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BioAssay: AID 449770

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine 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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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
AID: 449770
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (humanM4 PAM Ca Ach FS)
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: 4
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
449769Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4Confirmatory 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
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
All data were collected by Millipore GPCRProfiler 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
Fixed Vanderbilt Compound Dose with Full Acetylcholine Dose Response Assay Format Vanderbilt University compounds were added to the assay plate in order to achieve 30uM concentration across the eight wells in duplicate (concentrations described here reflect the final concentration during the addition of ACh dose response). 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 (This assay added Vanderbilt compounds to respective wells#then fluorescence measurements were collected to determine agonist activities).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 full dose response of Acetylcholine to respective wells then fluorescence measurements were collected to determine agonist activities for control purposes and to determine any shift in ACh potencies in wells containing Vanderbilt compounds).
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.'
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 hM4 Value 1 Rep 1 (1e-07μM**)value from vehicle control for hM4 cells at concentration 1 for replicate 1Float
2Veh hM4 Value 2 Rep 1 (1e-06μM**)value from vehicle control for hM4 cells at concentration 2 for replicate 1Float
3Veh hM4 Value 3 Rep 1 (1e-05μM**)value from vehicle control for hM4 cells at concentration 3 for replicate 1Float
4Veh hM4 Value 4 Rep 1 (0.0001μM**)value from vehicle control for hM4 cells at concentration 4 for replicate 1Float
5Veh hM4 Value 5 Rep 1 (0.001μM**)value from vehicle control for hM4 cells at concentration 5 for replicate 1Float
6Veh hM4 Value 6 Rep 1 (0.01μM**)value from vehicle control for hM4 cells at concentration 6 for replicate 1Float
7Veh hM4 Value 7 Rep 1 (0.1μM**)value from vehicle control for hM4 cells at concentration 7 for replicate 1Float
8Veh hM4 Value 8 Rep 1 (1μM**)value from vehicle control for hM4 cells at concentration 8 for replicate 1Float
9Cmpd hM4 Value 1 Rep 1 (1e-07μM**)value from test compound for hM4 cells at concentration 1 for replicate 1Float
10Cmpd hM4 Value 2 Rep 1 (1e-06μM**)value from test compound for hM4 cells at concentration 2 for replicate 1Float
11Cmpd hM4 Value 3 Rep 1 (1e-05μM**)value from test compound for hM4 cells at concentration 3 for replicate 1Float
12Cmpd hM4 Value 4 Rep 1 (0.0001μM**)value from test compound for hM4 cells at concentration 4 for replicate 1Float
13Cmpd hM4 Value 5 Rep 1 (0.001μM**)value from test compound for hM4 cells at concentration 5 for replicate 1Float
14Cmpd hM4 Value 6 Rep 1 (0.01μM**)value from test compound for hM4 cells at concentration 6 for replicate 1Float
15Cmpd hM4 Value 7 Rep 1 (0.1μM**)value from test compound for hM4 cells at concentration 7 for replicate 1Float
16Cmpd hM4 Value 8 Rep 1 (1μM**)value from test compound for hM4 cells at concentration 8 for replicate 1Float
17Veh hM4 Value 1 Rep 2 (1e-07μM**)value from vehicle control for hM4 cells at concentration 1 for replicate 2Float
18Veh hM4 Value 2 Rep 2 (1e-06μM**)value from vehicle control for hM4 cells at concentration 2 for replicate 2Float
19Veh hM4 Value 3 Rep 2 (1e-05μM**)value from vehicle control for hM4 cells at concentration 3 for replicate 2Float
20Veh hM4 Value 4 Rep 2 (0.0001μM**)value from vehicle control for hM4 cells at concentration 4 for replicate 2Float
21Veh hM4 Value 5 Rep 2 (0.001μM**)value from vehicle control for hM4 cells at concentration 5 for replicate 2Float
22Veh hM4 Value 6 Rep 2 (0.01μM**)value from vehicle control for hM4 cells at concentration 6 for replicate 2Float
23Veh hM4 Value 7 Rep 2 (0.1μM**)value from vehicle control for hM4 cells at concentration 7 for replicate 2Float
24Veh hM4 Value 8 Rep 2 (1μM**)value from vehicle control for hM4 cells at concentration 8 for replicate 2Float
25Cmpd hM4 Value 1 Rep 2 (1e-07μM**)value from test compound for hM4 cells at concentration 1 for replicate 2Float
26Cmpd hM4 Value 2 Rep 2 (1e-06μM**)value from test compound for hM4 cells at concentration 2 for replicate 2Float
27Cmpd hM4 Value 3 Rep 2 (1e-05μM**)value from test compound for hM4 cells at concentration 3 for replicate 2Float
28Cmpd hM4 Value 4 Rep 2 (0.0001μM**)value from test compound for rM4 cells at concentration 4 for replicate 2Float
