Interaction Studies of Multiple Binding Sites on M₄ Muscarinic Acetylcholine Receptors

Abstract

This study investigated the reciprocal cross-interactions between two distinct allosteric sites on the M 4 muscarinic acetylcholine receptor (mAChR) in the absence or presence of different orthosteric ligands. Initial studies revealed that two novel benzimidazole allosteric modulators, 17-␤-hydroxy-17-␣-ethy nyl-delta(4)-androstano [3,2-b] (McN-A-343), or xanomeline] revealed low degrees of negative cooperativity between WIN 62,577 and each agonist, whereas stronger negative cooperativity was observed against atropine. It is interesting that when these experiments were repeated using the prototypical modulators heptane-1,7-bis-(dimethyl-3Ј-phthalimidopropyl)-ammonium bromide (C 7 /3-phth), alcuronium, or brucine (which act at a separate allosteric site), WIN 62,577 exhibited negative cooperativity with each modulator when the orthosteric site was unoccupied, but this switched to neutral cooperativity when the receptor was occupied by [ 3 H]QNB. Dissociation kinetic experiments using [ 3 H]NMS and combination of C 7 /3-phth with WIN 62,577 also provided evidence for neutral cooperativity between the two allosteric sites when the orthosteric site is occupied. Together, these results provide insight into the nature of the interaction between two distinct allosteric sites on the M 4 mAChR and how this interaction is perturbed upon occupancy of the orthosteric site.Muscarinic acetylcholine receptors (mAChRs) are prototypical members of the family A G protein-coupled receptor superfamily and mediate the majority of the actions of acetylcholine (ACh) in both the peripheral and the central nervous systems. Although these receptors are a focus of intense research as potential therapeutic targets, a significant challenge is the issue of high sequence conservation within the orthosteric domain across all five mAChR subtypes (Hulme et al., 1990;Wess, 1993). Such sequence conservation can account for the current paucity of orthosteric mAChR agonists and antagonists that display high selectivity for one mAChR subtype to the relative exclusion of all others. This problem is particularly pertinent to studies of the central nervous system, which is known to express all five subtypes of mAChR (Ehlert et al., 1995). Recent studies using mAChR knockout mice have highlighted the role of specific mAChRs in central disorders such as cognitive dysfunction, schizophrenia, and a variety of pain states (Gomeza et al., 2001;Hamilton et al., 2001;Wess et al., 2003), and thus, the ability to better target drugs to each of the mAChRs is of ongoing therapeutic relevance. 1 Current affiliation: Metabolic Pharmaceuticals Ltd., Baker Heart Research Institute, Prahran, Victoria, Australia.Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.024711.ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; C 7 /3-phth, heptane-1,7-bis-(dimethyl-3Ј-phthalimidopropyl)-ammonium bromide; DMEM, Dulbecco's modified Eagle's medium; NMS, N-methylscopolamine; ACh, acetylcholine; CH...

Molecular Mechanisms of Action and In Vivo Validation of an M4 Muscarinic Acetylcholine Receptor Allosteric Modulator with Potential Antipsychotic Properties

“…This finding is in general agreement with our prior mutagenesis study (Chan et al, 2008), which suggested an engagement of the modulator with extracellular loop regions of the M 4 mAChR and an important function of D 432 in the third extracellular loop; this residue has been implicated in the binding of the prototypical modulator, gallamine, to the M 4 mAChR (Gnagey et al, 1999). In contrast, the interaction between LY2033298 and atropine was negatively cooperative, whereas that between LY2033298 and WIN51708 was neutrally cooperative; both findings clearly indicate that LY2033298 does not interact with either the orthosteric site or a second allosteric site on the M 4 mAChR recognized by staurosporine, KT5720, and various WIN compounds (Lanzafame et al, 2006;Lazareno et al, 2000Lazareno et al, , 2002.…”

“…Preliminary mutagenesis experiments with LY2033298 also implicated extracellular loop regions in the actions of the modulator (Chan et al, 2008). However, there is another class of 'atypical' modulators, such as WIN51708, WIN62577, staurosporine, and KT5720, that bind to a second, currently unresolved, allosteric site on mAChRs (Lanzafame et al, 2006;Lazareno et al, 2000Lazareno et al, , 2002. Thus, to gain more definitive insight into which allosteric site is involved in the binding of LY2033298, we exploited the ability of the modulator to also act as an agonist and performed functional interaction studies between this compound and either the classic orthosteric antagonist, atropine, the 'prototypical-site' allosteric modulator, C 7 /3-phth, or the 'second-site' allosteric modulator, WIN51708.…”

Roof and Floor of the Muscarinic Binding Pocket: Variations in the Binding Modes of Orthosteric Ligands

“…It differs from a recent ab initio modeling prediction (Peng et al, 2006). Dissimilar docking of NMS and QNB might affect not only the kinetics of formation and breakdown but also the functional properties of the receptor-ligand complexes, perhaps accounting for differences in cooperativity between NMS, QNB, and allosteric ligands (Lanzafame et al, 2006) and affecting the potential for inverse agonism (Schwartz et al, 2006). The shorter side chain of ACh also fails to penetrate far into the TM domain and is unlikely to interact strongly with TM5 in the ground state of the receptor.…”

Comparison of Receptor Binding Characteristics of Commonly Used Muscarinic Antagonists in Human Bladder Detrusor and Mucosa

“…Radioligand binding with the muscarinic receptor ligand [ 3 H]QNB (specific activity, 37 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) was performed as described previously using membranes from the human detrusor muscle and mucosa (Mansfield et al, 2005) and from CHO cells (Lanzafame et al, 2006).…”