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BioAssay: AID 1767

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M2

The M1 muscarinic receptor is thought to be an important therapeutic target in schizophrenia. A cell-based fluorometric calcium assay was developed for high throughput screening. This assay was used to identify compounds with high selectivity for the M1 receptor subtype that act at an allosteric site on the receptor, thus providing increased specificity for this single receptor subtype. It is anticipated that these compounds will provide important tools for the study of muscarinic receptor function in the CNS. ..more
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 Tested Compounds
 Tested Compounds
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Inconclusive(4)
 
 
 Tested Substances
 Tested Substances
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Inconclusive(4)
 
 
AID: 1767
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JC001_NMS_M2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-15

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening depositor-specified cross reference: Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen
1488Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation AssayOther depositor-specified cross reference: Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation Assay
1798Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe SummarySummary1 depositor-specified cross reference
1470Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS binding at M1Confirmatory same project related to Summary assay
1508Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M5Confirmatory same project related to Summary assay
1741Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Counterscreen with M4 ReceptorOther same project related to Summary assay
1743Discovery of novel allosteric modulators of the M1 muscarinic receptor: Y381A Mutant M1 ReceptorConfirmatory same project related to Summary assay
1744Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Activity against Muscarinic PanelConfirmatory same project related to Summary assay
1757Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M4Confirmatory same project related to Summary assay
1764Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M3Confirmatory same project related to Summary assay
1788Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Ancillary ActivityOther same project related to Summary assay
Description:
Assay Provider: P. Jeffrey Conn
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of novel allosteric modulators of the M1 muscarinic receptor
Grant Number: 1 R03 MH077606-01

The M1 muscarinic receptor is thought to be an important therapeutic target in schizophrenia. A cell-based fluorometric calcium assay was developed for high throughput screening. This assay was used to identify compounds with high selectivity for the M1 receptor subtype that act at an allosteric site on the receptor, thus providing increased specificity for this single receptor subtype. It is anticipated that these compounds will provide important tools for the study of muscarinic receptor function in the CNS.

Agents that enhance cholinergic transmission or activate muscarinic acetylcholine receptors (mAChRs) have been developed to ameliorate the loss of cognitive function in patients with Alzheimer's Disease (AD). While cholinergic agents have been partially successful in improving cognitive function in AD patients, the most exciting findings coming from clinical studies with these agents have been the demonstration of efficacy in reducing psychotic symptoms in patients with AD and other neurodegenerative disorders. Interestingly, the M1/M4 preferring mAChR agonist, xanomeline, also induces a robust antipsychotic effect in schizophrenic patients, suggesting that mAChR agonists may have broad utility in reducing psychotic symptoms in patients suffering from schizophrenia and certain neurodegenerative disorders.

Evidence suggests that the antipsychotic effects of cholinergic agents may be mediated by the M1 mAChR subtype. However, previous compounds developed to selectively activate M1 receptors have failed in clinical development due to a lack of true specificity for M1 and adverse effects associated with activation of other mAChR subtypes. Furthermore, the lack of highly selective compounds has made it impossible to definitively determine whether the behavioral and clinical effects of these compounds are mediated by M1 and the M4 receptor subtype is also a viable candidate for mediating the antipsychotic effects.

Previous attempts to develop agonists and antagonists that are highly selective for M1 or other specific mAChR subtypes have failed because of the high conservation of the ACh binding site and difficulty in developing compounds that are truly specific. However, in recent years, major advances have been made in discovery of highly selective antagonists of other G protein-coupled receptors (GPCRs) that act at allosteric sites rather than the orthosteric neurotransmitter binding site [1, 2]. These compounds induce a noncompetitive blockade of receptor function and tend to be highly selective for the targeted receptor. Even more promising for discovery of M1-selective agonists, novel compounds have now been discovered that act at an allosteric site on M1 receptor rather than the orthosteric ACh-binding site to induce a robust activation of the receptor and provide high receptor subtype specificity [3, 4].