29Cmpd hM4 Value 5 Rep 2 (0.001μM**)value from test compound for hM4 cells at concentration 5 for replicate 2Float
30Cmpd hM4 Value 6 Rep 2 (0.01μM**)value from test compound for hM4 cells at concentration 6 for replicate 2Float
31Cmpd hM4 Value 7 Rep 2 (0.1μM**)value from test compound for hM4 cells at concentration 7 for replicate 2Float
32Cmpd hM4 Value 8 Rep 2 (1μM**)value from test compound for hM4 cells at concentration 8 for replicate 2Float
33Veh hM4 Value 1 Rep 3 (1e-07μM**)value from vehicle control for hM4 cells at concentration 1 for replicate3Float
34Veh hM4 Value 2 Rep 3 (1e-06μM**)value from vehicle control for hM4 cells at concentration 2 for replicate3Float
35Veh hM4 Value 3 Rep 3 (1e-05μM**)value from vehicle control for hM4 cells at concentration 3 for replicate3Float
36Veh hM4 Value 4 Rep 3 (0.0001μM**)value from vehicle control for hM4 cells at concentration 4 for replicate3Float
37Veh hM4 Value 5 Rep 3 (0.001μM**)value from vehicle control for hM4 cells at concentration 5 for replicate3Float
38Veh hM4 Value 6 Rep 3 (0.01μM**)value from vehicle control for hM4 cells at concentration 6 for replicate3Float
39Veh hM4 Value 7 Rep 3 (0.1μM**)value from vehicle control for hM4 cells at concentration 7 for replicate3Float
40Veh hM4 Value 8 Rep 3 (1μM**)value from vehicle control for hM4 cells at concentration 8 for replicate3Float
41Veh hM4 Value 1 Rep 4 (1e-07μM**)value from vehicle for hM4 cells at concentration 1 for replicate 4Float
42Veh hM4 Value 2 Rep 4 (1e-06μM**)value from vehicle for hM4 cells at concentration 2 for replicate 4Float
43Veh hM4 Value 3 Rep 4 (1e-05μM**)value from vehicle for hM4 cells at concentration 3 for replicate 4Float
44Veh hM4 Value 4 Rep 4 (0.0001μM**)value from vehicle for hM4 cells at concentration 4 for replicate 4Float
45Veh hM4 Value 5 Rep 4 (0.001μM**)value from vehicle for hM4 cells at concentration 5 for replicate 4Float
46Veh hM4 Value 6 Rep 4 (0.01μM**)value from vehicle for hM4 cells at concentration 6 for replicate 4Float
47Veh hM4 Value 7 Rep 4 (0.1μM**)value from vehicle for hM4 cells at concentration 7 for replicate 4Float
48Veh hM4 Value 8 Rep 4 (1μM**)value from vehicle for hM4 cells at concentration 8 for replicate 4Float
49Veh hM4 Bottombottom fit value for curve from vehicle controlFloat
50Veh hM4 Toptop fit value for curve from vehicle controlFloat
51Veh hM4 LogEC50calculated LogEC50 value from all replicates for vehicle controlFloat
52Veh hM4 EC50calculated EC50 value from all replicates for vehicle controlFloatμM
53Veh hM4 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for vehicle controlFloat
54Veh hM4 Std Error TopCalculated standard error for the top for the sigmoidal fit for vehicle controlFloat
55Veh hM4 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for vehicle controlFloat
56Veh hM4 95% CI BottomBottom 95% confidence interval for sigmoidal fit for vehicle controlString
57Veh hM4 95% CI TopTop 95% confidence interval for sigmoidal fit for vehicle controlString
58Veh hM4 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for vehicle controlString
59Veh hM4 95% CI EC50EC50 95% confidence interval for sigmoidal fit for vehicle controlString
60Veh hM4 Deg FreedomDegrees of freedom in sigmoidal fit for vehicle controlInteger
61Veh hM4 R-SquaredR-squared value for sigmoidal fit for vehicle controlFloat
62Veh hM4 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for vehicle controlFloat
63Cmpd hM4 Bottombottom fit value for curve from vehicle controlFloat
64Cmpd hM4 Toptop fit value for curve from vehicle controlFloat
65Cmpd hM4 LogEC50calculated LogEC50 value from all replicates for the compoundFloat
66Cmpd hM4 EC50*calculated EC50 value from all replicates for the compoundFloatμM
67Cmpd hM4 Std Error BottomCalculated standard error for the bottom for the sigmoidal fit for the compoundFloat
68Cmpd hM4 Std Error TopCalculated standard error for the top for the sigmoidal fit for the compoundFloat
69Cmpd hM4 Std Error LogEC50Calculated standard error for the logEC50 value for the sigmoidal fit for the compoundFloat
70Cmpd hM4 95% CI BottomBottom 95% confidence interval for sigmoidal fit for the compoundString
71Cmpd hM4 95% CI TopTop 95% confidence interval for sigmoidal fit for the compoundString
72Cmpd hM4 95% CI LogEC50LogEC50 95% confidence interval for sigmoidal fit for the compoundString
73Cmpd hM4 95% CI EC50EC50 95% confidence interval for sigmoidal fit for the compoundString
74Cmpd hM4 Deg FreedomDegrees of freedom in sigmoidal fit for the compoundInteger
75Cmpd hM4 R-SquaredR-squared value for sigmoidal fit for the compoundFloat
76Cmpd hM4 Abs Sum of SquaresAbsolute sum of squares for sigmoidal fit for the compoundFloat
77VUIDVanderbilt internal identifierString

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

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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