A subunit-selective negative allosteric modulator of GluN1/GluN3A glycine-activated receptor

The ionotropic glutamate N-methyl-d-aspartate (NMDA) receptor gene family includes 7 homologous subunits (GluN1, GluN2A-D, and GluN3A-B). The coassembly of GluN1 and GluN2 form the well-studied NMDA receptor, which requires binding of both glutamate and glycine to be activated. However, glycine alone can activate receptors assembled from the GluN1/GluN3 subunits, though the role of these glycine-activated receptors in the central nervous system remains poorly understood. We have previously identified a negative allosteric modulator (EU1180-438) that was a selective inhibitor of GluN3-containing receptors over NMDA, AMPA, kainate, GABA, inhibitory glycine, and other receptors. This compound was equally potent for both GluN3A- and GluN3B-containing receptors. Here we describe 2 novel negative allosteric modulators, EU1180-560 and EU1180-590, that are selective for GluN1/GluN3A over GluN1/GluN3B receptors. EU1180-560 and EU1180-590 inhibit GluN1/GluN3A receptors with an IC₅₀ value of approximately 2.6 μM and 3.7 μM, respectively, with no detectable effect on GluN1/GluN3B receptors. Both compounds produce noncompetitive, voltage-independent inhibition, consistent with a negative allosteric modulation mechanism. These compounds also have little to no effect on GluN1/GluN2-containing NMDA, AMPA, GABA, glycine, P2X, and kainate receptors. EU1180-590 additionally shows promising brain penetration. These results emphasize the potential utility of a GluN1/GluN3A subunit-selective allosteric modulator for use as experimental tool compounds to advance our understanding of the physiological role of GluN1/GluN3A receptors in the brain. These data also demonstrate the feasibility for development of GluN3A-selective therapeutic agents for potential treatment of neurological and neuropsychiatric diseases. SIGNIFICANCE STATEMENT: A novel negative allosteric modulator that acts specifically at GluN1/GluN3A receptors was identified. This compound should serve as a useful tool in future studies designed to elucidate the functional role of GluN1/GluN3A receptors in the central nervous system.