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BioAssay: AID 1743

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Y381A Mutant M1 Receptor

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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AID: 1743
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JC001_Mut1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-12

Data Table ( Complete ):           View Active Data    View All Data
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
1488Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation AssayOther depositor-specified cross reference
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
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
1767Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M2Confirmatory 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 for their ability to modulate calcium flux in a cell line expressing the Y381A mutant M1 receptor.

Experimental Protocol:
Stable CHO-K1 cell lines expressing rat Y381A mutant M1 receptor were plated into 96-well Costar optical bottom assay plates at a concentration of 70K cells/well in 100 microliters of Ham's F-12 media. Cells were incubated overnight at 37 degrees C (5% CO2). The following day, medium was removed from cells and they were incubated in 50 microliters of Fluo-4 AM diluted in assay buffer (HBSS containing 20 mM HEPES and 2.5 mM probenecid, pH 7.4) for 1 hour at 37 degrees C. Dye was removed and replaced with 45 microliters of assay buffer. Test compounds were serially diluted in DMSO-matched assay buffer at 2X concentration and 45 microliters was applied to a cell-containing assay plate in an automated format using the Flexstation (Molecular Devices). Calcium flux was measured over time as an increase in fluorescence (fold over basal), and the max minus min values for each well were copied into a custom excel template, which normalized the data as a % of maximum response elicited by a high concentration of N-desmethyl-clozapine (NDMC). This data was then copied into Prism (version 4.0c), which fit the data to a sigmoidal dose-response model with variable slopes using no constraints or weighting.
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
1R1 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 1Float
2R1 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 1Float
3R1 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 1Float
4R1 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 1Float
5R1 Value5 (1μM**)normalized activity at 1 micromolar for replicate 1Float
6R1 Value6 (3μM**)normalized activity at 3 micromolar for replicate 1Float
7R1 Value7 (10μM**)normalized activity at 10 micromolar for replicate 1Float
8R1 Value8 (30μM**)normalized activity at 30 micromolar for replicate 1Float
9R2 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 2Float
10R2 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 2Float
11R2 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 2Float
12R2 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 2Float
13R2 Value5 (1μM**)normalized activity at 1 micromolar for replicate 2Float
14R2 Value6 (3μM**)normalized activity at 3 micromolar for replicate 2Float
15R2 Value7 (10μM**)normalized activity at 10 micromolar for replicate 2Float
16R2 Value8 (30μM**)normalized activity at 30 micromolar for replicate 2Float
17R3 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 3Float
18R3 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 3Float
19R3 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 3Float
20R3 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 3Float
21R3 Value5 (1μM**)normalized activity at 1 micromolar for replicate 3Float
22R3 Value6 (3μM**)normalized activity at 3 micromolar for replicate 3Float
23R3 Value7 (10μM**)normalized activity at 10 micromolar for replicate 3Float
24R3 Value8 (30μM**)normalized activity at 30 micromolar for replicate 3Float
25BottomPrism best fit value for bottom of curveFloat
26TopPrism best fit value for top of curveFloat
27LogEC50Prism best fit value for LogEC50Float
28HillSlopePrism best fit value for Hill SlopeFloat
