Crystal structure and pharmacological characterization of a novel N-methyl-D-aspartate (NMDA) receptor antagonist at the GluN1 glycine binding site.
NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-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 Kb 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.
From the Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Trine
T
Kvist
Thomas Bielefeldt
TB
Steffensen
Jeremy R
JR
Greenwood
Fatemeh
F
Mehrzad Tabrizi
Kasper B
KB
Hansen
Michael
M
Gajhede
Darryl S
DS
Pickering
Stephen F
SF
Traynelis
Jette Sandholm
JS
Kastrup
Hans
H
Bräuner-Osborne
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011810:Quinoxalines / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000819:agonists* / Q000737:chemistry / Q000235:genetics; D014230:Triazoles / Q000737:chemistry* / Q000494:pharmacology; D014982:Xenopus laevis
C585833:1-thioxo-1,2-dihydro(1,2,4)triazolo(4,3-a)quinoxalin-4(5H)-one; D002352:Carrier Proteins; C121673:Gprin1 protein, mouse; D009419:Nerve Tissue Proteins; D011810:Quinoxalines; D016194:Receptors, N-Methyl-D-Aspartate; D014230:Triazoles
Bioisoster; Competitive Antagonist; Crystal Structure; Glycine Site; Ion Channels; Ligand-binding Protein; Molecular Pharmacology; NMDA Receptor; Neurotransmitter Receptors; Schild Analysis