Discovery of novel allosteric modulators of the M1 muscarinic receptor: Agonist NMS competition at M2
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
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 against the muscarinic M2 receptor for their ability to modulate [3H]N-methyl-scopolamine ([3H]-NMS) binding.
Membranes were prepared from M2-expressing CHO cells as described (Shirey et al., 2008). Binding reactions contained 0.1 nM [3H]-NMS, 20 ug of membranes and compound or vehicle (0.3% DMSO, final, to define total binding) or 1uM atropine (to define nonspecific binding) in a total volume of 500 ul. The KD of [3H]-NMS was determined empirically to be 0.21 nM. Compounds were serially diluted in DMSO, then diluted in assay buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) to give a final DMSO concentration of 0.3% in the binding reaction. Binding reactions were incubated for 2 hours at room temperature on a Lab-Line Titer plate shaker at setting 7 (~750 rpm). Reactions were stopped and membranes collected onto 96-well Barex microplates with GF/B filter (1um pore size) using a Brandel harvester and washed 3X with ice-cold harvesting buffer (50mM Tris-HCl, 0.9% NaCl, pH 7.4). Filter plates were dried overnight and counted in a PerkinElmer TopCount scintillation counter (PerkinElmer Life and Analytical Sciences). For acetylcholine affinity experiments, the KI of an acetylcholine competition curve was determined in the absence and presence of fixed concentrations (3 to 30uM, final) of test compound. Actual [3H]-NMS concentrations were back-calculated after counting aliquots of 5X [3H]-NMS added to the reaction. Radioligand depletion was routinely kept to approximately 5% or less.
Shirey JK, Xiang Z, Orton D, Brady AE, Johnson KA, Williams R, Ayala JE,Rodriguez AL, Wess J, Weaver D, Niswender CM, Conn PJ., "An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission." Nat Chem Biol. 2008 Jan;4(1):42-50. PMID: 18059262
Data from the Perkin Elmer Topcount scintillation plate reader was normalized using the maximum specific binding of the radioligand. The data were input into GraphPad Prism version 5.01 and fitted to a one site competition model using least squares fit with no constraints and no weighting. Atropine was used to determine non-specific binding. Compounds at concentrations upto 30 micromolar did not fit a full sigmoidal dose-response curve and hence, reliable descriptive statistics could not be determined. The 'Score' was assigned as '30' to reflect dose dependency and the 'Outcome' was assigned as 'Inconclusive.'
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* Activity Concentration. ** Test Concentration.
Data Table (Concise)