Background and purpose: M 1 muscarinic ACh receptors (mAChRs) represent an attractive drug target for the treatment of cognitive deficits associated with diseases such as Alzheimer's disease and schizophrenia. However, the discovery of subtypeselective mAChR agonists has been hampered by the high degree of conservation of the orthosteric ACh-binding site among mAChR subtypes. The advent of functional screening assays has enabled the identification of agonists such as AC-42 (4-nbutyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine), which bind to an allosteric site and selectively activate the M 1 mAChR subtype. However, studies with this compound have been limited to recombinantly expressed mAChRs. Experimental approach: In this study, we have compared the pharmacological profile of AC-42 and a close structural analogue, 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) at human recombinant, and rat native, mAChRs by calcium mobilization, inositol phosphate accumulation and both in vitro and in vivo electrophysiology. Key results: Calcium mobilization and inositol phosphate accumulation assays revealed that both AC-42 and 77-LH-28-1 display high selectivity to activate the M 1 mAChR over other mAChR subtypes. Furthermore, 77-LH-28-1, but not AC-42, acted as an agonist at rat hippocampal M 1 receptors, as demonstrated by its ability to increase cell firing and initiate gamma frequency network oscillations. Finally, 77-LH-28-1 stimulated cell firing in the rat hippocampus in vivo following subcutaneous administration. Conclusions and implications: These data suggest that 77-LH-28-1 is a potent, selective, bioavailable and brain-penetrant agonist at the M 1 mAChR and therefore that it represents a better tool than AC-42, with which to study the pharmacology of the M 1 mAChR. (2008) 154, 1104-1115 doi:10.1038/bjp.2008 published online 5 May 2008 Keywords: muscarinic receptors; selective agonist; allosteric; AC-42; 77-LH-28-1; calcium mobilization; inositol phosphate; cell firing; network oscillations There is a wide array of pharmacological tools with which to study mAChRs. For example, N-methyl scopolamine, quinuclidinylbenzilate, pirenzepine and darifenacin are among numerous mAChR antagonists, and ACh and oxotremorine-M among mAChR agonists, which have been used in unlabelled and radiolabelled forms to characterize the localization, pharmacology and function of mAChRs. Unfortunately, most of these pharmacological tools exhibit poor selectivity between mAChR subtypes (Caulfield and Birdsall, 1998;Ellis, 2002). Those agents that do display high degrees of mAChR subtype selectivity are few in number and when discovered are often shown to interact with an allosteric, rather than the orthosteric, site as exemplified by the highly selective M 1 receptor peptide antagonist MT-7 (muscarinic toxin 7; Olianas et al., 2000). British Journal of PharmacologyTherefore, the identification of selective M 1 mAChR agonists would represent a significant advance in mAChR pharmacology and could offer t...

The M 2 muscarinic acetylcholine receptor (mAChR) possesses at least one binding site for allosteric modulators that is dependent on the residues 172 EDGE 175 , Tyr 177 , and Thr 423 . However, the contribution of these residues to actions of allosteric agonists, as opposed to modulators, is unknown. We created mutant M 2 mAChRs in which the charge of the 172 EDGE 175 sequence had been neutralized and each Tyr 177 and Thr 423 was substituted with alanine. Radioligand binding experiments revealed that these mutations had a profound inhibitory effect on the prototypical modulators gallamine, alcuronium, and heptane-1,7-bis-[dimethyl-3Ј-phthalimidopropyl]-ammonium bromide (C 7 /3-phth) but minimal effects on the orthosteric antagonist Additional mutagenesis experiments found that these effects were predominantly mediated by Tyr 177 and Thr 423 , rather than the 172 EDGE 175 sequence. The functional interaction between each of the allosteric agonists and ACh was characterized by high negative cooperativity but was consistent with an increased allosteric agonist affinity at the combined EDGE-YT mutant M 2 mAChR. This study has thus revealed a differential role of critical allosteric site residues on the binding and function of allosteric agonists versus allosteric modulators of M 2 mAChRs.Muscarinic acetylcholine receptors (mAChRs) are a group of five family A G protein-coupled receptors (GPCRs) distributed throughout the body (Hulme et al., 1990;Christopoulos, 2007). Drugs targeting mAChRs are currently used in the treatment of chronic obstructive pulmonary disease and urinary incontinence. These receptors also represent potential therapeutic targets for conditions such as Alzheimer's disease, schizophrenia, and irritable bowel syndrome (Felder et al., 2000;Eglen et al., 2001). To date, however, the widespread development of highly efficacious mAChR therapeutics with acceptable side effect profiles has been limited by a relative lack of ligands with sufficient subtype selectivity (Felder et al., 2000;Eglen et al., 2001).Allosteric modulation of GPCRs represents a novel therapeutic avenue for overcoming difficulties associated with selective drug targeting (Christopoulos, 2002;May et al., 2007), and may be particularly amenable to mAChRs. Functional and radioligand binding studies have provided evidence for at least two allosteric binding sites on each mAChR subtype (Ellis et al., 1991;Christopoulos et al., 1998;

1 This study characterises the binding of a novel nonpeptide antagonist radioligand, [ 3 H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX 1 ) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 3 Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX 1 receptor, with orexin-A displaying a Bfive-fold higher affinity than orexin-B (K i values of 3187158 and 15167597 nM, respectively). 4 SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX 1 receptor in both whole cell (K i values 99718, 5778.3 and 1974.5 nM, respectively) and membrane (K i values 3873.6, 2774.1 and 4.570.2 nM, respectively) formats. 5 Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX 1 receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6 These studies indicate that [ 3 H]SB-674042 is a specific, high-affinity radioligand for the OX 1 receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX 1 receptors.