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BioAssay: AID 449765

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) : Activity with human M4

Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) ..more
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AID: 449765
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (hM4 Calcium CRC Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
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: 4
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.
Data collected by Millipore GPCR Profiler Screening

Assay Design
Percentage activations were determined by comparing well data to Emax values upon addition of reference agonists after five minute incubation of vehicle or Vanderbilt compounds. These samples were run using a 'Single Addition' assay protocol for the first and second assay run.

Compound Preparation
Master stock solution
Unless specified otherwise, all sample compounds are diluted in 100% anhydrous DMSO including all serial dilutions. If sample compounds were provided in a different solvent all master stock dilutions are performed in the specified solvent. All control wells contain identical solvent final concentrations as sample compound wells.

Compound plate for assay
Sample compounds were transferred from a master stock solution into a daughter plate that was used in the assay. Each sample compound was diluted into assay buffer (1x HBSS with 20mM HEPES and 2.5mM Probenecid) at an appropriate concentration to obtain final concentrations specified by Vanderbilt University.

Calcium Flux Assay
Vanderbilt University compounds were added to the assay plate in order to achieve an eight-point, four-fold serial dose response with a top concentration of 30uM (concentrations described here reflect the final concentration during the addition of EC20 ACh). Reference agonist was handled as mentioned above serving as assay control. The reference agonist was handled as described above for Emax. Read for 180 seconds using the FLIPRTETRA. At the completion of the first 'Single Addition' assay run, assay plate is removed from the FLIPRTetra and placed at 25 degrees C for 7 minutes. Read for 180 seconds using the FLIPRTETRA (This assay added EC20 Acetylcholine to the respective wells then fluorescence measurements were collected to determine Vanderbilt compounds increasing the g-protein activation relative to EC20 ACh).

Data Processing
All plates were subjected to appropriate baseline corrections. Once baseline corrections were processed, maximum fluorescence values were exported and data manipulated to calculate percentage activation, percentage inhibition, Z', EC50, and IC50. Dose response curves were generated using GraphPad Prism. The curves were fit by utilizing Sigmodial dose response (variable slope)fitting with the bottom parameter fixed at '0.' Compounds with fits having R squared greater than 0.8 were considered 'Active' and assigned a 'Score' of '100.' 'Inactive' compounds were scored as '0.'
Result Definitions
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OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Rep1 Conc1 (29.9916μM**)replicate 1 at compound concentration 1Float%
2Rep1 Conc2 (7.49894μM**)replicate 1 at compound concentration 2Float%
3Rep1 Conc3 (1.87499μM**)replicate 1 at compound concentration 3Float%
4Rep1 Conc4 (0.468813μM**)replicate 1 at compound concentration 4Float%
5Rep1 Conc5 (0.11722μM**)replicate 1 at compound concentration 5Float%
6Rep1 Conc6 (0.029309μM**)replicate 1 at compound concentration 6Float%
7Rep1 Conc7 (0.007328μM**)replicate 1 at compound concentration 7Float%
8Rep1 Conc8 (0.001832μM**)replicate 1 at compound concentration 8Float%
9Rep2 Conc1 (29.9916μM**)replicate 2 at compound concentration 1Float%
10Rep2 Conc2 (7.49894μM**)replicate 2 at compound concentration 2Float%
11Rep2 Conc3 (1.87499μM**)replicate 2 at compound concentration 3Float%
12Rep2 Conc4 (0.468813μM**)replicate 2 at compound concentration 4Float%
13Rep2 Conc5 (0.11722μM**)replicate 2 at compound concentration 5Float%
14Rep2 Conc6 (0.029309μM**)replicate 2 at compound concentration 6Float%
15Rep2 Conc7 (0.007328μM**)replicate 2 at compound concentration 7Float%
16Rep2 Conc8 (0.001832μM**)replicate 2 at compound concentration 8Float%
17Bottombottom of fitted curve using all replicatesFloat
18Toptop of fitted curve using all replicatesFloat
19LogEC50log of the EC50 valueFloat
20Hillslopehillslope from fitted curveFloat
21EC50*calculated EC50 value based on all replictesFloatμM
22Std Error Bottomstandard error of bottom of fitted curveFloat
23Std Error Topstandard error of top of fitted curveFloat
24Std Error LogEC50standard error of logEC0 valueFloat
25Std Error Hillslopestandard error of hillslopeFloat
2695%CI Bottom95% confidence intervals for bottom of fitted curve String
2795%CI Top95% confidence intervals for top of fitted curve String
2895%CI LogEC5095% confidence intervals of logEC50 valueString
2995%CI Hillslope95% confidence intervals for hillslopeString
3095%CI EC5095% confidence intervals for EC50 valueString
31Deg Freedomdegrees of freedomInteger
32R squaredfit statistic Float
33VUIDVanderbilt internal identifierString

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

Data Table (Concise)