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BioAssay: AID 651878

Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: CRC 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(12)
 
 
Probe(1)
 
 
Active(9)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(12)
 
 
Probe(1)
 
 
Active(9)
 
 
Inactive(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 651878
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: 2012-12-06
Hold-until Date: 2013-07-12
Modify Date: 2013-07-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 9
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 2.5 min and then stimulated with the EC20 of acetylcholine for 50 seconds. 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' (PAM).
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average_EC50_uM*Calculated Avg EC50 for the 3 replicatesFloatμM
2SD_EC50_uMCalculated Stand. Dev of EC50 for the 3 replicatesFloatμM
3SEM_EC50_uMCalculated SEM for EC50 for the 3 replicatesFloatμM
4Average_%_ACh_MaxCalculated Average for Avg. Max Ach concentrationFloat%
5SD_Average_%_ACh_MaxCalculated Std. Deviation for the Avg. Max Ach concentrationFloat%
6SEM_Average_%_ACh_MaxCalculated S.E.M. for the Avg. Max Ach ConcentrationFloat%
7Result_CategoryString
8Value_at_30_uM_1 (31.62μM**)Value with M5 for replicate 1 at 0.01 micromolarFloatμM
9Value_at_10_uM_1 (10μM**)Value with M5 for replicate 1 at 0.0316 micromolarFloatμM
10Value_at_3_uM_1 (3.16μM**)Value with M5 for replicate 1 at 0.1 micromolarFloatμM
11Value_at_1_uM_1 (1μM**)Value with M5 for replicate 1 at 0.316 micromolarFloatμM
12Value_at_0.3_uM_1 (0.32μM**)Value with M5 for replicate 1 at 1 micromolarFloatμM
13Value_at_0.1_uM_1 (0.1μM**)Value with M5 for replicate 1 at 3.16 micromolarFloatμM
14Value_at_0.03_uM_1 (0.03μM**)Value with M5 for replicate 1 at 10 micromolarFloatμM
15Value_at_0.01_uM_1 (0.01μM**)Value with M5 for replicate 1 at 31.6 micromolarFloatμM
16EC50_uM_1Calculate EC50 for Replicate 1FloatμM
17%_ACh_Max_1Calculated Max Ach Concentration for Replicate 1Float%
18Value_at_30_uM_2 (31.62μM**)Value with M5 for replicate 2 at 0.01 micromolarFloatμM
19Value_at_10_uM_2 (10μM**)Value with M5 for replicate 2 at 0.0316 micromolarFloatμM
20Value_at_3_uM_2 (3.16μM**)Value with M5 for replicate 2 at 0.1 micromolarFloatμM
21Value_at_1_uM_2 (1μM**)Value with M5 for replicate 2 at 0.316 micromolarFloatμM
22Value_at_0.3_uM_2 (0.32μM**)Value with M5 for replicate 2 at 1 micromolarFloatμM
23Value_at_0.1_uM_2 (0.1μM**)Value with M5 for replicate 2 at 3.16 micromolarFloatμM
24Value_at_0.03_uM_2 (0.03μM**)Value with M5 for replicate 2 at 10 micromolarFloatμM
25Value_at_0.01_uM_2 (0.01μM**)Value with M5 for replicate 2 at 31.6 micromolarFloatμM
26EC50_uM_2Calculate EC50 for Replicate 2FloatμM
27%_ACh_Max_2Calculated Max Ach Concentration for Replicate 2Float%
28Value_at_30_uM_3 (31.62μM**)Value with M5 for replicate 3 at 0.01 micromolarFloatμM
29Value_at_10_uM_3 (10μM**)Value with M5 for replicate 3 at 0.0316 micromolarFloatμM
30Value_at_3_uM_3 (3.16μM**)Value with M5 for replicate 3 at 0.1 micromolarFloatμM
31Value_at_1_uM_3 (1μM**)Value with M5 for replicate 3 at 0.316 micromolarFloatμM
32Value_at_0.3_uM_3 (0.32μM**)Value with M5 for replicate 3 at 1 micromolarFloatμM
33Value_at_0.1_uM_3 (0.1μM**)Value with M5 for replicate 3 at 3.16 micromolarFloatμM
34Value_at_0.03_uM_3 (0.03μM**)Value with M5 for replicate 3 at 10 micromolarFloatμM
35Value_at_0.01_uM_3 (0.01μM**)Value with M5 for replicate 3 at 31.6 micromolarFloatμM
36EC50_uM_3Calculate EC50 for Replicate 3FloatμM
37%_ACh_Max_3Calculated Max Ach Concentration for Replicate 3Float%

* Activity Concentration. ** Test Concentration.

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