AC-260584, an orally bioavailable M(1) muscarinic receptor allosteric agonist, improves cognitive performance in an animal model.

The recent discovery of allosteric potentiators and agonists of the muscarinic M(1) receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC(50) 7.6-7.7) and efficacious (90-98% of carbachol) muscarinic M(1) receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M(1) receptor over the M(2), M(3), M(4) and M(5) muscarinic receptor subtypes. Using GTPgammaS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M(1) receptor activation since it was not observed in M(1) knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M(1) receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M(1) selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.

Differential regulation of muscarinic M1 receptors by orthosteric and allosteric ligands.

BACKGROUND: Activation of muscarinic M1 receptors is mediated via interaction of orthosteric agonists with the acetylcholine binding site or via interaction of allosteric agonists with different site(s) on the receptor. The focus of the present study was to determine if M1 receptors activated by allosteric agonists undergo the same regulatory fate as M1 receptors activated by orthosteric agonists. RESULTS: The orthosteric agonists carbachol, oxotremorine-M and pilocarpine were compared to the allosteric agonists AC-42, AC-260584, N-desmethylclozapine and xanomeline. All ligands activated M1 receptors and stimulated interaction of the receptors with beta-arrestin-1. All ligands reduced cell surface binding and induced the loss of total receptor binding. Receptor internalization was blocked by treatment with hypertonic sucrose indicating that all ligands induced formation of clathrin coated vesicles. However, internalized receptors recycled to the cell surface following removal of orthosteric, but not allosteric agonists. Whereas all ligands induced loss of cell surface receptor binding, no intracellular vesicles could be observed after treatment with AC-260584 or xanomeline. Brief stimulation of M1 receptors with AC-260584 or xanomeline resulted in persistent activation of M1 receptors, suggesting that continual receptor signaling might impede or delay receptor endocytosis into intracellular vesicles. CONCLUSION: These results indicate that allosteric agonists differ from orthosteric ligands and among each other in their ability to induce different regulatory pathways. Thus, signaling and regulatory pathways induced by different allosteric ligands are ligand specific.

Structural requirements of transmembrane domain 3 for activation by the M1 muscarinic receptor agonists AC-42, AC-260584, clozapine, and N-desmethylclozapine: evidence for three distinct modes of receptor activation.

Transmembrane domain 3 (TM3) plays a crucial role mediating muscarinic acetylcholine receptor activation by acetylcholine, carbachol, and other muscarinic agonists. We compared the effects of point mutations throughout TM3 on the interactions of carbachol, 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), a potent structural analog of AC-42 called 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), N-desmethylclozapine, and clozapine with the M(1) muscarinic receptor. The binding and activation profiles of these ligands fell into three distinct patterns; one exemplified by orthosteric compounds like carbachol, another by structural analogs of AC-42, and a third by structural analogs of N-desmethylclozapine. All mutations tested severely reduced carbachol binding and activation of M(1). In contrast, the agonist actions of AC-42 and AC-260584 were greatly potentiated by the W101A mutation, slightly reduced by Y106A, and slightly increased by S109A. Clozapine and N-desmethylclozapine displayed substantially increased maximum responses at the Y106A and W101A mutants, slightly lower activity at S109A, but no substantial changes in potency. At L102A and N110A, agonist responses to AC-42, AC-260584, clozapine, and N-desmethylclozapine were all substantially reduced, but usually less than carbachol. D105A showed no functional responses to all ligands. Displacement and dissociation rate experiments demonstrated clear allosteric properties of AC-42 and AC-260584 but not for N-desmethylclozapine and clozapine, indicating that they may contact different residues than carbachol to activate M(1) but occupy substantially overlapping spaces, in contrast to AC-42 and AC-260584, which occupy separable spaces. These results show that M(1) receptors can be activated in at least three distinct ways and that there is no requirement for potent muscarinic agonists to mimic acetylcholine interactions with TM3.