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BioAssay: AID 1923

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

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: 1923
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4_analog_potency)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-09-04
Modify Date: 2010-07-23

Data Table ( Complete ):           Active    All
Sequence: cholinergic receptor, muscarinic 4 [Rattus norvegicus]
Description ..   

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 32
Depositor Specified Assays
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screenscreening
643Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation Screenother
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.
The purpose of this assay was to test the lead compound for potency against rM4/Gqi5 in a calcium mobilization assay.

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 37degrees 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 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). All compounds showed dose-dependency and therefore were assigned 'Outcome' = 'Active' and 'Score' = '100'.
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Replicate 1First independent determination of PAM EC50FloatμM
2EC50 Replicate 2Second independent determination of PAM EC50FloatμM
3EC50 Replicate 3Third independent determination of PAM EC50FloatμM
4EC50 Mean*Averaged EC50 from Replicates 1-3FloatμM
5EC50 SDStandard Deviation of EC50 Replicates 1-3Float
6EC50 95%CI95% Confidence Intervals for EC50 Replicates 1-3String
7Degrees of FreedomDegrees of Freedom for EC50 Replicates 1-3Integer
8R SquaredR Squared value for EC50 Replicates 1-3Float

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

Data Table (Concise)