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BioAssay: AID 1928

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5

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: 1928
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4 analog foldshift selectivity hM5)
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 ):           All
Target
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 M5 in a calcium mobilization assay.

Assay Info: CHO-K1 cells stably expressing human M5 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 hM5 Value 1 Rep 1 (1e-06μM**)value from vehicle control for hM5 cells at concentration 1 for replicate 1Float
2Veh hM5 Value 2 Rep 1 (1e-05μM**)value from vehicle control for hM5 cells at concentration 2 for replicate 1Float
3Veh hM5 Value 3 Rep 1 (0.0001μM**)value from vehicle control for hM5 cells at concentration 3 for replicate 1Float
4Veh hM5 Value 4 Rep 1 (0.000316228μM**)value from vehicle control for hM5 cells at concentration 4 for replicate 1Float
5Veh hM5 Value 5 Rep 1 (0.001μM**)value from vehicle control for hM5 cells at concentration 5 for replicate 1Float
6Veh hM5 Value 6 Rep 1 (0.00316228μM**)value from vehicle control for hM5 cells at concentration 6 for replicate 1Float
7Veh hM5 Value 7 Rep 1 (0.01μM**)value from vehicle control for hM5 cells at concentration 7 for replicate 1Float
8Veh hM5 Value 8 Rep 1 (1μM**)value from vehicle control for hM5 cells at concentration 8 for replicate 1Float
9Cmpd hM5 Value 1 Rep 1 (1e-06μM**)value from test compound for hM5 cells at concentration 1 for replicate 1Float
10Cmpd hM5 Value 2 Rep 1 (1e-05μM**)value from test compound for hM5 cells at concentration 2 for replicate 1Float
11Cmpd hM5 Value 3 Rep 1 (0.0001μM**)value from test compound for hM5 cells at concentration 3 for replicate 1Float
12Cmpd hM5 Value 4 Rep 1 (0.000316228μM**)value from test compound for hM5 cells at concentration 4 for replicate 1Float
13Cmpd hM5 Value 5 Rep 1 (0.001μM**)value from test compound for hM5 cells at concentration 5 for replicate 1Float
14Cmpd hM5 Value 6 Rep 1 (0.00316228μM**)value from test compound for hM5 cells at concentration 6 for replicate 1Float
15Cmpd hM5 Value 7 Rep 1 (0.01μM**)value from test compound for hM5 cells at concentration 7 for replicate 1Float
16Cmpd hM5 Value 8 Rep 1 (1μM**)value from test compound for hM5 cells at concentration 8 for replicate 1Float
17Veh hM5 Value 1 Rep 2 (1e-06μM**)value from vehicle control for hM5 cells at concentration 1 for replicate 2Float
18Veh hM5 Value 2 Rep 2 (1e-05μM**)value from vehicle control for hM5 cells at concentration 2 for replicate 2Float
19Veh hM5 Value 3 Rep 2 (0.0001μM**)value from vehicle control for hM5 cells at concentration 3 for replicate 2Float
20Veh hM5 Value 4 Rep 2 (0.000316228μM**)value from vehicle control for hM5 cells at concentration 4 for replicate 2Float
21Veh hM5 Value 5 Rep 2 (0.001μM**)value from vehicle control for hM5 cells at concentration 5 for replicate 2Float
22Veh hM5 Value 6 Rep 2 (0.00316228μM**)value from vehicle control for hM5 cells at concentration 6 for replicate 2Float
23Veh hM5 Value 7 Rep 2 (0.01μM**)value from vehicle control for hM5 cells at concentration 7 for replicate 2Float
24Veh hM5 Value 8 Rep 2 (1μM**)value from vehicle control for hM5 cells at concentration 8 for replicate 2Float
25Cmpd hM5 Value 1 Rep 2 (1e-06μM**)value from test compound for hM5 cells at concentration 1 for replicate 2Float
26Cmpd hM5 Value 2 Rep 2 (1e-05μM**)value from test compound for hM5 cells at concentration 2 for replicate 2Float
27Cmpd hM5 Value 3 Rep 2 (0.0001μM**)value from test compound for hM5 cells at concentration 3 for replicate 2Float
28Cmpd hM5 Value 4 Rep 2 (0.000316228μM**)value from test compound for hM5 cells at concentration 4 for replicate 2Float
29Cmpd hM5 Value 5 Rep 2 (0.001μM**)value from test compound for hM5 cells at concentration 5 for replicate 2Float
30Cmpd hM5 Value 6 Rep 2 (0.00316228μM**)value from test compound for hM5 cells at concentration 6 for replicate 2Float
31Cmpd hM5 Value 7 Rep 2 (0.01μM**)value from test compound for hM5 cells at concentration 7 for replicate 2Float
32Cmpd hM5 Value 8 Rep 2 (1μM**)value from test compound for hM5 cells at concentration 8 for replicate 2Float
33Veh hM5 Value 1 Rep 3 (1e-06μM**)value from vehicle control for hM5 cells at concentration 1 for replicate3Float
