A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor

Mol Pharmacol. 2007 Aug;72(2):291-302. doi: 10.1124/mol.106.033746. Epub 2007 May 3.

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.

MeSH terms

  • Allosteric Site
  • Animals
  • Benzeneacetamides / metabolism*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Ligands
  • Muscarinic Antagonists / metabolism*
  • N-Methylscopolamine / metabolism
  • Obidoxime Chloride / pharmacology
  • Piperidines / metabolism*
  • Radioligand Assay
  • Receptor, Muscarinic M2 / chemistry
  • Receptor, Muscarinic M2 / metabolism*

Substances

  • 4-(N-(7-(3-(1-carbamoyl-1,1-diphenylmethyl)pyrrolidin-1-yl)hept-1-yl)-N-(n-propyl)amino)-1-(2,6-dimethoxybenzyl)piperidine
  • Benzeneacetamides
  • Ligands
  • Muscarinic Antagonists
  • Piperidines
  • Receptor, Muscarinic M2
  • Obidoxime Chloride
  • N-Methylscopolamine