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BioAssay: AID 449767

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine

Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) ..more
 Tested Compounds
 Tested Compounds
 Tested Substances
 Tested Substances
AID: 449767
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4 PAM Ca ACh Foldshift Summary)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-07-23

Data Table ( Complete ):           Active    All
Sequence: muscarinic acetylcholine receptor M4 [Rattus norvegicus]
Description ..   

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 22
Depositor Specified Assays
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenscreeningDiscovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen
623Voltage-Dependent Potassium Channel Beta Subunit (KvBeta) Positive Modulator: Primary ScreenscreeningDiscovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation Screen
2616Chemical optimization of in vitro pharmacology and DMPK properties of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) Series with Greatly Improved Human Receptor Activitysummary
Assay Provider: Colleen Niswender
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM)
Grant Number: MH077607-1

To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems. Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia. In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M2 and M3 mAChRs. Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. Clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent. In follow-up studies in rats, xanomeline displayed an antipsychotic-like profile comparable to clozapine. However, a long standing question concerned whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia. This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction. These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression, suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients. However, multiple studies suggest that M1 may also play an important role in the antipsychotic effects of mAChR agonists and that the relative contributions of M1 and M4 to the antipsychotic efficacy of xanomeline or antipsychotic-like effects of this compound in animal models are not known. However, highly selective centrally penetrant activators of either M1 or M4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors.
Assay Info: CHO-K1 cells stably expressing rat M4 and Gqi5 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). The degree of potentiation or foldshift of the ACh CRC induced by the test compound was calculated by dividing the EC50 for ACh plus vehicle by the EC50 for ACh in the presence of test compound. Foldshift values less than 3 were considered 'Inactive' with a 'Score' of '0'. Foldshift values of greater than 3 were assigned as 'Active.' For values of 3-30, a 'Score' of '50' was assigned and for values greater than 30 a 'Score' of '100' was assigned.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1VUIDVanderbilt internal identifierString
2rM4 Foldshift Rep1 DescriptorDescriptor for Replicate 1String
3rM4 Foldshift Rep1 (30μM**)Value for Replicate 1FloatμM
4rM4 Foldshift Rep2 DescriptorDescriptor for Replicate 2String
5rM4 Foldshift Rep2 (30μM**)Value for Replicate 2FloatμM
6rM4 Foldshift Rep3 DescriptorDescriptor for Replicate 3String
7rM4 Foldshift Rep3 (30μM**)Value for Replicate 3FloatμM
8rM4 Foldshift Rep4 DescriptorDescriptor for Replicate 4String
9rM4 Foldshift Rep4 (30μM**)Value for Replicate 4FloatμM
10rM4 Foldshift Rep5 DescriptorDescriptor for Replicate 5String
11rM4 Foldshift Rep5 (30μM**)Value for Replicate 5FloatμM
12rM4 Foldshift Rep6 DescriptorDescriptor for Replicate 6String
13rM4 Foldshift Rep6 (30μM**)Value for Replicate 6FloatμM
14rM4 Foldshift Mean DescriptorDescriptor for Mean ValueString
15rM4 Foldshift Mean (30μM**)Mean Value of all ReplicatesFloatμM

** Test Concentration.
Additional Information
Grant Number: MH077607-1

Data Table (Concise)