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
BioActive Compounds: 1938
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.
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.
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  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.
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.
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.
Data Table (Concise)