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

Theravance, Inc., South San Francisco, California

THRX-160209 is a potent antagonist at the M₂ 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 M₂ receptor subtype (apparent pK_I = 9.51 ± 0.22) that was several orders of magnitude greater than its two monovalent components (apparent pK_I values < 6.0), 2) specificity of THRX-160209 for the M₂ receptor subtype compared with the closely related M₄ (apparent pK_I = 8.78 ± 0.24) and M₁,M₃, and M₅ receptors (apparent pK_I values 8.0), and 3) acceleration (>10-fold) of the dissociation rate of tritium-labeled THRX-160209 from M₂ 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 [³H]N-methyl scopolamine (NMS). The effects of THRX-160209 on retardation of [³H]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-[7-[4-[(4S)-4-methyl-1,3-oxazolidin-2-yl]phenoxy]heptyl]-1,2-oxazole (W84) and gallamine. Taken together, these data are consistent with the hypothesis that THRX-160209 binds in a multivalent manner to the M₂ receptor, simultaneously occupying the orthosteric site and a spatially distinct allosteric site.

Received for publication January 8, 2007.

Accepted for publication May 3, 2007.

Address correspondence to: Tod Steinfeld, Department of Molecular and Cellular Biology, Theravance, Inc., 901 Gateway Blvd, South San Francisco, California. E-mail: tsteinfeld{at}theravance.com

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