Abstract

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.

Highlights

  • The glycine-binding GluN1 and GluN3 subunits of NMDA receptors have distinctive selectivity profiles

  • Pharmacological Characterization of TK40—We aimed at exploiting the differences in the GluN1 and GluN3 orthosteric binding sites to generate antagonists that can discriminate between the glycine-binding GluN1 and GluN3 subunits

  • Virtual screening can successfully identify competitive antagonists that can discriminate between GluN1 and GluN3 subunits of NMDA receptors

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Summary

Background

The glycine-binding GluN1 and GluN3 subunits of NMDA receptors have distinctive selectivity profiles. 5,7Dichlorokynurenic acid (5,7-DCKA) [17] is a widely used glycine site antagonist in studies of the physiological roles of NMDA receptors due to its high potency and selectivity for the GluN1 subunit [1, 15, 16]. Despite binding of the same endogenous ligand to both GluN1 and GluN3 subunits, glycine has been reported to bind with a 650-fold higher affinity at the isolated GluN3A LBD over the isolated GluN1 LBD, indicating that the glycine-binding site of GluN3 is different from that of GluN1 [23] At present, it remains unclear how these binding affinities determined at the soluble LBD translate into potencies at full-length receptors. We report here the pharmacological characterization of this novel antagonist at NMDA receptor subtypes and its binding mode using x-ray crystallography

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