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BioAssay: AID 1932

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1

Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM) ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Inactive(6)
 
 
 Tested Substances
 Tested Substances
All(6)
 
 
Inactive(6)
 
 
AID: 1932
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4 analog foldshift selectivity rM1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2009-09-04
Modify Date: 2010-07-23

Data Table ( Complete ):           All
Target
Sequence: cholinergic receptor, muscarinic 1 [Rattus norvegicus]
Description ..   
Protein Family: Serpentine type 7TM GPCR chemoreceptor Srx

Gene:CHRM1     Related Protein 3D Structures     More BioActivity Data..
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
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
Description:
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.
Protocol
The purpose of this assay was to test the lead compound for fold-shift potency using acetylcholine against human M1 in a calcium mobilization assay.

Assay Info: CHO-K1 cells stably expressing rat M1 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 absence of left-shift of the Acetylcholine CRC in the presense of test compound demonstrates lack of PAM activity at this off-target muscarinic receptor subtypes.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Veh rM1 Value 1 Rep 1 (1e-07μM**)value from vehicle control for rM1 cells at concentration 1 for replicate 1Float
2Veh rM1 Value 2 Rep 1 (1e-06μM**)value from vehicle control for rM1 cells at concentration 2 for replicate 1Float
3Veh rM1 Value 3 Rep 1 (1e-05μM**)value from vehicle control for rM1 cells at concentration 3 for replicate 1Float
4Veh rM1 Value 4 Rep 1 (0.0001μM**)value from vehicle control for rM1 cells at concentration 4 for replicate 1Float
5Veh rM1 Value 5 Rep 1 (0.001μM**)value from vehicle control for rM1 cells at concentration 5 for replicate 1Float
6Veh rM1 Value 6 Rep 1 (0.01μM**)value from vehicle control for rM1 cells at concentration 6 for replicate 1Float
7Veh rM1 Value 7 Rep 1 (0.1μM**)value from vehicle control for rM1 cells at concentration 7 for replicate 1Float
8Veh rM1 Value 8 Rep 1 (1μM**)value from vehicle control for rM1 cells at concentration 8 for replicate 1Float
9Cmpd rM1 Value 1 Rep 1 (1e-07μM**)value from test compound for rM1 cells at concentration 1 for replicate 1Float
10Cmpd rM1 Value 2 Rep 1 (1e-06μM**)value from test compound for rM1 cells at concentration 2 for replicate 1Float
11Cmpd rM1 Value 3 Rep 1 (1e-05μM**)value from test compound for rM1 cells at concentration 3 for replicate 1Float
12Cmpd rM1 Value 4 Rep 1 (0.0001μM**)value from test compound for rM1 cells at concentration 4 for replicate 1Float
13Cmpd rM1 Value 5 Rep 1 (0.001μM**)value from test compound for rM1 cells at concentration 5 for replicate 1Float
14Cmpd rM1 Value 6 Rep 1 (0.01μM**)value from test compound for rM1 cells at concentration 6 for replicate 1Float
15Cmpd rM1 Value 7 Rep 1 (0.1μM**)value from test compound for rM1 cells at concentration 7 for replicate 1Float
16Cmpd rM1 Value 8 Rep 1 (1μM**)value from test compound for rM1 cells at concentration 8 for replicate 1Float
17Veh rM1 Value 1 Rep 2 (1e-07μM**)value from vehicle control for rM1 cells at concentration 1 for replicate 2Float
18Veh rM1 Value 2 Rep 2 (1e-06μM**)value from vehicle control for rM1 cells at concentration 2 for replicate 2Float
19Veh rM1 Value 3 Rep 2 (1e-05μM**)value from vehicle control for rM1 cells at concentration 3 for replicate 2Float
20Veh rM1 Value 4 Rep 2 (0.0001μM**)value from vehicle control for rM1 cells at concentration 4 for replicate 2Float
21Veh rM1 Value 5 Rep 2 (0.001μM**)value from vehicle control for rM1 cells at concentration 5 for replicate 2Float
22Veh rM1 Value 6 Rep 2 (0.01μM**)value from vehicle control for rM1 cells at concentration 6 for replicate 2Float
23Veh rM1 Value 7 Rep 2 (0.1μM**)value from vehicle control for rM1 cells at concentration 7 for replicate 2Float
24Veh rM1 Value 8 Rep 2 (1μM**)value from vehicle control for rM1 cells at concentration 8 for replicate 2Float
25Cmpd rM1 Value 1 Rep 2 (1e-07μM**)value from test compound for rM1 cells at concentration 1 for replicate 2Float
26Cmpd rM1 Value 2 Rep 2 (1e-06μM**)value from test compound for rM1 cells at concentration 2 for replicate 2Float
27Cmpd rM1 Value 3 Rep 2 (1e-05μM**)value from test compound for rM1 cells at concentration 3 for replicate 2Float
28Cmpd rM1 Value 4 Rep 2 (0.0001μM**)value from test compound for rM1 cells at concentration 4 for replicate 2Float
29Cmpd rM1 Value 5 Rep 2 (0.001μM**)value from test compound for rM1 cells at concentration 5 for replicate 2Float
30Cmpd rM1 Value 6 Rep 2 (0.01μM**)value from test compound for rM1 cells at concentration 6 for replicate 2Float
