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BioAssay: AID 1741

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Counterscreen with M4 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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 Tested Compounds
 Tested Compounds
All(1665)
 
 
Active(150)
 
 
Inactive(1195)
 
 
Inconclusive(320)
 
 
 Tested Substances
 Tested Substances
All(1665)
 
 
Active(150)
 
 
Inactive(1195)
 
 
Inconclusive(320)
 
 
AID: 1741
Data Source: Vanderbilt Screening Center for GPCRs, Ion Channels and Transporters (JC001_Confirm_M4)
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2009-05-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 150
Related Experiments
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AIDNameTypeProbeComment
626Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary ScreenScreening depositor-specified cross reference: Primary Assay with M1
1488Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation AssayOther depositor-specified cross reference: Confirmation Assay with M1
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
1798Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe SummarySummary1 depositor-specified cross reference
652178Confirmed Agonists of Novel Allosteric Modulators of the M1 Muscarinic ReceptorOther 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
1743Discovery of novel allosteric modulators of the M1 muscarinic receptor: Y381A Mutant M1 ReceptorConfirmatory 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. 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
The purpose of this experiment was to counterscreen in duplicate compounds that scored as 'Active' in the primary assay (Pubchem AID 626) against the closely related muscarinic M4 receptor.

Assay Protocol:
1. Hamster Ovary (CHO) cells containing M4 receptor and Gqi5 were plated at 25,000 cells/well in assay media (DMEM (Invitrogen), 20 mM HEPES, 10% FBS, 2 mM L-glutamine, 1X Non-essential Amino Acids (NEAA), 1 mM Na pyruvate) 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 mM HEPES, 2.5 mM Probenecid, pH 7.4) containing 2.0 uM 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 nm excitation and 540 nm emission filters.
6. 10 micromolar test compound in assay buffer + 0.1 percent DMSO was added at 5 seconds simultaneously the plate was kinetically imaged.
7. Subsequently, 0.8 nanomolar acetylcholine (EC20) 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'.
5. Compounds selected with 'Score' of '100' and 'Outcome' of 'Active' had values that differed in duplicate from the mean DMSO vehicle distribution at 99.7% confidence level. Compounds with 'Score' of '50' and 'Outcome' of 'Discrepant' different from the mean DMSO vehicle in only one of the two replicates.
6. All calculations were done on a per plate basis using Pipeline Pilot with the R statistics package.
Comment
These data reflect the observations from confirmation screening where the number of independent observations or sample size is two (n = 2). 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
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Value1minimum fluorescence intensity value subtracted from the maximum fluorescence intensity value within the observed time range for the first compound replicateFloat
2DMSO_mean1mean of 'Value' for the DMSO negative control population calculated on a per-plate basis for the first testFloat
3DMSO_stddev1standard deviation of 'Value' for the DMSO negative control population calculated on a per-plate basis for the first testFloat
4EC20_mean1mean of 'Value' for the EC20 control population calculated on a per-plate basis for the first testFloat
5EC20_stddev1standard deviation of 'Value' for the EC20 control population calculated on a per-plate basis for the first testFloat
6ECmax_mean1mean of 'Value' for the ECmax positive control population calculated on a per-plate basis for the first testFloat
7ECmax_stddev1standard deviation of 'Value' for the ECmax positive control population calculated on a per-plate basis for the first testFloat
8Value2minimum fluorescence intensity value subtracted from the maximum fluorescence intensity value within the observed time range for the second compound replicateFloat
9DMSO_mean2mean of 'Value' for the DMSO negative control population calculated on a per-plate basis for the second testFloat
10DMSO_stddev2standard deviation of 'Value' for the DMSO negative control population calculated on a per-plate basis for the second testFloat
11EC20_mean2mean of 'Value' for the EC20 control population calculated on a per-plate basis for the second testFloat
12EC20_stddev2standard deviation of 'Value' for the EC20 control population calculated on a per-plate basis for the second testFloat
13ECmax_mean2mean of 'Value' for the ECmax positive control population calculated on a per-plate basis for the second testFloat
14ECmax_stddev2standard deviation of 'Value' for the ECmax positive control population calculated on a per-plate basis for the second testFloat
Additional Information
Grant Number: 1 R03 MH077606-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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