Inhibition of GluN2B-containing <i>N</i> -methyl- <scp>D-</scp> aspartate receptors by radiprodil

N-methyl-D-aspartate (NMDA) receptors mediate a slow, Ca2+-permeable component of excitatory synaptic transmission in the brain and participate in neuronal development and synaptic plasticity. Most NMDA receptors are tetrameric assemblies of two GluN1 and two GluN2 subunits encoded by five genes (GRIN1 and GRIN2A-GRIN2D), which produce GluN1 and GluN2A-GluN2D subunits. NMDA receptors that contain the GluN2B subunit have unique pharmacological properties, being inhibited by multiple structurally distinct series of biaryl compounds with high potency and selectivity. These agents are of considerable therapeutic interest, given the numerous roles that GluN2B-containing NMDA receptors play in normal brain function and pathological situations. Among GluN2B-selective negative allosteric modulators, radiprodil inhibits NMDA receptors that contain GluN2B with high potency and selectivity and appears to be safe in humans. Here, we evaluate the structural determinants of radiprodil binding to the heterodimeric GluN1-GluN2B amino terminal domain by X-ray crystallography and explore the molecular mechanism of inhibition. A large number of de novo variants have been identified in the GRIN gene family in patients with various neurological and neuropsychiatric conditions, including autism, intellectual disability, epilepsy, language disorders and movement disorders. We show that radiprodil is an effective antagonist at >80% of human disease-associated GRIN1 and GRIN2B missense variants tested in vitro (22/27, equally or more effective as wild-type receptors), including variants in the pore-forming region, linker regions and elsewhere that uniformly increase NMDA receptor-mediated charge transfer. We show that radiprodil blocks synaptic GluN2B receptors in brain slices acutely isolated from a knock-in mouse line harbouring the gain-of-function variant GluN2B-Ser810Arg associated with early-onset epileptic encephalopathy and intractable seizures in patients. In addition, radiprodil delays the onset of seizures (458 ± 90 s, versus 207 ± 23 s in the vehicle group) in response to in vivo administration of the chemoconvulsant pentylenetetrazole. These data support the potential utility of GluN2B-selective antagonists, such as radiprodil, for clinical treatments of neurological conditions where clinical aetiologies might involve increased current mediated by GluN2B-containing NMDA receptors.