31Cmpd rM1 Value 7 Rep 2 (0.1μM**)value from test compound for rM1 cells at concentration 7 for replicate 2Float
32Cmpd rM1 Value 8 Rep 2 (1μM**)value from test compound for rM1 cells at concentration 8 for replicate 2Float
33Veh rM1 Value 1 Rep 3 (1e-07μM**)value from vehicle control for rM1 cells at concentration 1 for replicate3Float
34Veh rM1 Value 2 Rep 3 (1e-06μM**)value from vehicle control for rM1 cells at concentration 2 for replicate3Float
35Veh rM1 Value 3 Rep 3 (1e-05μM**)value from vehicle control for rM1 cells at concentration 3 for replicate3Float
36Veh rM1 Value 4 Rep 3 (0.0001μM**)value from vehicle control for rM1 cells at concentration 4 for replicate3Float
37Veh rM1 Value 5 Rep 3 (0.001μM**)value from vehicle control for rM1 cells at concentration 5 for replicate3Float
38Veh rM1 Value 6 Rep 3 (0.01μM**)value from vehicle control for rM1 cells at concentration 6 for replicate3Float
39Veh rM1 Value 7 Rep 3 (0.1μM**)value from vehicle control for rM1 cells at concentration 7 for replicate3Float
40Veh rM1 Value 8 Rep 3 (1μM**)value from vehicle control for rM1 cells at concentration 8 for replicate3Float
41Cmpd rM1 Value 1 Rep 3 (1e-07μM**)value from test compound for rM1 cells at concentration 1 for replicate3Float
42Cmpd rM1 Value 2 Rep 3 (1e-06μM**)value from test compound for rM1 cells at concentration 2 for replicate3Float
43Cmpd rM1 Value 3 Rep 3 (1e-05μM**)value from test compound for rM1 cells at concentration 3 for replicate3Float
44Cmpd rM1 Value 4 Rep 3 (0.0001μM**)value from test compound for rM1 cells at concentration 4 for replicate3Float
45Cmpd rM1 Value 5 Rep 3 (0.001μM**)value from test compound for rM1 cells at concentration 5 for replicate3Float
46Cmpd rM1 Value 6 Rep 3 (0.01μM**)value from test compound for rM1 cells at concentration 6 for replicate3Float
47Cmpd rM1 Value 7 Rep 3 (0.1μM**)value from test compound for rM1 cells at concentration 7 for replicate3Float
48Cmpd rM1 Value 8 Rep 3 (1μM**)value from test compound for rM1 cells at concentration 8 for replicate3Float
49Veh rM1 Bottombottom fit value for curve from vehicle controlFloat
50Veh rM1 Toptop fit value for curve from vehicle controlFloat
51Veh rM1 LogEC50calculated LogEC50 value from all replicates for vehicle controlFloat
52Veh rM1 EC50calculated EC50 value from all replicates for vehicle controlFloatμM
53Veh rM1 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for vehicle controlFloat
54Veh rM1 Std Error TopCalculated standard error for the top for the sigmoidal fit for vehicle controlFloat
55Veh rM1 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for vehicle controlFloat
56Veh rM1 95% CI BottomBottom 95% confidence interval for sigmoidal fit for vehicle controlString
57Veh rM1 95% CI TopTop 95% confidence interval for sigmoidal fit for vehicle controlString
58Veh rM1 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for vehicle controlString
59Veh rM1 95% CI EC50EC50 95% confidence interval for sigmoidal fit for vehicle controlString
60Veh rM1 Deg FreedomDegrees of freedom in sigmoidal fit for vehicle controlInteger
61Veh rM1 R-SquaredR-squared value for sigmoidal fit for vehicle controlFloat
62Veh rM1 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for vehicle controlFloat
63Veh rM1 Number of ValuesNumber of values used in calculating the sigmoidal fit for vehicle controlInteger
64Cmpd rM1 Bottombottom fit value for curve from vehicle controlFloat
65Cmpd rM1 Toptop fit value for curve from vehicle controlFloat
66Cmpd rM1 LogEC50calculated LogEC50 value from all replicates for the compoundFloat
67Cmpd rM1 EC50*calculated EC50 value from all replicates for the compoundFloatμM
68Cmpd rM1 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for the compoundFloat
69Cmpd rM1 Std Error TopCalculated standard error for the top for the sigmoidal fit for the compoundFloat
70Cmpd rM1 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for the compoundFloat
71Cmpd rM1 95% CI BottomBottom 95% confidence interval for sigmoidal fit for the compoundString
72Cmpd rM1 95% CI TopTop 95% confidence interval for sigmoidal fit for the compoundString
73Cmpd rM1 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for the compoundString
74Cmpd rM1 95% CI EC50EC50 95% confidence interval for sigmoidal fit for the compoundString
75Cmpd rM1 Deg FreedomDegrees of freedom in sigmoidal fit for the compoundInteger
76Cmpd rM1 R-SquaredR-squared value for sigmoidal fit for the compoundFloat
77Cmpd rM1 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for the compoundFloat
78Cmpd rM1 Number of ValuesNumber of values used in calculating the sigmoidal fit for the compoundInteger

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

Data Table (Concise)
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