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BioAssay: AID 651935

Discovery of Selective Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor (M5) in CHO cells expressing Rattus Norvegicus M4 receptor

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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AID: 651935
Data Source: Vanderbilt Specialized Chemistry Center (Selectivity Studies for Muscarinic Receptor 5 (M5) PAM in Ra..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-12-12
Hold-until Date: 2013-07-12
Modify Date: 2013-07-12

Data Table ( Complete ):           All
Target
Tested Compound:
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 rat M1, human M2/Gqi5, human M3, rat M4/Gqi5, or 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).

These compounds had EC50s less than 10 micromolar for the muscarinic M5 receptor in the calcium flux assay, and therefore, 'Outcome' was assigned as 'Active'. Compound SID 85285464 had no selectivity preference for M5 compared to the other subtypes and was assigned 'Score' of '50'. Compound SID 85285486 was more than 60-fold selective for M5 over the other subtypes, and thus assigned a 'Score' of '100'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Average_EC50_uM_rM4*Calculated Avg EC50FloatμM
2Result_Category_rM4String
3Value_at_30_uM_1_rM4 (30μM**)Avg Value of compound + Ach at 30uMFloatμM
4Value_at_10_uM_1_rM4 (10μM**)Avg. Value of compound + Ach at 10 uMFloatμM
5Value_at_3_uM_1_rM4 (3μM**)Avg Value of compound + Ach at 3 uMFloatμM
6Value_at_1_uM_1_rM4 (1μM**)Avg Value of compound + Ach at 1 uMFloatμM
7Value_at_0.3_uM_1_rM4 (0.3μM**)Avg Value of compound + Ach at 0.3 uMFloatμM
8Value_at_0.1_uM_1_rM4 (0.1μM**)Avg. Value of compound + Ach at 0.1 uMFloatμM
9Value_at_0.03_uM_1_rM4 (0.03μM**)Avg. Value of compound + Ach at 0.3 uMFloatμM
10Value_at_0.01_uM_1_rM4 (0.01μM**)Avg. Value of compound + Ach at 0.01 uMFloatμM
11Value_at_uM_0.003_rM4 (0.003μM**)Avg. Value of compound + Ach at 0.003 UmFloatμM
12Value_at_0.001_uM_1_rM4 (0.001μM**)Avg. Value of compound + Ach at 0.001 uMFloatμM

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

Data Table (Concise)
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