Structure-Function Studies of Allosteric Agonism at M₂Muscarinic Acetylcholine Receptors

Abstract

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;

Characterization of a CNS penetrant, selective M1muscarinic receptor agonist, 77‐LH‐28‐1

“…Intuitively, one would expect that the high degree of mAChR selectivity of 77-LH-28-1 is derived from an interaction with the M 1 mAChR via an allosteric binding site, as has been described previously for AC-42 (Langmead et al, 2006). The interaction of 77-LH-28-1 with the M 2 mAChR has been recently examined, and, in agreement with the studies presented here, 77-LH-28-1 displayed little or no agonist activity at this receptor subtype (May et al, 2007). However, it does interact with the M 2 mAChR with low affinity (in agreement with the calcium mobilization data presented in Table 1) and it significantly retards the rate of [ 3 H]N-methyl scopolamine dissociation from the M 2 mAChR, indicative of an allosteric mode of action (May et al, 2007).…”

“…The interaction of 77-LH-28-1 with the M 2 mAChR has been recently examined, and, in agreement with the studies presented here, 77-LH-28-1 displayed little or no agonist activity at this receptor subtype (May et al, 2007). However, it does interact with the M 2 mAChR with low affinity (in agreement with the calcium mobilization data presented in Table 1) and it significantly retards the rate of [ 3 H]N-methyl scopolamine dissociation from the M 2 mAChR, indicative of an allosteric mode of action (May et al, 2007). Further studies are required to fully define the mechanism of action by which 77-LH-28-1 activates the M 1 mAChR.…”

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

“…Compounds such as AC-42, 77-LH-28-1, N-desmethylclozapine, TBPB, and McN-A-343 have been earlier classified as 'allosteric agonists' at M 1 and/or M 2 mAChRs (Birdsall et al, 1983;Jones et al, 2008;May et al, 2007a;Spalding et al, 2006;Sur et al, 2003). However, the interaction between each of these ligands and orthosteric mAChR ligands is antagonistic, suggesting that they either interact with both the orthosteric and an allosteric site on the mAChR, or otherwise are highly negatively cooperative with orthosteric ligands.…”

“…LY2033298 binds to the M 4 mAChR at a Region that Overlaps with the 'Prototypical' Allosteric-Binding Site Extensive mutagenesis studies have determined a function for residues in the second and third extracellular loops and at the top of transmembrane domain VII to be important for the binding of prototypical allosteric modulators such as C 7 /3-phth and gallamine at mAChRs (Ellis et al, 1993;Huang et al, 2005;Krejci and Tucek, 2001;Leppik et al, 1994;May et al, 2007a;Prilla et al, 2006;Voigtlander et al, 2003). Preliminary mutagenesis experiments with LY2033298 also implicated extracellular loop regions in the actions of the modulator (Chan et al, 2008).…”

Parkinson's Disease Therapeutics: New Developments and Challenges Since the Introduction of Levodopa

“…These agents provide new opportunities to determine the physiological roles of individual mAChRs in the basal ganglia circuitry and to assess the antiparkinsonian efficacy of highly selective mAChR antagonists (Conn et al, 2009a, b), but to do so the full specificity profile of these compounds must be characterized in vivo. In this regard, recent in vivo studies indicate that the allosteric modulators display favorable pharmacokinetic properties and bloodbrain barrier permeability, and have confirmed their potential therapeutic benefits in rodent models of Alzheimer's disease and schizophrenia (Caccamo et al, 2006;May et al, 2007;Brady et al, 2008;Chan et al, 2008;Shekhar et al, 2008;Conn et al, 2009a, b;Bridges et al, 2010;Digby et al, 2010 A 2A receptor antagonists (Schwarzschild et al, 2006;Menon and Stacy, 2008;Morelli et al, 2010;Shah and Hodgson, 2010). Adenosine is a ubiquitous purine with signaling properties that mediate its effects through four subtypes of G-protein-coupled adenosine receptors: A 1 , A 2A , A 2B , and A 3 (Schwarzschild et al, 2006;Menon and Stacy, 2008;Morelli et al, 2007Morelli et al, , 2009Morelli et al, , 2010Shah and Hodgson, 2010).…”