29EC50*Prism best fit value for EC50FloatμM
30Std. Error BottomCalculated standard error for the Bottom valueFloat
31Std. ErrorTopCalculated standard error for the Top valueFloat
32Std. Error LogEC50Calculated standard error for the LogEC50 valueFloat
33Std. Error HillSlopeCalculated standard error for the HillSlope valueFloat
3495% Confidence Intervals BottomPrism-calculated 95% confidence intervals for the Bottom valueString
3595% Confidence Intervals TopPrism-calculated 95% confidence intervals for the Top valueString
3695% Confidence Intervals LogEC50Prism-calculated 95% confidence intervals for the LogEC50 valueString
3795% Confidence Intervals HillSlopePrism-calculated 95% confidence intervals for the HillSlope valueString
3895% Confidence Intervals EC50Prism-calculated 95% confidence intervals for the EC50 valueString
39Degrees of FreedomNumber of degrees of freedom in this modelInteger
40RsquaredCoefficient of determination for this data setFloat
41AbsSumOfSquaresAbsolute sum of squares of the residualsFloat
42StddevResidualsStandard deviation of the residualsFloat
43NumPointsNumber of points analyzedInteger
44TBPB R1 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 1 for TBPBFloat
45TBPB R1 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 1 for TBPBFloat
46TBPB R1 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 1 for TBPBFloat
47TBPB R1 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 1 for TBPBFloat
48TBPB R1 Value5 (1μM**)normalized activity at 1 micromolar for replicate 1 for TBPBFloat
49TBPB R1 Value6 (3μM**)normalized activity at 3 micromolar for replicate 1 for TBPBFloat
50TBPB R1 Value7 (10μM**)normalized activity at 10 micromolar for replicate 1 for TBPBFloat
51TBPB R1 Value8 (30μM**)normalized activity at 30 micromolar for replicate 1 for TBPBFloat
52TBPB R2 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 2 for TBPBFloat
53TBPB R2 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 2 for TBPBFloat
54TBPB R2 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 2 for TBPBFloat
55TBPB R2 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 2 for TBPBFloat
56TBPB R2 Value5 (1μM**)normalized activity at 1 micromolar for replicate 2 for TBPBFloat
57TBPB R2 Value6 (3μM**)normalized activity at 3 micromolar for replicate 2 for TBPBFloat
58TBPB R2 Value7 (10μM**)normalized activity at 10 micromolar for replicate 2 for TBPBFloat
59TBPB R2 Value8 (30μM**)normalized activity at 30 micromolar for replicate 2 for TBPBFloat
60TBPB R3 Value1 (0.01μM**)normalized activity at 0.01 micromolar for replicate 3 for TBPBFloat
61TBPB R3 Value2 (0.03μM**)normalized activity at 0.03 micromolar for replicate 3 for TBPBFloat
62TBPB R3 Value3 (0.1μM**)normalized activity at 0.1 micromolar for replicate 3 for TBPBFloat
63TBPB R3 Value4 (0.301μM**)normalized activity at 0.301 micromolar for replicate 3 for TBPBFloat
64TBPB R3 Value5 (1μM**)normalized activity at 1 micromolar for replicate 3 for TBPBFloat
65TBPB R3 Value6 (3μM**)normalized activity at 3 micromolar for replicate 3 for TBPBFloat
66TBPB R3 Value7 (10μM**)normalized activity at 10 micromolar for replicate 3 for TBPBFloat
67TBPB R3 Value8 (30μM**)normalized activity at 30 micromolar for replicate 3 for TBPBFloat
68TBPB FitPrism reported "ambiguous" for a sigmoidal dose-response fit meaning that single unique parameters were not found.String
69TBPB BottomPrism best fit value for bottom of curve for TBPBString
70TBPB TopPrism best fit value for top of curve for TBPBString
71TBPB LogEC50Prism best fit value for LogEC50 for TBPBString
72TBPB HillSlopePrism best fit value for Hill Slope for TBPBString
73TBPB EC50Prism best fit value for EC50 for TBPBString
74TBPB Std. Error BottomCalculated standard error for the Bottom value for TBPBString
75TBPB Std. ErrorTopCalculated standard error for the Top value for TBPBString
76TBPB Std. Error LogEC50Calculated standard error for the LogEC50 value for TBPBString
77TBPB Std. Error HillSlopeCalculated standard error for the HillSlope value for TBPBString
78TBPB 95% Confidence Intervals BottomPrism-calculated 95% confidence intervals for the Bottom value for TBPBString
