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Author Notes:

To whom correspondence should be addressed: Dept. of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, DK-2100 Copenhagen, Denmark., Tel.: 45-3917-9659; E-mail: hbo@sund.ku.dk


Research Funding:

This work was supported by the Lundbeck Foundation; the GluTarget Programme of Excellence at the University of Copenhagen; the Danish Ministry of Science, Innovation, and Higher Education EliteForsk Programme; the Augustinus Foundation DanScatt; and the Danish Council for Independent Research-Medical Sciences.


  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • Crystal Structure
  • Ion Channels
  • Ligand-binding Protein
  • Molecular Pharmacology
  • Neurotransmitter Receptors
  • NMDA Receptor
  • Schild Analysis
  • Bioisoster
  • Competitive Antagonist
  • Glycine Site
  • CORE

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-D-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site


Journal Title:

Journal of Biological Chemistry


Volume 288, Number 46


, Pages 33124-33135

Type of Work:

Article | Final Publisher PDF


NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of gl ycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit- selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a] quinoxalin- 4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with K b values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed > 100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Copyright information:

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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