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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 ):           View Active Data    View All Data
BioActive Compounds: 1938
Related Experiments
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AIDNameTypeProbeComment
628Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Primary ScreenScreening depositor-specified cross reference
677Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Confirmation ScreenOther depositor-specified cross reference
678Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Secondary Assay 1Other depositor-specified cross reference
859Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response AssayConfirmatory depositor-specified cross reference
860Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response CounterscreenConfirmatory depositor-specified cross reference
1470Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS binding at M1Confirmatory depositor-specified cross reference
1488Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation AssayOther depositor-specified cross reference
1508Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M5Confirmatory depositor-specified cross reference
1741Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Counterscreen with M4 ReceptorOther depositor-specified cross reference
1743Discovery of novel allosteric modulators of the M1 muscarinic receptor: Y381A Mutant M1 ReceptorConfirmatory depositor-specified cross reference
1744Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Activity against Muscarinic PanelConfirmatory depositor-specified cross reference
1757Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M4Confirmatory depositor-specified cross reference
1764Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M3Confirmatory depositor-specified cross reference
1767Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M2Confirmatory depositor-specified cross reference
1788Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Ancillary ActivityOther depositor-specified cross reference
1798Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe SummarySummary1 depositor-specified cross reference
1921Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator: Ancillary ActivityOther depositor-specified cross reference
1923Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog PotencyConfirmatory depositor-specified cross reference
1928Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at hM5Confirmatory depositor-specified cross reference
1929Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Foldshift Selectivity with hM3Confirmatory depositor-specified cross reference
1930Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Fold-shift Selectivity with hM2Confirmatory depositor-specified cross reference
1932Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM1Confirmatory depositor-specified cross reference
1938Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): Analog Dose Response with rM4Confirmatory depositor-specified cross reference
1939Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator (PAM): NMS Competition at rM4Confirmatory depositor-specified cross reference
2416Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5Summary depositor-specified cross reference
2425Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M1Confirmatory depositor-specified cross reference
2433Discovery of novel allosteric modulators of the M1 muscarinic receptor: PAM Calcium Assay Dose-Response with M5Other depositor-specified cross reference
2434Discovery of novel allosteric modulators of the M1 muscarinic receptor: Acetylcholine Fold-shift Activity of PAMSConfirmatory 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
2651Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: PAM Calcium Assay SARConfirmatory depositor-specified cross reference
435021Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Activity for AnalogsOther depositor-specified cross reference
449767Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of AcetylcholineOther depositor-specified cross reference
449769Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Analog Fold-shift Selectivity at rM4Confirmatory depositor-specified cross reference
449770Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM): Foldshift of Acetylcholine with human M4Confirmatory depositor-specified cross reference
652178Confirmed Agonists of Novel Allosteric Modulators of the M1 Muscarinic ReceptorOther depositor-specified cross reference
2186Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Fold-shift AssayConfirmatory same project related to Summary assay
2192Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine CompetitionConfirmatory same project related to Summary assay
2194Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: [3H]N-methylscopolamine Competition with AcetylcholineConfirmatory same project related to Summary assay
2198Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: PAM SAR with Muscarinic M5Other same project related to Summary assay
2204Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: Calcium Flux AssayConfirmatory same project related to Summary assay
2206Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with Muscarinic M1Other same project related to Summary assay
2665Discovery of Novel Allosteric Modulators of the Muscarinic Receptor M5: SAR with AcetylcholineScreening same project related to Summary assay
651776Discovery of Novel Positive Allosteric Modulators (PAM) of the Muscarinic Receptor M5: Fold-shift AssayConfirmatory same project related to Summary assay
625Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Primary ScreenScreening same project related to Summary assay
643Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation ScreenOther 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: 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)
Data Table ( Complete ):     View Active Data    View All Data
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