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BioAssay: AID 588745

Human M3 PAM Extended Characterization CounterScreen (CRC)

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 more ..
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 Tested Compounds
 Tested Compounds
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Inactive(9)
 
 
 Tested Substances
 Tested Substances
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Inactive(9)
 
 
AID: 588745
Data Source: Vanderbilt Specialized Chemistry Center (hM3_PAM_EC_CounterScreen)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-30
Hold-until Date: 2012-10-25
Modify Date: 2012-10-27

Data Table ( Complete ):           All
Target
Sequence: muscarinic acetylcholine receptor M3 [Homo sapiens]
Description ..   
Protein Family: Serpentine type 7TM GPCR chemoreceptor Srx

Gene:CHRM3     Related Protein 3D Structures     More BioActivity Data..
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
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 ActivitysummaryDiscovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM).
Description:
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. 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.
Protocol
hM3 Muscarinic Receptor CounterScreen

Cell line creation and culture of the human M3/CHO line.
Human M3 (hM3) CHO cells were a gift from the laboratory of Allan Levey (Emory University). hM3/CHO cells were cultured in Ham's F-12; 10% FBS, 20mM HEPES, 50mug/mL G418 (Mediatech, Inc., Herndon, VA). All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise noted.

Potency determinations.

Assays were performed within the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center. Human M3/CHO cells (15,000 cells/20 mul/well) were plated in black-walled, clear-bottomed, TC treated, 384 well plates (Greiner Bio-One, Monroe, North Carolina) in Ham's F-12, 10% FBS, 20 mM HEPES. The cells were grown overnight at 37 degrees C in the presence of 5% CO2. The next day, the medium was removed and replaced with 20 microL of 2.3 microM Fluo-4, AM (Invitrogen, Carlsbad, CA) prepared as a 2.3 mM stock in DMSO and mixed in a 1:1 ratio with 10% (w/v) pluronic acid F-127 and diluted in Assay Buffer (Hank's balanced salt solution, 20 mM HEPES and 2.5 mM Probenecid (Sigma-Aldrich, St. Louis, MO)) for 45 minutes at 37 degrees C.
Dye was removed and replaced with 20 microL of Assay Buffer. Test compounds were transferred to daughter plates using an Echo acoustic plate reformatter (Labcyte, Sunnyvale, CA) and then diluted into Assay Buffer to generate a 2x stock in 0.6% DMSO (0.3% final).

Acetylcholine EC20 and 80 were prepared at a 5X stock solution in assay buffer prior to addition to assay plates. Ca2+ mobilization was measured at 37 degrees C using a Functional Drug Screening System 6000 (FDSS6000, Hamamatsu, Japan) kinetic plate reader according to the following protocol. Cells were preincubated with test compound (or vehicle) for 144 seconds prior to the addition of an EC20 concentration of the agonist, acetylcholine (ACh). 86 seconds after this addition, an EC80 concentration of ACh was added. Control wells also received a maximal ACh concentration (1 mM) for eventual response normalization. The signal amplitude was first normalized to baseline and then as a percentage of the maximal response to acetylcholine. Microsoft XLfit (IDBS, Bridgewater, NJ) was utilized for curves fitting and EC50 value determination using a four point logistical equation.

Compounds showing dose-dependency were assigned 'Outcome' = 'Active', EC50='Value', and % ACh max='Value".

Compound concentrations (log [M]):

-9.29,-8.82,-8.34,-7.86,-7.39,-6.91,-6.43,-5.95,-5.48,-5.00,-4.52
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Potency*Potency value in micromolarFloatμM
2Potency Upper CLPotency Upper Confidence Limit in micromolarFloatμM
3Potency Lower CLPotency Lower Confidence Limit in micromolarFloatμM
4Ach MaxPercent Maximum Acetylcholine ResponseFloat%
5Ach Max Upper CLPercent Maximum Acetylcholine Response Upper Confidence LimitFloat%
6Ach Max Lower CLPercent Maximum Acetylcholine Response Lower Confidence LimitFloat%
7Value_at_0.000508_uM (0.000508μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
8Value_at_0.001524_uM (0.001524μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
9Value_at_0.004572_uM (0.004572μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
10Value_at_0.013717_uM (0.013717μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
11Value_at_0.041152_uM (0.041152μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
12Value_at_0.123457_uM (0.123457μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
13Value_at_0.37037_uM (0.37037μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
14Value_at_1.111111_uM (1.11111μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
15Value_at_3.333333_uM (3.33333μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
16Value_at_10_uM (10μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
17Value_at_30_uM (30μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
18Value_at_0.000623_uM (0.000623μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
19Value_at_0.001869_uM (0.001869μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
20Value_at_0.005606_uM (0.005606μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
21Value_at_0.016818_uM (0.016818μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
22Value_at_0.050453_uM (0.050453μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
23Value_at_0.151358_uM (0.151358μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
24Value_at_0.454074_uM (0.454074μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
25Value_at_1.362222_uM (1.36222μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
26Value_at_4.086667_uM (4.08667μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
27Value_at_12.26_uM (12.26μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
28Value_at_36.78_uM (36.78μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
29Value_at_0.000634_uM (0.000634μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
30Value_at_0.001902_uM (0.001902μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
31Value_at_0.005706_uM (0.005706μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
32Value_at_0.017119_uM (0.017119μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
33Value_at_0.051358_uM (0.051358μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
34Value_at_0.154074_uM (0.154074μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
35Value_at_0.462222_uM (0.462222μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
36Value_at_1.386667_uM (1.38667μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
37Value_at_4.16_uM (4.16μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
38Value_at_12.48_uM (12.48μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
39Value_at_37.44_uM (37.44μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
40Value_at_0.000767_uM (0.000767μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
41Value_at_0.002301_uM (0.002301μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
42Value_at_0.006904_uM (0.006904μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
43Value_at_0.020713_uM (0.020713μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
44Value_at_0.06214_uM (0.06214μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
45Value_at_0.18642_uM (0.18642μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
46Value_at_0.559259_uM (0.559259μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
47Value_at_1.677778_uM (1.67778μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
48Value_at_5.033333_uM (5.03333μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
49Value_at_15.1_uM (15.1μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float
50Value_at_45.3_uM (45.3μM**)Normalized fluorescence corrected for baseline and expressed as percent of measured Ach Max value (see Protocol).Float

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

Data Table (Concise)
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