1.May, L.T. and A. Christopoulos, Allosteric modulators of G-protein-coupled receptors. Curr Opin Pharmacol, 2003. 3(5): p. 551-6.
2.Gasparini, F., R. Kuhn, and J.P. Pin, Allosteric modulators of group1 metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic perspectives. Curr Opin Pharmacol, 2002. 2(1): p. 43-9.
3.Spalding, T.A., et al., Discovery of an ectopic activation site on the M(1) muscarinic receptor. Mol Pharmacol, 2002. 61(6): p. 1297-302.
4.Sur, C., et al., N-desmethylclozapine, an allosteric agonist at muscarinic 1 receptor, potentiates Nmethyl-D-aspartate receptor activity. Proc Natl Acad Sci USA, 2003. 100(23): p. 13674-9.
Protocol
The purpose of this screen was to test compounds of interest against the muscarinic M2 receptor for their ability to modulate [3H]N-methyl-scopolamine ([3H]-NMS) binding.
Experimental Protocol:
Membranes were prepared from M2-expressing CHO cells as described (Shirey et al., 2008). Binding reactions contained 0.1 nM [3H]-NMS, 20 ug of membranes and compound or vehicle (0.3% DMSO, final, to define total binding) or 1uM atropine (to define nonspecific binding) in a total volume of 500 ul. The KD of [3H]-NMS was determined empirically to be 0.21 nM. Compounds were serially diluted in DMSO, then diluted in assay buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) to give a final DMSO concentration of 0.3% in the binding reaction. Binding reactions were incubated for 2 hours at room temperature on a Lab-Line Titer plate shaker at setting 7 (~750 rpm). Reactions were stopped and membranes collected onto 96-well Barex microplates with GF/B filter (1um pore size) using a Brandel harvester and washed 3X with ice-cold harvesting buffer (50mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). For acetylcholine affinity experiments, the KI of an acetylcholine competition curve was determined in the absence and presence of fixed concentrations (3 to 30uM, final) of test compound. Actual [3H]-NMS concentrations were back-calculated after counting aliquots of 5X [3H]-NMS added to the reaction. Radioligand depletion was routinely kept to approximately 5% or less.
Shirey JK, Xiang Z, Orton D, Brady AE, Johnson KA, Williams R, Ayala JE,Rodriguez AL, Wess J, Weaver D, Niswender CM, Conn PJ., "An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission." Nat Chem Biol. 2008 Jan;4(1):42-50. PMID: 18059262
Data Fitting:
Data from the Perkin Elmer Topcount scintillation plate reader was normalized using the maximum specific binding of the radioligand. The data were input into GraphPad Prism version 5.01 and fitted to a one site competition model using least squares fit with no constraints and no weighting. Atropine was used to determine non-specific binding. Compounds at concentrations upto 30 micromolar did not fit a full sigmoidal dose-response curve and hence, reliable descriptive statistics could not be determined. The 'Score' was assigned as '30' to reflect dose dependency and the 'Outcome' was assigned as 'Inconclusive.'
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Value 1 Replicate 1 (0.00151μM**)Normalized activity at 0.00151 micromolar for replicate 1Float
2Value 2 Replicate 1 (0.00457μM**)Normalized activity at 0.00457 micromolar for replicate 1Float
3Value 3 Replicate 1 (0.0138μM**)Normalized activity at 0.0138 micromolar for replicate 1Float
4Value 4 Replicate 1 (0.0407μM**)Normalized activity at 0.0407 micromolar for replicate 1Float
5Value 5 Replicate 1 (0.123μM**)Normalized activity at 0.123 micromolar for replicate 1Float
6Value 6 Replicate 1 (0.371μM**)Normalized activity at 0.371 micromolar for replicate 1Float
7Value 7 Replicate 1 (1.12μM**)Normalized activity at 1.12 micromolar for replicate 1Float
8Value 8 Replicate 1 (3.31μM**)Normalized activity at 3.31 micromolar for replicate 1Float
9Value 9 Replicate 1 (10μM**)Normalized activity at 10 micromolar for replicate 1Float
10Value 10 Replicate 1 (30μM**)Normalized activity at 30 micromolar for replicate 1Float
