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BioAssay: AID 449769

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4

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(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 449769
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4 analog foldshift selectivity rM4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2010-07-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening depositor-specified cross reference
643Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation ScreenOther depositor-specified cross reference
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 depositor-specified cross reference
625Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Primary ScreenScreening same project related to Summary assay
1921Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator: Ancillary ActivityOther same project related to Summary assay
1923Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog PotencyConfirmatory same project related to Summary assay
1928Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5Confirmatory same project related to Summary assay
1929Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Foldshift Selectivity with hM3Confirmatory same project related to Summary assay
1930Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Fold-shift Selectivity with hM2Confirmatory same project related to Summary assay
1932Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1Confirmatory same project related to Summary assay
1938Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): Analog Dose Response with rM4Confirmatory same project related to Summary assay
1939Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition at rM4Confirmatory same project related to Summary assay
449767Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of AcetylcholineOther same project related to Summary assay
449770Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine with human M4Confirmatory same project related to Summary assay
588743Chemical optimization of the highly selective mAChR 4 (M4) Positive Allosteric Modulator (PAM) SeriesConfirmatory same project related to Summary assay
588744Human M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588745Human M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588746Human M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588747Human M5 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588748Rat M2 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588749Rat M1 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588750Rat M3 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588751Rat M4 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588752Rat M5 PAM Extended Characterization CounterScreen (CRC)Confirmatory same project related to Summary assay
588755Human M4 PAM Extended Characterization Fold ShiftOther same project related to Summary assay
588758ML253 Competition in Radioligand Binding assays (Riserca)Other same project related to Summary assay
623924Chemical 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 Activity (hM1 CounterScreen)Confirmatory same project related to Summary assay
623925Chemical 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 Activity (hM2 CounterScreen)Confirmatory same project related to Summary assay
623926Chemical 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 Activity (hM3 CounterScreen)Confirmatory same project related to Summary assay
623938Chemical 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 Activity (hM4 Calcium Potency)Confirmatory1 same project related to Summary assay
623939Chemical 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 Activity (hM5 CounterScreen)Confirmatory same project related to Summary assay
623940Chemical 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 Activity (rM1 CounterScreen)Confirmatory same project related to Summary assay
623941Chemical 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 Activity (rM2 CounterScreen)Confirmatory same project related to Summary assay
623943Chemical 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 Activity (rM3 CounterScreen)Confirmatory same project related to Summary assay
623945Chemical 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 Activity (rM4 CounterScreen)Confirmatory1 same project related to Summary assay
