1 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 1FloatμM
10Cmpd Conc4 Rep1 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 1FloatμM
11Cmpd Conc5 Rep1 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 1FloatμM
12Cmpd Conc6 Rep1 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 1FloatμM
13Cmpd Conc7 Rep1 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 1FloatμM
14Cmpd Conc8 Rep1 (1μM**)Value for compound + Ach at 1.0 uM replicate 1FloatμM
15EC50_uM_Rep1Calculated EC50 for replicate 1FloatμM
16Fold_Shift_Rep1Calculated Fold Shift for Replicate 1.Float
17Cmpd Conc1 Rep2 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 2FloatμM
18Cmpd Conc2 Rep2 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 2FloatμM
19Cmpd Conc3 Rep2 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 2FloatμM
20Cmpd Conc4 Rep2 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 2FloatμM
21Cmpd Conc5 Rep2 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 2FloatμM
22Cmpd Conc6 Rep2 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 2FloatμM
23Cmpd Conc7 Rep2 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 2FloatμM
24Cmpd Conc8 Rep2 (1μM**)Value for compound + Ach at 1 uM replicate 2FloatμM
25EC50_uM_Rep2Calculated EC50 for replicate 2FloatμM
26Fold_Shift_Rep2Calculated Fold Shift for Replicate 2Float
27Cmpd Conc1 Rep3 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 3FloatμM
28Cmpd Conc2 Rep3 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 3FloatμM
29Cmpd Conc3 Rep3 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 3FloatμM
30Cmpd Conc4 Rep3 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 3FloatμM
31Cmpd Conc5 Rep3 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 3FloatμM
32Cmpd Conc6 Rep3 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 3FloatμM
33Cmpd Conc7 Rep3 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 3FloatμM
34Cmpd Conc8 Rep3 (1μM**)Value for compound + Ach at 1 uM replicate 3FloatμM
35EC50_uM_Rep3Calculated EC50 for replicate 3FloatμM
36Fold_Shift_Rep3Calculated Fold Shift for replicate 3Float
37Average_EC50_uM*Calculated Avg EC50 for the first 3 replicatesFloatμM
38SD_EC50_uMCalculated Stand. Dev of EC50 for the first 3 replicatesFloatμM
39SEM_EC50_uMCalculated SEM for EC50 for the first 3 replicatesFloatμM
40Average_Fold_ShiftCalculated Average Fold Shift for the first 3 replicatesFloat
41SD_Fold_ShiftCalculated Stand. Dev of Fold Shifts for the first 3 replicatesFloat
42SEM_Fold_ShiftCalculated S.E.M. of Fold shifts for the first 3 replicatesFloat

* Activity Concentration. ** Test Concentration.

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