A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M₂ Muscarinic Receptor

Abstract

THRX-160209 is a potent antagonist at the M 2 muscarinic acetylcholine (ACh) receptor subtype that was designed using a multivalent strategy, simultaneously targeting the orthosteric site and a nearby site known to bind allosteric ligands. In this report, we describe three characteristics of THRX-160209 binding that are consistent with a multivalent interaction: 1) an apparent affinity of the multivalent ligand for the M 2 receptor subtype (apparent pK I ϭ 9.51 Ϯ 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK I values Ͻ 6.0), 2) specificity of THRX-160209 for the M 2 receptor subtype compared with the closely related M 4 (apparent pK I ϭ 8.78 Ϯ 0.24) and M 1 , M 3 , and M 5 receptors (apparent pK I values Յ 8.0), and 3) acceleration (Ͼ10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M 2 receptors by competing monovalent ligands that are known to interact with either the orthosteric site (e.g., atropine) or a well characterized allosteric site (e.g., obidoxime) on the receptor. In complementary kinetic studies assessing allosteric modulation of the receptor, unlabeled THRX-160209 retarded dissociation of [3 H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [ 3 H]NMS dissociation were competitively inhibited by obidoxime, suggesting that obidoxime and THRX-160209 bind to an overlapping region coincident with other typical muscarinic allosteric agents, such as 3-methyl-5-W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M 2 receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.The muscarinic receptor family consists of five subtypes that are found in smooth and cardiac muscle, epithelial and endothelial cells, secretory cells, neurons, and inflammatory cells (Caulfield, 1993;Racke and Matthiesen, 2004). These receptors represent attractive targets for drug design in a variety of therapeutic areas, including overactive bladder, chronic obstructive pulmonary disease, and Parkinson's disease (Eglen et al., 2001;Katzenschlager et al., 2003;Barnes, 2004;Hegde et al., 2004). However, most currently marketed, muscarinic receptor-targeted therapies exhibit significant side effects, some of which are due to potent drug interactions at undesirable muscarinic receptor subtypes outside the intended organ system. High sequence homology within the orthosteric site, across the five receptor subtypes, makes the synthesis of tissue-specific or subtype-specific drugs challenging (Hulme et al., 1990). A drug with specificity for a given subtype or tissue is predicted to have increased efficacy but decreased side effects relative to a drug with equal affinity for all subtypes (Gainetdinov and Caron, 1999).Muscarinic receptors are known to be subject to modulation by ligands that do not bind to the ACh binding pocket (the "orthosteric site") (Ellis et al., 1991). Targeting these "secondary" or "allosteri...

Cooperativity Has Empirical and Ultimate Levels of Explanation

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Discovery of a Novel Class of Negative Allosteric Modulator of the Dopamine D2 Receptor Through Fragmentation of a Bitopic Ligand

“…3,5 Such an approach has been successfully exploited to target GPCRs within the muscarinic acetylcholine receptor (mAChR) and adenosine receptor families. [6][7][8][9] In addition, recent studies have demonstrated that ligands that selectively target the M 1 and M 2 mAChRs may achieve their receptor-subtype-selectivity through a hitherto unappreciated bitopic mode of interaction. 10,11 One approach to explore the binding mode of bitopic ligands is to isolate both orthosteric and allosteric fragments from the parent structure.…”

Molecular Mechanisms of Bitopic Ligand Engagement with the M1 Muscarinic Acetylcholine Receptor

“…For example, although the M 1 mAChR-selective agonist N-desmethylclozapine was originally suggested to be allosteric, definitive evidence for an allosteric interaction with the receptor is lacking (10 -12). Moreover, novel bitopic ligands have recently been described, which concomitantly associate with both the orthosteric binding site and an allosteric binding site (13)(14)(15)(16)(17)(18)(19); it is possible that previous studies of selective agonists have classified such bitopic ligands as purely allosteric.…”