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BioAssay: AID 626

Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen

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
All(63675)
 
 
Active(1938)
 
 
Inactive(61737)
 
 
 Tested Substances
 Tested Substances
All(63682)
 
 
Active(1938)
 
 
Inactive(61744)
 
 
 Related BioAssays
 Related BioAssays
AID: 626
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (VMLSCN00000006)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-03-21
Modify Date: 2007-09-19

Data Table ( Complete ):           Active    All
BioActive Compounds: 1938
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
628Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Primary Screenscreening
677Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Confirmation Screenother
678Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Secondary Assay 1other
859Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Assayconfirmatory
860Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Counterscreenconfirmatory
1470Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS binding at M1confirmatory
1488Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation Assayother
1508Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M5confirmatory
1741Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Counterscreen with M4 Receptorother
1743Discovery of novel allosteric modulators of the M1 muscarinic receptor: Y381A Mutant M1 Receptorconfirmatory
1744Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Activity against Muscarinic Panelconfirmatory
1757Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M4confirmatory
1764Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M3confirmatory
1767Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M2confirmatory
1788Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Ancillary Activityother
1798Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe Summarysummary1
1921Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator: Ancillary Activityother
1923Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Potencyconfirmatory
1928Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5confirmatory
1929Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Foldshift Selectivity with hM3confirmatory
1930Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Fold-shift Selectivity with hM2confirmatory
1932Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1confirmatory
1938Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): Analog Dose Response with rM4confirmatory
1939Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition at rM4confirmatory
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5summary
2425Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M1confirmatory
2433Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M5other
2434Discovery of novel allosteric modulators of the M1 muscarinic receptor: Acetylcholine Fold-shift Activity of PAMSconfirmatory
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
2651Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SARconfirmatory
435021Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Activity for Analogsother
449767Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholineother
449769Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4confirmatory
449770Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine with human M4confirmatory
652178Confirmed Agonists of Novel Allosteric Modulators of the M1 Muscarinic Receptorother
Description:
Assay Provider: P. Jeffery 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
Assay Protocol:
1. Hamster Ovary (CHO) cells containing M1 receptor (ATCC #CRL-1985) were plated at 10,000 cells/well in assay media (F12 (Ham), 10% FBS, 2 millimolar GlutaMAX (Invitrogen), 20mM HEPES) in 384 well plates.
2. The plates were incubated overnight at 37 degrees C in 5% CO2.
3. Media was removed and assay buffer (Hanks Balanced Salt Solution, 20 millimolar HEPES, 2.5 millimolar Probenecid, pH 7.4) containing 4.0 micromolar Fluo4-AM dye (Invitrogen) was added.
4. Cells were incubated for 45 minutes (37 degrees C, 5% CO2) for dye loading.
5. Cell plates were loaded into the Hamamatsu FDSS equipped with 480 nanometer excitation and 540 nanometer emission filters
6. 10micromolar test compound in assay buffer + 0.1 percent DMSO was added at 5 seconds simultaneously the plate was kinetically imaged.
7. Subsequently, 8 nanomolar acetylcholine (EC80) in assay buffer was added at 197 seconds and imaging continued for a total of 4 minutes acquisition time.
8. 0.1% DMSO, compound vehicle, and 80nM acetylcholine (ECMAX) were added to each plate as controls.

Data Processing:
1. Minimum and maximum fluorescence intensities were selected from the time window ranging from the initial imaging of the cell plate (0 seconds) to 196 seconds.
2. The minimum fluorescence intensity was subtracted from the maximum fluorescence intensity to give 'Value'.
3. Bscore [1] was calculated using 'Value' normalized for row and column effects on a per plate basis
4. 'Value' and 'BScore' were treated as Gaussian distributions.
5. Compounds selected with 'Score' of 100' and 'Outcome' of Active had values that differed from the mean sample distribution at 99.7% confidence level.
6. All calculations were done on a per plate basis using Pipeline Pilot with the R statistics package.

References:
1. Malo N, Hanley JA, Cerquozzi S, Pelletier J, Nadon R. Statistical practice in high-throughput screening data analysis. Nat Biotechnol. 2006 Feb;24(2):167-75.
Comment
These data reflect the observations from primary screening where the number of independent observations or sample size is one (n = 1). Possible artifacts include, but are not limited to, dust in or on the microtiter plate, compounds that fluoresce, and compounds or conditions that cause a change in intracellular calcium levels.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Valueminimum fluorescence intensity value subtracted from the maximum fluorescence intensity value within the observed time rangeFloat
2Bscoreplate-based row and column median normalization of 'Value'Float
3Value_meanmean of 'Value' per plateFloat
4Value_stddevstandard deviation of 'Value' per plateFloat
5Bscore_meanmean of 'Bscore' per plateFloat
6Bscore_stddevstandard deviation of 'Bscore' per plateFloat

Data Table (Concise)
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