11Value 1 Replicate 2 (0.00151μM**)Normalized activity at 0.00151 micromolar for replicate 2Float
12Value 2 Replicate 2 (0.00457μM**)Normalized activity at 0.00457 micromolar for replicate 2Float
13Value 3 Replicate 2 (0.0138μM**)Normalized activity at 0.0138 micromolar for replicate 2Float
14Value 4 Replicate 2 (0.0407μM**)Normalized activity at 0.0407 micromolar for replicate 2Float
15Value 5 Replicate 2 (0.123μM**)Normalized activity at 0.123 micromolar for replicate 2Float
16Value 6 Replicate 2 (0.371μM**)Normalized activity at 0.371 micromolar for replicate 2Float
17Value 7 Replicate 2 (1.12μM**)Normalized activity at 1.12 micromolar for replicate 2Float
18Value 8 Replicate 2 (3.31μM**)Normalized activity at 3.31 micromolar for replicate 2Float
19Value 9 Replicate 2 (10μM**)Normalized activity at 10 micromolar for replicate 2Float
20Value 10 Replicate 2 (30μM**)Normalized activity at 30 micromolar for replicate 2Float
21Value 1 Replicate 3 (0.00151μM**)Normalized activity at 0.00151 micromolar for replicate 3Float
22Value 2 Replicate 3 (0.00457μM**)Normalized activity at 0.00457 micromolar for replicate 3Float
23Value 3 Replicate 3 (0.0138μM**)Normalized activity at 0.0138 micromolar for replicate 3Float
24Value 4 Replicate 3 (0.0407μM**)Normalized activity at 0.0407 micromolar for replicate 3Float
25Value 5 Replicate 3 (0.123μM**)Normalized activity at 0.123 micromolar for replicate 3Float
26Value 6 Replicate 3 (0.371μM**)Normalized activity at 0.371 micromolar for replicate 3Float
27Value 7 Replicate 3 (1.12μM**)Normalized activity at 1.12 micromolar for replicate 3Float
28Value 8 Replicate 3 (3.31μM**)Normalized activity at 3.31 micromolar for replicate 3Float
29Value 9 Replicate 3 (10μM**)Normalized activity at 10 micromolar for replicate 3Float
30Value 10 Replicate 3 (30μM**)Normalized activity at 30 micromolar for replicate 3Float
31Value 1 Replicate 4 (0.00151μM**)Normalized activity at 0.00151 micromolar for replicate 4Float
32Value 2 Replicate 4 (0.00457μM**)Normalized activity at 0.00457 micromolar for replicate 4Float
33Value 3 Replicate 4 (0.0138μM**)Normalized activity at 0.0138 micromolar for replicate 4Float
34Value 4 Replicate 4 (0.0407μM**)Normalized activity at 0.0407 micromolar for replicate 4Float
35Value 5 Replicate 4 (0.123μM**)Normalized activity at 0.123 micromolar for replicate 4Float
36Value 6 Replicate 4 (0.371μM**)Normalized activity at 0.371 micromolar for replicate 4Float
37Value 7 Replicate 4 (1.12μM**)Normalized activity at 1.12 micromolar for replicate 4Float
38Value 8 Replicate 4 (3.31μM**)Normalized activity at 3.31 micromolar for replicate 4Float
39Value 9 Replicate 4 (10μM**)Normalized activity at 10 micromolar for replicate 4Float
40Value 10 Replicate 4 (30μM**)Normalized activity at 30 micromolar for replicate 4Float
41BottomBest fit value for bottom of curveFloat
42TopBest fit value for top of curveFloat
43LogEC50Best fit value for LogEC50Float
44EC50*Best fit value for EC50FloatμM
45KIBest fit value for KIFloat
46Std. Error BottomCalculated standard error for the Bottom valueFloat
47Std. ErrorTopCalculated standard error for the Top valueFloat
48Std. Error LogEC50Calculated standard error for the LogEC50 valueFloat
4995% Confidence Intervals BottomCalculated 95% confidence intervals for the Bottom valueString
5095% Confidence Intervals TopCalculated 95% confidence intervals for the Top valueString
5195% Confidence Intervals LogEC50Calculated 95% confidence intervals for the LogEC50 valueString
5295% Confidence Intervals EC50Calculated 95% confidence intervals for the EC50 valueString
5395% Confidence Intervals KICalculated 95% confidence intervals for the KI valueString
54Degrees of FreedomNumber of degrees of freedom in this modelInteger
55RsquaredCoefficient of determination for this data setFloat
56AbsSumOfSquaresAbsolute sum of squares of the residualsFloat
57StddevResidualsStandard deviation of the residualsFloat
