Sequence: cholinergic receptor, muscarinic 4 [Rattus norvegicus]

Gene:CHRM4     Conserved Domain     Related Protein 3D Structures

AID	Name	Type	Comment
626	Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen	screening
643	Discovery of Novel Allosteric Agonists of the M4 Muscarinic Receptor: Confirmation Screen	other
2616	Chemical 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	summary

Assay Provider: Colleen Niswender
Assay Provider Affiliation: Vanderbilt University
Grant Title: Discovery of a Highly Selective in vitro and in vivo M4 Positive Allosteric Modulator(PAM)
Grant Number: MH077607-1

To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M1-M5) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems. Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia. In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M2 and M3 mAChRs. Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChR subtypes, development of chemical agents that are selective for a single subtype has been largely unsuccessful, and in the absence of highly selective activators of M4, it has been impossible to test the role of selective M4 activation. Clinical trials with xanomeline, a M1/M4-preferring orthosteric agonist, demonstrated efficacy as both a cognition-enhancing agent and an antipsychotic agent. In follow-up studies in rats, xanomeline displayed an antipsychotic-like profile comparable to clozapine. However, a long standing question concerned whether or not the antipsychotic efficacy or antipsychotic-like activity in animal models is mediated by activation of M1, M4, or a combination of both receptors. Data from mAChR knockout mice led to the suggestion that a selective M1 agonist would be beneficial for cognition, whereas an M4 agonist would provide antipsychotic activity for the treatment of schizophrenia. This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction. These data, coupled with findings that schizophrenic patients have altered hippocampal M4 but not M1 receptor expression, suggest that selective activators of M4 may provide a novel treatment strategy for schizophrenia patients. However, multiple studies suggest that M1 may also play an important role in the antipsychotic effects of mAChR agonists and that the relative contributions of M1 and M4 to the antipsychotic efficacy of xanomeline or antipsychotic-like effects of this compound in animal models are not known. However, highly selective centrally penetrant activators of either M1 or M4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors.

The purpose of this experiment was to test compounds of interest for their ability to modulate [3H]N-methyl-scopolamine ([3H]-NMS) binding at the rat M4 muscarinic receptor.

Competition binding assays were carried out essentially as described in (Shirey et al., 2008) with 25 mg of membrane protein prepared from CHO-K1 cells stably expressing rat M4 and 0.1nM [3H]-N-methylscopolamine (GE Healthcare) in a final volume of 1mL. Non-specific binding was determined in the presence of 1 mM atropine. A full atropine CRC was determined as a positive control. The compounds did not affect binding of the orthosteric ligand [3H]-NMS suggesting an allosteric modulation for the lead compounds. 'Outcome' was assigned as 'Inactive' and 'Score' was assigned as "0'.

Grant Number: MH077607-1