623946Chemical 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 Activity (rM5 CounterScreen)Confirmatory same project related to Summary assay
623948Chemical 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 Activity (hM4 Fold Shift)Other1 same project related to Summary assay
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 rat m4 in a calcium mobilization assay.
Assay Info: CHO-K1 cells stably expressing rat M4 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 left-shift of the Acetylcholine CRC in the presence of test compound demonstrates PAM activity at this muscarinic receptor subtype. Compounds were assigned as 'Active' and scored as '100'.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Test Type: In vitro/In vivo
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Veh rM4 Value 1 Rep 1 (1e-07μM**)value from vehicle control for rM4 cells at concentration 1 for replicate 1Float
2Veh rM4 Value 2 Rep 1 (1e-06μM**)value from vehicle control for rM4 cells at concentration 2 for replicate 1Float
3Veh rM4 Value 3 Rep 1 (1e-05μM**)value from vehicle control for rM4 cells at concentration 3 for replicate 1Float
4Veh rM4 Value 4 Rep 1 (0.0001μM**)value from vehicle control for rM4 cells at concentration 4 for replicate 1Float
5Veh rM4 Value 5 Rep 1 (0.001μM**)value from vehicle control for rM4 cells at concentration 5 for replicate 1Float
6Veh rM4 Value 6 Rep 1 (0.01μM**)value from vehicle control for rM4 cells at concentration 6 for replicate 1Float
7Veh rM4 Value 7 Rep 1 (0.1μM**)value from vehicle control for rM4 cells at concentration 7 for replicate 1Float
8Veh rM4 Value 8 Rep 1 (1μM**)value from vehicle control for rM4 cells at concentration 8 for replicate 1Float
9Cmpd rM4 Value 1 Rep 1 (1e-07μM**)value from test compound for rM4 cells at concentration 1 for replicate 1Float
10Cmpd rM4 Value 2 Rep 1 (1e-06μM**)value from test compound for rM4 cells at concentration 2 for replicate 1Float
11Cmpd rM4 Value 3 Rep 1 (1e-05μM**)value from test compound for rM4 cells at concentration 3 for replicate 1Float
12Cmpd rM4 Value 4 Rep 1 (0.0001μM**)value from test compound for rM4 cells at concentration 4 for replicate 1Float
13Cmpd rM4 Value 5 Rep 1 (0.001μM**)value from test compound for rM4 cells at concentration 5 for replicate 1Float
14Cmpd rM4 Value 6 Rep 1 (0.01μM**)value from test compound for rM4 cells at concentration 6 for replicate 1Float
15Cmpd rM4 Value 7 Rep 1 (0.1μM**)value from test compound for rM4 cells at concentration 7 for replicate 1Float
16Cmpd rM4 Value 8 Rep 1 (1μM**)value from test compound for rM4 cells at concentration 8 for replicate 1Float
17Veh rM4 Value 1 Rep 2 (1e-07μM**)value from vehicle control for rM4 cells at concentration 1 for replicate 2Float
18Veh rM4 Value 2 Rep 2 (1e-06μM**)value from vehicle control for rM4 cells at concentration 2 for replicate 2Float
19Veh rM4 Value 3 Rep 2 (1e-05μM**)value from vehicle control for rM4 cells at concentration 3 for replicate 2Float
20Veh rM4 Value 4 Rep 2 (0.0001μM**)value from vehicle control for rM4 cells at concentration 4 for replicate 2Float
21Veh rM4 Value 5 Rep 2 (0.001μM**)value from vehicle control for rM4 cells at concentration 5 for replicate 2Float
22Veh rM4 Value 6 Rep 2 (0.01μM**)value from vehicle control for rM4 cells at concentration 6 for replicate 2Float
23Veh rM4 Value 7 Rep 2 (0.1μM**)value from vehicle control for rM4 cells at concentration 7 for replicate 2Float
24Veh rM4 Value 8 Rep 2 (1μM**)value from vehicle control for rM4 cells at concentration 8 for replicate 2Float
25Cmpd rM4 Value 1 Rep 2 (1e-07μM**)value from test compound for rM4 cells at concentration 1 for replicate 2Float
26Cmpd rM4 Value 2 Rep 2 (1e-06μM**)value from test compound for rM4 cells at concentration 2 for replicate 2Float
27Cmpd rM4 Value 3 Rep 2 (1e-05μM**)value from test compound for rM4 cells at concentration 3 for replicate 2Float
28Cmpd rM4 Value 4 Rep 2 (0.0001μM**)value from test compound for rM4 cells at concentration 4 for replicate 2Float
29Cmpd rM4 Value 5 Rep 2 (0.001μM**)value from test compound for rM4 cells at concentration 5 for replicate 2Float
30Cmpd rM4 Value 6 Rep 2 (0.01μM**)value from test compound for rM4 cells at concentration 6 for replicate 2Float
31Cmpd rM4 Value 7 Rep 2 (0.1μM**)value from test compound for rM4 cells at concentration 7 for replicate 2Float
32Cmpd rM4 Value 8 Rep 2 (1μM**)value from test compound for rM4 cells at concentration 8 for replicate 2Float
