Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry

J Biomol Screen. 2006 Mar;11(2):194-207. doi: 10.1177/1087057105284340. Epub 2006 Feb 20.


Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

MeSH terms

  • Acetylcholine
  • Allosteric Regulation
  • Allosteric Site
  • Animals
  • Cell Line
  • Dose-Response Relationship, Drug
  • Guinea Pigs
  • Insecta
  • Ligands
  • Mass Spectrometry / methods*
  • Molecular Structure
  • Muscarinic Antagonists / pharmacology*
  • Protein Binding / drug effects*
  • Radioligand Assay
  • Receptor, Muscarinic M2 / chemistry*
  • Receptor, Muscarinic M2 / isolation & purification
  • Receptors, G-Protein-Coupled / antagonists & inhibitors*


  • Ligands
  • Muscarinic Antagonists
  • Receptor, Muscarinic M2
  • Receptors, G-Protein-Coupled
  • Acetylcholine