AID	Name	Type	Comment
626	Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen	screening
1488	Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Confirmation Assay	other
1798	Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist Probe Summary	summary

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.

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.

Grant Number: 1 R03 MH077606-01