79TBPB 95% Confidence Intervals TopPrism-calculated 95% confidence intervals for the Top value for TBPBString
80TBPB 95% Confidence Intervals LogEC50Prism-calculated 95% confidence intervals for the LogEC50 value for TBPBString
81TBPB 95% Confidence Intervals HillSlopePrism-calculated 95% confidence intervals for the HillSlope value for TBPBString
82TBPB 95% Confidence Intervals EC50Prism-calculated 95% confidence intervals for the EC50 value for TBPBString
83TBPB Degrees of FreedomNumber of degrees of freedom in this model for TBPBFloat
84TBPB RsquaredCoefficient of determination for this data set for TBPBFloat
85TBPB AbsSumOfSquaresAbsolute sum of squares of the residuals for TBPBFloat
86TBPB StddevResidualsStandard deviation of the residuals for TBPBFloat
87TBPB NumPointsNumber of points analyzed for TBPBInteger
88ACh R1 Value1 (0.005μM**)normalized activity at 0.005 micromolar for replicate 1 of acetylcholineFloat
89ACh R1 Value2 (0.05μM**)normalized activity at 0.05 micromolar for replicate 1 of acetylcholineFloat
90ACh R1 Value3 (0.5μM**)normalized activity at 0.5 micromolar for replicate 1 of acetylcholineFloat
91ACh R1 Value4 (5μM**)normalized activity at 5 micromolar for replicate 1 of acetylcholineFloat
92ACh R1 Value5 (50μM**)normalized activity at 50 micromolar for replicate 1 of acetylcholineFloat
93ACh R1 Value6 (500μM**)normalized activity at 500 micromolar for replicate 1 of acetylcholineFloat
94ACh R1 Value7 (5000μM**)normalized activity at 5000 micromolar for replicate 1 of acetylcholineFloat
95ACh R2 Value1 (0.005μM**)normalized activity at 0.005 micromolar for replicate 2 of acetylcholineFloat
96ACh R2 Value2 (0.05μM**)normalized activity at 0.05 micromolar for replicate 2 of acetylcholineFloat
97ACh R2 Value3 (0.5μM**)normalized activity at 0.5 micromolar for replicate 2 of acetylcholineFloat
98ACh R2 Value4 (5μM**)normalized activity at 5 micromolar for replicate 2 of acetylcholineFloat
99ACh R2 Value5 (50μM**)normalized activity at 50 micromolar for replicate 2 of acetylcholineFloat
100ACh R2 Value6 (500μM**)normalized activity at 500 micromolar for replicate 2 of acetylcholineFloat
101ACh R2 Value7 (5000μM**)normalized activity at 5000 micromolar for replicate 2 of acetylcholineFloat
102Ach BottomPrism best fit value for bottom of curve for acetylcholineFloat
103Ach TopPrism best fit value for top of curve for acetylcholineFloat
104Ach LogEC50Prism best fit value for LogEC50 for acetylcholineFloat
105Ach HillSlopePrism best fit value for Hill Slope for acetylcholineFloat
106Ach EC50Prism best fit value for EC50 for acetylcholineFloat
107Ach Std. Error BottomCalculated standard error for the Bottom value for acetylcholineFloat
108Ach Std. ErrorTopCalculated standard error for the Top value for acetylcholineFloat
109Ach Std. Error LogEC50Calculated standard error for the LogEC50 value for acetylcholineFloat
110Ach Std. Error HillSlopeCalculated standard error for the HillSlope value for acetylcholineFloat
111Ach 95% Confidence Intervals BottomPrism-calculated 95% confidence intervals for the Bottom value for acetylcholineString
112Ach 95% Confidence Intervals TopPrism-calculated 95% confidence intervals for the Top value for acetylcholineString
113Ach 95% Confidence Intervals LogEC50Prism-calculated 95% confidence intervals for the LogEC50 value for acetylcholineString
114Ach 95% Confidence Intervals HillSlopePrism-calculated 95% confidence intervals for the HillSlope value for acetylcholineString
115Ach 95% Confidence Intervals EC50Prism-calculated 95% confidence intervals for the EC50 value for acetylcholineString
116Ach Degrees of FreedomNumber of degrees of freedom in this model for acetylcholineInteger
117Ach RsquaredCoefficient of determination for this data set for acetylcholineFloat
118Ach AbsSumOfSquaresAbsolute sum of squares of the residuals for acetylcholineFloat
119Ach StddevResidualsStandard deviation of the residuals for acetylcholineFloat
120Ach NumPointsNumber of points analyzed for acetylcholineInteger

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

Data Table (Concise)
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