33Veh rM4 Value 1 Rep 3 (1e-07μM**)value from vehicle control for rM4 cells at concentration 1 for replicate3Float
34Veh rM4 Value 2 Rep 3 (1e-06μM**)value from vehicle control for rM4 cells at concentration 2 for replicate3Float
35Veh rM4 Value 3 Rep 3 (1e-05μM**)value from vehicle control for rM4 cells at concentration 3 for replicate3Float
36Veh rM4 Value 4 Rep 3 (0.0001μM**)value from vehicle control for rM4 cells at concentration 4 for replicate3Float
37Veh rM4 Value 5 Rep 3 (0.001μM**)value from vehicle control for rM4 cells at concentration 5 for replicate3Float
38Veh rM4 Value 6 Rep 3 (0.01μM**)value from vehicle control for rM4 cells at concentration 6 for replicate3Float
39Veh rM4 Value 7 Rep 3 (0.1μM**)value from vehicle control for rM4 cells at concentration 7 for replicate3Float
40Veh rM4 Value 8 Rep 3 (1μM**)value from vehicle control for rM4 cells at concentration 8 for replicate3Float
41Cmpd rM4 Value 1 Rep 3 (1e-07μM**)value from test compound for rM4 cells at concentration 1 for replicate3Float
42Cmpd rM4 Value 2 Rep 3 (1e-06μM**)value from test compound for rM4 cells at concentration 2 for replicate3Float
43Cmpd rM4 Value 3 Rep 3 (1e-05μM**)value from test compound for rM4 cells at concentration 3 for replicate3Float
44Cmpd rM4 Value 4 Rep 3 (0.0001μM**)value from test compound for rM4 cells at concentration 4 for replicate3Float
45Cmpd rM4 Value 5 Rep 3 (0.001μM**)value from test compound for rM4 cells at concentration 5 for replicate3Float
46Cmpd rM4 Value 6 Rep 3 (0.01μM**)value from test compound for rM4 cells at concentration 6 for replicate3Float
47Cmpd rM4 Value 7 Rep 3 (0.1μM**)value from test compound for rM4 cells at concentration 7 for replicate3Float
48Cmpd rM4 Value 8 Rep 3 (1μM**)value from test compound for rM4 cells at concentration 8 for replicate3Float
49Veh rM4 Bottombottom fit value for curve from vehicle controlFloat
50Veh rM4 Toptop fit value for curve from vehicle controlFloat
51Veh rM4 LogEC50calculated LogEC50 value from all replicates for vehicle controlFloat
52Veh rM4 EC50calculated EC50 value from all replicates for vehicle controlFloatμM
53Veh rM4 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for vehicle controlFloat
54Veh rM4 Std Error TopCalculated standard error for the top for the sigmoidal fit for vehicle controlFloat
55Veh rM4 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for vehicle controlFloat
56Veh rM4 95% CI BottomBottom 95% confidence interval for sigmoidal fit for vehicle controlString
57Veh rM4 95% CI TopTop 95% confidence interval for sigmoidal fit for vehicle controlString
58Veh rM4 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for vehicle controlString
59Veh rM4 95% CI EC50EC50 95% confidence interval for sigmoidal fit for vehicle controlString
60Veh rM4 Deg FreedomDegrees of freedom in sigmoidal fit for vehicle controlInteger
61Veh rM4 R-SquaredR-squared value for sigmoidal fit for vehicle controlFloat
62Veh rM4 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for vehicle controlFloat
63Veh rM4 Number of ValuesNumber of values used in calculating the sigmoidal fit for vehicle controlInteger
64Cmpd rM4 Bottombottom fit value for curve from vehicle controlFloat
65Cmpd rM4 Toptop fit value for curve from vehicle controlFloat
66Cmpd rM4 LogEC50calculated LogEC50 value from all replicates for the compoundFloat
67Cmpd rM4 EC50*calculated EC50 value from all replicates for the compoundFloatμM
68Cmpd rM4 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for the compoundFloat
69Cmpd rM4 Std Error TopCalculated standard error for the top for the sigmoidal fit for the compoundFloat
70Cmpd rM4 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for the compoundFloat
71Cmpd rM4 95% CI BottomBottom 95% confidence interval for sigmoidal fit for the compoundString
72Cmpd rM4 95% CI TopTop 95% confidence interval for sigmoidal fit for the compoundString
73Cmpd rM4 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for the compoundString
74Cmpd rM4 95% CI EC50EC50 95% confidence interval for sigmoidal fit for the compoundString
75Cmpd rM4 Deg FreedomDegrees of freedom in sigmoidal fit for the compoundInteger
76Cmpd rM4 R-SquaredR-squared value for sigmoidal fit for the compoundFloat
77Cmpd rM4 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for the compoundFloat
78Cmpd rM4 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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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