58NumPointsNumber of points analyzedFloat
59Mean Atropine1 (1.7e-05μM**)Mean normalized activity for atropine at 0.000017 micromolarFloat
60Mean Atropine2 (5.1e-05μM**)Mean normalized activity for atropine at 0.000051 micromolarFloat
61Mean Atropine3 (0.00151μM**)Mean normalized activity for atropine at 0.000151 micromolarFloat
62Mean Atropine4 (0.000457μM**)Mean normalized activity for atropine at 0.000457 micromolarFloat
63Mean Atropine5 (0.00138μM**)Mean normalized activity for atropine at 0.00138 micromolarFloat
64Mean Atropine6 (0.004μM**)Mean normalized activity for atropine at 0.004 micromolarFloat
65Mean Atropine7 (0.012μM**)Mean normalized activity for atropine at 0.012 micromolarFloat
66Mean Atropine8 (0.037μM**)Mean normalized activity for atropine at 0.037micromolarFloat
67Mean Atropine9 (0.112μM**)Mean normalized activity for atropine at 0.112 micromolarFloat
68Mean Atropine10 (0.331μM**)Mean normalized activity for atropine at 0.331 micromolarFloat
69Mean Atropine11 (1μM**)Mean normalized activity for atropine at 1.0 micromolarFloat
70StdDev Atropine1Standard deviation of mean normalized activity for atropine at 0.000017 micromolarFloat
71StdDev Atropine2Standard deviation of mean normalized activity for atropine at 0.000051 micromolarFloat
72StdDev Atropine3Standard deviation of mean normalized activity for atropine at 0.000151 micromolarFloat
73StdDev Atropine4Standard deviation of mean normalized activity for atropine at 0.000457 micromolarFloat
74StdDev Atropine5Standard deviation of mean normalized activity for atropine at 0.00138 micromolarFloat
75StdDev Atropine6Standard deviation of mean normalized activity for atropine at 0.004 micromolarFloat
76StdDev Atropine7Standard deviation of mean normalized activity for atropine at 0.012 micromolarFloat
77StdDev Atropine8Standard deviation of mean normalized activity for atropine at 0.037micromolarFloat
78StdDev Atropine9Standard deviation of mean normalized activity for atropine at 0.112 micromolarFloat
79StdDev Atropine10Standard deviation of mean normalized activity for atropine at 0.331 micromolarFloat
80StdDev Atropine11Standard deviation of mean normalized activity for atropine at 1.0 micromolarFloat
81AtropineBottomBest fit value for bottom of curve for the atropine controlsFloat
82AtropineTopBest fit value for top of curve for the atropine controlsFloat
83AtropineLogEC50Best fit value for LogEC50 for the atropine controlsFloat
84AtropineEC50Best fit value for EC50 for the atropine controlsFloat
85Atropine Std. Error BottomCalculated standard error for the Bottom value for the atropine controlsFloat
86Atropine Std. ErrorTopCalculated standard error for the LogEC50 value for the atropine controlsFloat
87Atropine Std. Error LogEC50Calculated 95% confidence intervals for the Bottom value for the atropine controlsFloat
88Atropine 95% Confidence Intervals BottomCalculated 95% confidence intervals for the Bottom value for the atropine controlsString
89Atropine 95% Confidence Intervals TopCalculated 95% confidence intervals for the Top value for the atropine controlsString
90Atropine 95% Confidence Intervals LogEC50Calculated 95% confidence intervals for the LogEC50 value for the atropine controlsString
91Atropine 95% Confidence Intervals EC50Calculated 95% confidence intervals for the EC50 value for the atropine controlsString
92Atropine Degrees of FreedomNumber of degrees of freedom in this model for the atropine controlsInteger
93AtropineRsquaredCoefficient of determination for this data set for the atropine controlsFloat
94AtropineAbsSumOfSquaresAbsolute sum of squares of the residuals for the atropine controlsFloat
95AtropineStddevResidualsStandard deviation of the residuals for the atropine controlsFloat
96AtropineNumPointsNumber of points analyzed for the atropine controlsInteger

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH077606-01

Data Table (Concise)
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