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BioAssay: AID 2198

Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: PAM SAR with Muscarinic M5

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(18)
 
 
Active(14)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(18)
 
 
Active(14)
 
 
Inactive(4)
 
 
AID: 2198
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (M5 PAM SAR M5)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-10
Modify Date: 2010-02-25

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 14
Related Experiments
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AIDNameTypeComment
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5Summarydepositor-specified cross reference
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with AcetylcholineScreeningdepositor-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
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
651776Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift AssayConfirmatorysame 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
CHO-K1 cells stably transfected with 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 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% DMSO. This stock was then added to the assay plate for a final DMSO concentration of 0.3%. Acetylcholine EC20 was 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 submaximal concentration (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).
For compounds with an average EC50 greater than 30uM, 'Outcome' was assigned as 'Inactive' and 'Score' was assigned as '0'. For compounds with an average EC50 less than 30uM, 'Outcome' was assigned as 'Active'. The 'Score' for compounds with average EC50s greater than or equal to 1uM was assigned as '50' and for less than 1uM as '100'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Replicate 1 QualifierQualifier for Replicate 1 ValueString
2Replicate 1EC50 value from Replicate 1Float
3Replicate 2 QualifierQualifier for Replicate 2 ValueString
4Replicate 2EC50 value from Replicate 2Float
5Replicate 3 QualifierQualifier for Replicate 3String
6Replicate 3EC50 value from Replicate 3Float
7Replicate 4EC50 value from Replicate 4Float
8Replicate 5EC50 value from Replicate 5Float
9EC50 QualifierQualifier for EC50 ValueString
10EC50Average EC50 valueFloat
11EC50 95%CI95% Confidence Interval for the Average EC50String
12R SquaredCalculated R Squared Value for the Average FitString
13VUIDUnique internal registration numberString
Additional Information
Grant Number: MH077606

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