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BioAssay: AID 1939

Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition 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)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Inactive(2)
 
 
AID: 1939
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (rM4_NMS_rM4)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-09-08

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 experiment was to test compounds of interest for their ability to modulate [3H]N-methyl-scopolamine ([3H]-NMS) binding at the rat M4 muscarinic receptor.

Competition binding assays were carried out essentially as described in (Shirey et al., 2008) with 25 mg of membrane protein prepared from CHO-K1 cells stably expressing rat M4 and 0.1nM [3H]-N-methylscopolamine (GE Healthcare) in a final volume of 1mL. Non-specific binding was determined in the presence of 1 mM atropine. A full atropine CRC was determined as a positive control. The compounds did not affect binding of the orthosteric ligand [3H]-NMS suggesting an allosteric modulation for the lead compounds. 'Outcome' was assigned as 'Inactive' and 'Score' was assigned as "0'.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1Value 1 Replicate 1 (0.000512861μM**)Value for test compound at 0.000512 uM, replicate 1Float
2Value 2 Replicate 1 (0.00151356μM**)Value for test compound at 0.00151 uM, replicate 1Float
3Value 3 Replicate 1 (0.00457088μM**)Value for test compound at 0.00457 uM, replicate 1Float
4Value 4 Replicate 1 (0.0138038μM**)Value for test compound at 0.0138 uM, replicate 1Float
5Value 5 Replicate 1 (0.040738μM**)Value for test compound at 0.0407 uM, replicate 1Float
6Value 6 Replicate 1 (0.123027μM**)Value for test compound at 0.123 uM, replicate 1Float
7Value 7 Replicate 1 (0.371535μM**)Value for test compound at 0.371 uM, replicate 1Float
8Value 8 Replicate 1 (1.12202μM**)Value for test compound at 1.12 uM, replicate 1Float
9Value 9 Replicate 1 (3.31131μM**)Value for test compound at 3.31 uM, replicate 1Float
10Value 10 Replicate 1 (10μM**)Value for test compound at 10.0 uM, replicate 1Float
11Value 11 Replicate 1 (30.1995μM**)Value for test compound at 30.2 uM, replicate 1Float
12Value 1 Replicate 2 (0.000512861μM**)Value for test compound at 0.000512 uM, replicate 2Float
13Value 2 Replicate 2 (0.00151356μM**)Value for test compound at 0.00151 uM, replicate 2Float
14Value 3 Replicate 2 (0.00457088μM**)Value for test compound at 0.00457 uM, replicate 2Float
15Value 4 Replicate 2 (0.0138038μM**)Value for test compound at 0.0138 uM, replicate 2Float
16Value 5 Replicate 2 (0.040738μM**)Value for test compound at 0.0407 uM, replicate 2Float
17Value 6 Replicate 2 (0.123027μM**)Value for test compound at 0.123 uM, replicate 2Float
18Value 7 Replicate 2 (0.371535μM**)Value for test compound at 0.371 uM, replicate 2Float
19Value 8 Replicate 2 (1.12202μM**)Value for test compound at 1.12 uM, replicate 2Float
20Value 9 Replicate 2 (3.31131μM**)Value for test compound at 3.31 uM, replicate 2Float
21Value 10 Replicate 2 (10μM**)Value for test compound at 10.0 uM, replicate 2Float
22Value 11 Replicate 2 (30.1995μM**)Value for test compound at 30.2 uM, replicate 2Float
23BottomCalculated bottom of sigmoidal fitFloat
24TopCalculated top of sigmoidal fitFloat
25LogEC50Calculated logEC50 of sigmoidal fitFloat
26EC50_uM*Calculated EC50 of sigmoidal fitFloatμM
27KICalculated KI of sigmoidal fitFloat
28Std. Error BottomCalculated standard error for the bottom of the sigmoidal fitFloat
29Std. ErrorTopCalculated standard error for the top of the sigmoidal fitFloat
30Std. Error LogEC50Calculated standard error for the logEC50Float
3195% Confidence Intervals Bottom95% confidence interval for the bottomString
3295% Confidence Intervals Top95% confidence interval for the topString
