Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties

Neuropsychopharmacology. 2010 Mar;35(4):855-69. doi: 10.1038/npp.2009.194. Epub 2009 Nov 25.


We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / metabolism*
  • Acetylcholine / pharmacology
  • Allosteric Regulation / drug effects
  • Allosteric Regulation / physiology
  • Allosteric Site / drug effects
  • Allosteric Site / physiology
  • Animals
  • Antipsychotic Agents / chemistry
  • Antipsychotic Agents / pharmacology*
  • Avoidance Learning / drug effects
  • Avoidance Learning / physiology
  • Binding, Competitive / drug effects*
  • Cell Line
  • Corpus Striatum / drug effects
  • Corpus Striatum / metabolism
  • Cricetinae
  • Cricetulus
  • Dose-Response Relationship, Drug
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • In Vitro Techniques
  • Mice
  • Mice, Knockout
  • Models, Molecular
  • Multivariate Analysis
  • Muscarinic Antagonists / pharmacokinetics
  • N-Methylscopolamine / pharmacokinetics
  • Nicotinic Acids / chemistry
  • Nicotinic Acids / pharmacology
  • Parasympatholytics / pharmacokinetics
  • Phosphorylation / drug effects
  • Protein Transport / drug effects
  • Quinuclidinyl Benzilate / pharmacokinetics
  • Radioligand Assay / methods
  • Rats
  • Receptor, Muscarinic M4 / chemistry
  • Receptor, Muscarinic M4 / deficiency
  • Receptor, Muscarinic M4 / drug effects
  • Receptor, Muscarinic M4 / physiology*
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Thiophenes / chemistry
  • Thiophenes / pharmacology
  • Tritium / metabolism
  • Tritium / pharmacokinetics


  • 3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamide
  • Antipsychotic Agents
  • Muscarinic Antagonists
  • Nicotinic Acids
  • Parasympatholytics
  • Receptor, Muscarinic M4
  • Thiophenes
  • Tritium
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Quinuclidinyl Benzilate
  • Acetylcholine
  • N-Methylscopolamine