Structure-based discovery of antagonists for GluN3-containing N-methyl-d-aspartate receptors

Abstract

NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.

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

“…At higher glycine concentrations, the selective block by TK40 at GluN1 subunits is outcompeted, resulting in diminished receptor current (declining phase), which is in agreement with previous reports (41,42). For glycine-activated responses at wild-type GluN1/N3 receptors, we have previously reported that only negligible current responses (Ͻ10 nA) are observed at GluN1/N3A receptors upon glycine application and that glycine concentrations above 10 M result in diminished receptor current at GluN1/N3B receptors, thereby producing a bell-shaped concentration-response relationship (38). Despite the potentiation, we still observed bell-shaped glycine concentration-response profiles at both wild-type GluN1/N3A and GluN1/N3B receptors in the presence of 3 M TK40, with the receptor current diminished at glycine concentrations above 100 M (Fig.…”

“…We recently reported a virtual screen of GluN3A LBD that was based on three hybrid models of the GluN3A LBD in an antagonist-bound state (38). The models were virtually screened against a compound library of ϳ4 million commercially available compounds, of which 99 shortlisted compounds were obtained.…”

“…The models were virtually screened against a compound library of ϳ4 million commercially available compounds, of which 99 shortlisted compounds were obtained. The compounds were functionally screened at GluN3A and GluN3B subunits, and active hits were subsequently functionally evaluated at diheteromeric GluN1/N2 receptors using electrophysiological recordings (38). Using this approach, we identified TK40 (Fig.…”

“…Using this approach, we identified TK40 (Fig. 1A) as a potent antagonist at the GluN1/N2A receptor (38).…”

“…We have recently reported a method to isolate the GluN3 pharmacology of GluN1/N3 receptors by mutating the GluN1 subunit (i.e. GluN1(F484A/T518L)), which renders the GluN1 subunit insensitive to glycine and abolishes the concentration-dependent inhibitory component of glycine at GluN1/N3 receptors even at supersaturating glycine concentrations (38). Here, we exploit this mutated diheteromeric GluN1(F484A/T518L)/N3 receptor to characterize the interaction of TK40 with the GluN3 subunit.…”