title: 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 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 <or= 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.
authors.affiliation: Department of Molecular and Cellular Biology, Theravance, Inc., 901 Gateway Blvd, South San Francisco, California, USA. tsteinfeld@theravance.com
authors.forename: Tod
authors.initials: T
authors.lastname: Steinfeld
authors.forename: Mathai
authors.initials: M
authors.lastname: Mammen
authors.forename: Jacqueline A M
authors.initials: JA
authors.lastname: Smith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Wilson
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Jasper
mesh_terms: D000495:Allosteric Site; D000818:Animals; D044482:Benzeneacetamides / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D008024:Ligands; D018727:Muscarinic Antagonists / Q000378:metabolism*; D019832:N-Methylscopolamine / Q000378:metabolism; D009768:Obidoxime Chloride / Q000494:pharmacology; D010880:Piperidines / Q000378:metabolism*; D011869:Radioligand Assay; D043585:Receptor, Muscarinic M2 / Q000737:chemistry / Q000378:metabolism*
chemical_list: C522459:4-(N-(7-(3-(1-carbamoyl-1,1-diphenylmethyl)pyrrolidin-1-yl)hept-1-yl)-N-(n-propyl)amino)-1-(2,6-dimethoxybenzyl)piperidine; D044482:Benzeneacetamides; D008024:Ligands; D018727:Muscarinic Antagonists; D010880:Piperidines; D043585:Receptor, Muscarinic M2; D009768:Obidoxime Chloride; D019832:N-Methylscopolamine