3395% Confidence Intervals LogEC5095% confidence interval for the logEC50String
3495% Confidence Intervals EC5095% confidence interval for the EC50String
3595% Confidence Intervals KI95% confidence interval for the KIString
36Degrees of FreedomDegrees of freedom in the sigmoidal fitInteger
37RsquaredR-squared for the sigmoidal fitFloat
38AbsSumOfSquaresAbsolute sum of squares for the sigmoidal fitFloat
39StddevResidualsStandard deviation of the residuals for the sigmoidal fitFloat
40NumPointsNumber of points in the sigmoidal fitInteger
41Mean Atropine1 (1.7e-05μM**)Mean value for atropine at 0.0000170 uMFloat
42Mean Atropine2 (5.12e-05μM**)Mean value for atropine at 0.0000512 uMFloat
43Mean Atropine3 (0.000151μM**)Mean value for atropine at 0.000151 uMFloat
44Mean Atropine4 (0.000457μM**)Mean value for atropine at 0.000457 uMFloat
45Mean Atropine5 (0.00138μM**)Mean value for atropine at 0.00138 uMFloat
46Mean Atropine6 (0.00407μM**)Mean value for atropine at 0.00407 uMFloat
47Mean Atropine7 (0.0123μM**)Mean value for atropine at 0.00123 uMFloat
48Mean Atropine8 (0.0371μM**)Mean value for atropine at 0.0371 uMFloat
49Mean Atropine9 (0.112μM**)Mean value for atropine at 0.112 uMFloat
50Mean Atropine10 (0.331μM**)Mean value for atropine at 0331 uMFloat
51Mean Atropine11 (1μM**)Mean value for atropine at 1.00 uMFloat
52StdDev Atropine1Standard deviation for atropine at 0.0000170 uMFloat
53StdDev Atropine2Standard deviation for atropine at 0.0000512 uMFloat
54StdDev Atropine3Standard deviation for atropine at 0.000151 uMFloat
55StdDev Atropine4Standard deviation for atropine at 0.000457 uMFloat
56StdDev Atropine5Standard deviation for atropine at 0.00138 uMFloat
57StdDev Atropine6Standard deviation for atropine at 0.00407 uMFloat
58StdDev Atropine7Standard deviation for atropine at 0.00123 uMFloat
59StdDev Atropine8Standard deviation for atropine at 0.0371 uMFloat
60StdDev Atropine9Standard deviation for atropine at 0.112 uMFloat
61StdDev Atropine10Standard deviation for atropine at 0331 uMFloat
62StdDev Atropine11Standard deviation for atropine at 1.00 uMFloat
63AtropineBottomCalculated bottom for sigmoidal fit for atropineFloat
64AtropineTopCalculated top for sigmoidal fit for atropineFloat
65AtropineLogEC50Calculated logEC50 for sigmoidal fit for atropineFloat
66AtropineEC50Calculated EC50 for sigmoidal fit for atropineFloatμM
67AtropineKICalculcated KI for sigmoidal fit for atropineFloat
68Atropine Std. Error BottomCalculated standard error for the bottom of the sigmoidal fit for atropineFloat
69Atropine Std. ErrorTopCalculated standard error for the top of the sigmoidal fit for atropineFloat
70Atropine Std. Error LogEC50Calculated standard error for the logEC50 of the sigmoidal fit for atropineFloat
71Atropine 95% Confidence Intervals Bottom95% confidence intervals for the bottom of the sigmoidal curve fit for atropineString
72Atropine 95% Confidence Intervals Top95% confidence intervals for the top of the sigmoidal curve fit for atropineString
73Atropine 95% Confidence Intervals LogEC5095% confidence intervals for the logEC50 of the sigmoidal curve fit for atropineString
74Atropine 95% Confidence Intervals EC5095% confidence intervals for the EC50 of the sigmoidal curve fit for atropineString
75Atropine Degrees of FreedomDegrees of freedom in the sigmoidal fit for atropineInteger
76AtropineRsquaredR-squared for the sigmoidal fit for atropineFloat
77AtropineAbsSumOfSquaresAbsolute sum of squares for the sigmoidal fit for atropineFloat
78AtropineStddevResidualsStandard deviation of the residuals for the sigmoidal fit for atropineFloat
79AtropineNumPointsNumber of points in the sigmoidal fit for atropineInteger

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

Data Table (Concise)
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