34Veh hM5 Value 2 Rep 3 (1e-05μM**)value from vehicle control for hM5 cells at concentration 2 for replicate3Float
35Veh hM5 Value 3 Rep 3 (0.0001μM**)value from vehicle control for hM5 cells at concentration 3 for replicate3Float
36Veh hM5 Value 4 Rep 3 (0.000316228μM**)value from vehicle control for hM5 cells at concentration 4 for replicate3Float
37Veh hM5 Value 5 Rep 3 (0.001μM**)value from vehicle control for hM5 cells at concentration 5 for replicate3Float
38Veh hM5 Value 6 Rep 3 (0.00316228μM**)value from vehicle control for hM5 cells at concentration 6 for replicate3Float
39Veh hM5 Value 7 Rep 3 (0.01μM**)value from vehicle control for hM5 cells at concentration 7 for replicate3Float
40Veh hM5 Value 8 Rep 3 (1μM**)value from vehicle control for hM5 cells at concentration 8 for replicate3Float
41Cmpd hM5 Value 1 Rep 3 (1e-06μM**)value from test compound for hM5 cells at concentration 1 for replicate3Float
42Cmpd hM5 Value 2 Rep 3 (1e-05μM**)value from test compound for hM5 cells at concentration 2 for replicate3Float
43Cmpd hM5 Value 3 Rep 3 (0.0001μM**)value from test compound for hM5 cells at concentration 3 for replicate3Float
44Cmpd hM5 Value 4 Rep 3 (0.000316228μM**)value from test compound for hM5 cells at concentration 4 for replicate3Float
45Cmpd hM5 Value 5 Rep 3 (0.001μM**)value from test compound for hM5 cells at concentration 5 for replicate3Float
46Cmpd hM5 Value 6 Rep 3 (0.00316228μM**)value from test compound for hM5 cells at concentration 6 for replicate3Float
47Cmpd hM5 Value 7 Rep 3 (0.01μM**)value from test compound for hM5 cells at concentration 7 for replicate3Float
48Cmpd hM5 Value 8 Rep 3 (1μM**)value from test compound for hM5 cells at concentration 8 for replicate3Float
49Veh hM5 Bottombottom fit value for curve from vehicle controlFloat
50Veh hM5 Toptop fit value for curve from vehicle controlFloat
51Veh hM5 LogEC50calculated LogEC50 value from all replicates for vehicle controlFloat
52Veh hM5 EC50calculated EC50 value from all replicates for vehicle controlFloatμM
53Veh hM5 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for vehicle controlFloat
54Veh hM5 Std Error TopCalculated standard error for the top for the sigmoidal fit for vehicle controlFloat
55Veh hM5 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for vehicle controlFloat
56Veh hM5 95% CI BottomBottom 95% confidence interval for sigmoidal fit for vehicle controlString
57Veh hM5 95% CI TopTop 95% confidence interval for sigmoidal fit for vehicle controlString
58Veh hM5 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for vehicle controlString
59Veh hM5 95% CI EC50EC50 95% confidence interval for sigmoidal fit for vehicle controlString
60Veh hM5 Deg FreedomDegrees of freedom in sigmoidal fit for vehicle controlInteger
61Veh hM5 R-SquaredR-squared value for sigmoidal fit for vehicle controlFloat
62Veh hM5 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for vehicle controlFloat
63Veh hM5 Number of ValuesNumber of values used in calculating the sigmoidal fit for vehicle controlInteger
64Cmpd hM5 Bottombottom fit value for curve from vehicle controlFloat
65Cmpd hM5 Toptop fit value for curve from vehicle controlFloat
66Cmpd hM5 LogEC50calculated LogEC50 value from all replicates for the compoundFloat
67Cmpd hM5 EC50*calculated EC50 value from all replicates for the compoundFloatμM
68Cmpd hM5 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for the compoundFloat
69Cmpd hM5 Std Error TopCalculated standard error for the top for the sigmoidal fit for the compoundFloat
70Cmpd hM5 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for the compoundFloat
71Cmpd hM5 95% CI BottomBottom 95% confidence interval for sigmoidal fit for the compoundString
72Cmpd hM5 95% CI TopTop 95% confidence interval for sigmoidal fit for the compoundString
73Cmpd hM5 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for the compoundString
74Cmpd hM5 95% CI EC50EC50 95% confidence interval for sigmoidal fit for the compoundString
75Cmpd hM5 Deg FreedomDegrees of freedom in sigmoidal fit for the compoundInteger
76Cmpd hM5 R-SquaredR-squared value for sigmoidal fit for the compoundFloat
77Cmpd hM5 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for the compoundFloat
78Cmpd hM5 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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