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BioAssay: AID 2186

Discovery of Novel Allosteric Modulators 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
All(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 2186
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M5 PAM ACh foldshift Ca)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-12-08
Modify Date: 2010-07-12

Data Table ( Complete ):           Active    All
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 Compounds: 3
Depositor Specified Assays
AIDNameTypeComment
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5summary
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with Acetylcholinescreening
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
1Veh Conc1 Rep1 (1e-07μM**)Value for Ach at 0.0000001 uM replicate 1Float%
2Veh Conc2 Rep1 (1e-06μM**)Value for Ach at 0.000001 uM replicate 1Float%
3Veh Conc3 Rep1 (1e-05μM**)Value for Ach at 0.00001 uM replicate 1Float%
4Veh Conc4 Rep1 (0.0001μM**)Value for Ach at 0.0001 uM replicate 1Float%
5Veh Conc5 Rep1 (0.001μM**)Value for Ach at 0.001 uM replicate 1Float%
6Veh Conc6 Rep1 (0.01μM**)Value for Ach at 0.01 uM replicate 1Float%
7Veh Conc7 Rep1 (0.1μM**)Value for Ach at 0.1 uM replicate 1Float%
8Veh Conc8 Rep1 (1μM**)Value for Ach at 1 uM replicate 1Float%
9Veh Conc1 Rep2 (1e-07μM**)Value for Ach at 0.0000001 uM replicate 2Float%
10Veh Conc2 Rep2 (1e-06μM**)Value for Ach at 0.000001 uM replicate 2Float%
11Veh Conc3 Rep2 (1e-05μM**)Value for Ach at 0.00001 uM replicate 2Float%
12Veh Conc4 Rep2 (0.0001μM**)Value for Ach at 0.0001 uM replicate 2Float%
13Veh Conc5 Rep2 (0.001μM**)Value for Ach at 0.001 uM replicate 2Float%
14Veh Conc6 Rep2 (0.01μM**)Value for Ach at 0.01 uM replicate 2Float%
15Veh Conc7 Rep2 (0.1μM**)Value for Ach at 0.1 uM replicate 2Float%
16Veh Conc8 Rep2 (1μM**)Value for Ach at 1 uM replicate 2Float%
17Veh Conc1 Rep3 (1e-07μM**)Value for Ach at 0.0000001 uM replicate 3Float%
18Veh Conc2 Rep3 (1e-06μM**)Value for Ach at 0.000001 uM replicate 3Float%
19Veh Conc3 Rep3 (1e-05μM**)Value for Ach at 0.00001 uM replicate 3Float%
20Veh Conc4 Rep3 (0.0001μM**)Value for Ach at 0.0001 uM replicate 3Float%
21Veh Conc5 Rep3 (0.001μM**)Value for Ach at 0.001 uM replicate 3Float%
22Veh Conc6 Rep3 (0.01μM**)Value for Ach at 0.01 uM replicate 3Float%
23Veh Conc7 Rep3 (0.1μM**)Value for Ach at 0.1 uM replicate 3Float%
24Veh Conc8 Rep3 (1μM**)Value for Ach at 1 uM replicate 3Float%
25Veh Conc1 Rep4 (1e-07μM**)Value for Ach at 0.0000001 uM replicate 4Float%
26Veh Conc2 Rep4 (1e-06μM**)Value for Ach at 0.000001 uM replicate 4Float%
27Veh Conc3 Rep4 (1e-05μM**)Value for Ach at 0.00001 uM replicate 4Float%
28Veh Conc4 Rep4 (0.0001μM**)Value for Ach at 0.0001 uM replicate 4Float%
29Veh Conc5 Rep4 (0.001μM**)Value for Ach at 0.001 uM replicate 4Float%
30Veh Conc6 Rep4 (0.01μM**)Value for Ach at 0.01 uM replicate 4Float%
31Veh Conc7 Rep4 (0.1μM**)Value for Ach at 0.1 uM replicate 4Float%
32Veh Conc8 Rep4 (1μM**)Value for Ach at 1 uM replicate 4Float%
33Cmpd Conc1 Rep1 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 1Float%
34Cmpd Conc2 Rep1 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 1Float%
35Cmpd Conc3 Rep1 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 1Float%
36Cmpd Conc4 Rep1 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 1Float%
37Cmpd Conc5 Rep1 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 1Float%
38Cmpd Conc6 Rep1 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 1Float%
39Cmpd Conc7 Rep1 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 1Float%
40Cmpd Conc1 Rep1 (1μM**)Value for compound + Ach at 1.0 uM replicate 1Float%
41Cmpd Conc1 Rep2 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 2Float%
42Cmpd Conc2 Rep2 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 2Float%
43Cmpd Conc3 Rep2 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 2Float%
44Cmpd Conc4 Rep2 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 2Float%
45Cmpd Conc5 Rep2 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 2Float%
46Cmpd Conc6 Rep2 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 2Float%
47Cmpd Conc7 Rep2 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 2Float%
48Cmpd Conc8 Rep2 (1μM**)Value for compound + Ach at 1 uM replicate 2Float%
49Cmpd Conc1 Rep3 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 3Float%
50Cmpd Conc2 Rep3 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 3Float%
51Cmpd Conc3 Rep3 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 3Float%
52Cmpd Conc4 Rep3 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 3Float%
53Cmpd Conc5 Rep3 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 3Float%
54Cmpd Conc6 Rep3 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 3Float%
55Cmpd Conc7 Rep3 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 3Float%
56Cmpd Conc8 Rep3 (1μM**)Value for compound + Ach at 1 uM replicate 3Float%
57Cmpd Conc1 Rep4 (1e-07μM**)Value for compound + Ach at 0.0000001 uM replicate 4Float%
58Cmpd Conc2 Rep4 (1e-06μM**)Value for compound + Ach at 0.000001 uM replicate 4Float%
59Cmpd Conc3 Rep4 (1e-05μM**)Value for compound + Ach at 0.00001 uM replicate 4Float%
60Cmpd Conc4 Rep4 (0.0001μM**)Value for compound + Ach at 0.0001 uM replicate 4Float%
61Cmpd Conc5 Rep4 (0.001μM**)Value for compound + Ach at 0.001 uM replicate 4Float%
62Cmpd Conc6 Rep4 (0.01μM**)Value for compound + Ach at 0.01 uM replicate 4Float%
63Cmpd Conc7 Rep4 (0.1μM**)Value for compound + Ach at 0.1 uM replicate 4Float%
64Cmpd Conc8 Rep4 (1μM**)Value for compound + Ach at 1 uM replicate 4Float%
65Veh BottomCalculated bottom for Ach controlFloat
66Veh TopCalculated top for Ach controlFloat
67Veh LogEC50Calculated logEC50 for Ach controlFloat
68Veh HillslopeCalculated Hill slope for Ach controlFloat
69Veh EC50Calculated EC50 for Ach controlFloatμM
70Veh Std Error BottomStd Error of bottom for Ach controlFloat
71Veh Std Error TopStd Error of top for Ach controlFloat
72Veh Std Error LogEC50Std Error of LogEC50 for Ach controlFloat
73Veh Std Error HillslopeStd Error of Hill slope for Ach controlFloat
74Veh 95%CI Bottom95% confidence intervals for bottom for Ach controlString
75Veh 95%CI Top95% confidence intervals for top for Ach controlString
76Veh 95%CI LogEC5095% confidence intervals for LogEC50 for Ach controlString
77Veh 95%CI Hillslope95% confidence intervals for Hill slope for Ach controlString
78Veh 95%CI EC5095% confidence intervals for EC50 for Ach controlString
79Veh Degrees FreedomDegrees of freedom for EC50 calculation for Ach controlInteger
80Veh R squaredR-squared for EC50 calculation for Ach controlFloat
81Veh Abs Sum SquaresAbsolute sum of squares for EC50 calculation for Ach controlFloat
82Cmpd BottomCalculated bottom for compound + ACh controlFloat
83Cmpd TopCalculated top for compound + ACh controlFloat
84Cmpd LogEC50Calculated logEC50 for compound + ACh controlFloat
85Cmpd HillslopeCalculated Hill slope for compound + ACh controlFloat
86Cmpd EC50*Calculated EC50 for compound + ACh controlFloatμM
87Cmpd Std Error BottomStd Error of bottom for compound + ACh controlFloat
88Cmpd Std Error TopStd Error of top for compound + ACh controlFloat
89Cmpd Std Error LogEC50Std Error of LogEC50 for compound + ACh controlFloat
90Cmpd Std Error HillslopeStd Error of Hill slope for compound + ACh controlFloat
91Cmpd 95%CI Bottom95% confidence intervals for bottom for compound + ACh controlString
92Cmpd 95%CI Top95% confidence intervals for top for compound + ACh controlString
93Cmpd 95%CI LogEC5095% confidence intervals for LogEC50 for compound + ACh controlString
94Cmpd 95%CI Hillslope95% confidence intervals for Hill slope for compound + ACh controlString
95Cmpd 95%CI EC5095% confidence intervals for EC50 for compound + ACh controlString
96Cmpd Degrees FreedomDegrees of freedom for EC50 calculation for compound + ACh controlInteger
97Cmpd R squaredR-squared for EC50 calculation for compound + ACh controlFloat
98Cmpd Abs Sum SquaresAbsolute sum of squares for EC50 calculation for compound + ACh controlFloat

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

Data Table (Concise)
Classification
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