OBJECTIVE:GRIN-related disorders due to pathogenic variants in GRIN1, GRIN2A, GRIN2B, or GRIN2D genes are associated with altered N-methyl-D-aspartate receptor (NMDAR) function. Functional changes include gain (GoF) and loss of receptor function (LoF). Clinical reports describing the use of the NMDAR blocker memantine in GRIN-related disorders show a diverse and inconsistent spectrum of treatment responses. METHODS:To evaluate clinical responses to memantine, we collected retrospective data on 34 individuals with GRIN variants, including 20 unpublished and 14 published cases. Variants were reclassified following American College of Medical Genetics and Genomics guidelines, and six in vitro functional assays were used to assess receptor function. We compared individuals with pathogenic GoF versus LoF in terms of associated clinical improvements, memantine sensitivity, and variant localization within the gene. RESULTS:In 19 of the 34 variants, a pathogenic likely or possible GoF of the receptor was detected. Fourteen of 19 individuals (74%) benefited from memantine, comprising improvements in behavior (71%), development (50%), and seizure frequency (39%). Individuals with either LoF or a functionally indeterminate or no effect GRIN variant (15/34 individuals) showed significantly less benefit from memantine treatment but nevertheless rare adverse events (3/15). An increased distance of the variant from the memantine binding site was associated with a clinical benefit. SIGNIFICANCE:Our retrospective observational study outlines the importance of correct classification of GRIN variants with regard to pathogenicity and functional consequence prior to applying memantine or other precision medicine approaches in clinical trials. Furthermore, the distance from a GoF variant to the memantine binding site correlated with a positive treatment response and may, at least in part, explain different degrees of therapeutic benefit.

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.

GABAergic inhibitory interneurons balance brain activity by suppressing excessive excitatory signaling and shaping network oscillations. Despite this critical function, our understanding of how neural circuits regulate GABAergic systems remains limited. Although N-methyl-D-aspartate receptor (NMDAR) functionality is defined by the GluN2A-D subunits, the interneuron NMDAR subunit composition remains unknown. Here, we show that GluN2D-NMDARs reside at presynaptic terminals of hippocampal parvalbumin-interneurons, and that the GluN2D intracellular C-terminal contains presynaptic targeting information. GluN2D-NMDARs are activated by changes in basal glutamate to bidirectionally control GABA release. Importantly, inhibition of GABA release by GluN2C/D modulators and subanesthetic ketamine is absent in mice lacking GluN2D in parvalbumin-interneurons, suggesting that GluN2D-dependent presynaptic regulation of GABA release may underlie some of ketamine's effects. The GluN2C/D-selective inhibitor NAB14 recapitulates durable ketamine-induced enhancement of long-term potentiation (LTP) and recovery of reward motivation in a behavioral model of depression without ketamine's undesirable effects, suggesting that GluN2D-NMDARs could be a therapeutically relevant target.

Rare Mendelian disorders affect 300-400 million people globally. Although genetic testing has become widely adopted, gene-specific evidence for tailored variant interpretation remains scattered across resources. We present Gene Portals, a framework for gene-centered multimodal knowledge bases that co-localize expert-harmonized clinical data, functional assays, population variation, structural annotations and gene-specific ACMG/AMP specifications within a single resource. A modular interface integrates this unified evidence with VCEP-refined ACMG specifications to enable automated gene-specific variant classification, infer molecular mechanisms, and support cross-gene analyses. We demonstrate the framework's utility across five Gene portals spanning eleven neurodevelopmental disorder-associated genes, integrating data from 4,423 individuals with 2,838 unique variants, 36,149 ClinVar submissions, and 1,044 expert-curated molecular readouts. By organizing evidence that is otherwise dispersed across multiple sources into a unified, queryable framework, the SCN, GRIN, CACNA1A, SATB2 and SLC6A1 Gene Portals became widely used community resources and provide an extensible template for standardized rare-disease variant interpretation and mechanism-aware discovery.

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.

N-Methyl-d-aspartate receptors (NMDARs) are a family of ligand-gated ionotropic glutamate receptors that mediate a slow, calcium-permeable component to excitatory neurotransmission. The GluN2D subunit is enriched in GABAergic inhibitory interneurons in cortical tissue. Diminished levels of GABAergic inhibition contribute to multiple neuropsychiatric conditions, suggesting that enhancing inhibition might have therapeutic utility, thus making GluN2D modulation an attractive drug target. Here, we describe the actions of a GluN2C/GluN2D-selective positive allosteric modulator, (+)-EU1180-453, which has improved drug-like properties, such as increased aqueous solubility, in comparison to the first-in-class GluN2C/GluN2D-selective prototypical positive allosteric modulator, (+)-CIQ. (+)-EU1180-453 doubles the NMDAR response at lower concentrations and produces a greater degree of maximal potentiation at 30 µM compared with (+)-CIQ. Using in vitro electrophysiological recordings, we show that (+)-EU1180-453 potentiates triheteromeric NMDARs containing at least one GluN2C or GluN2D subunit and is active at both exon5-lacking and exon5-containing GluN1 splice variants. (+)-EU1180-453 increases glutamate efficacy for GluN2C/GluN2D-containing NMDARs both by prolonging the deactivation time and by potentiating the peak response amplitude. We show that (+)-EU1180-453 selectively increases synaptic NMDAR-mediated charge transfer onto postnatal day 11-15 CA1 stratum radiatum hippocampal interneurons but is without effect on CA1 pyramidal cells. This increased charge transfer enhances inhibitory output from GABAergic interneurons onto CA1 pyramidal cells in a GluN2D-dependent manner. (+)-EU1180-453 also shifts excitatory-to-inhibitory coupling towards increased inhibition and produces enhanced gamma-band power from carbachol-induced field potential oscillations in hippocampal slices. Thus, (+)-EU1180-453 can enhance overall circuit inhibition, which could prove therapeutically useful for the treatment of anxiety, depression, schizophrenia and other neuropsychiatric disorders. KEY POINTS: (+)EU-1180-453 is a GluN2C/GluN2D positive allosteric modulator and is active at triheteromeric receptors. (+)EU-1180-453 is active at exon5-containing and exon5-lacking GluN1-containing receptors. (+)EU-1180-453 selectively potentiates the interneuron network and can enhance carbachol-induced gamma-band power.

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that mediate a slow, Ca2+-permeable component of excitatory neurotransmission. Modulation of NMDAR function has the potential for disease modification as NMDAR dysfunction has been implicated in neurodevelopment, neuropsychiatric, neurologic, and neurodegenerative disorders. We recently described the thieno[2,3-day]pyrimidin-4-one (EU1622) class of positive allosteric modulators, including several potent and efficacious analogs. Here we have used electrophysiological recordings from Xenopus oocytes, human embryonic kidney cells, and cultured cerebellar and cortical neurons to determine the mechanisms of action of a representative member of this class of modulator. EU1622-240 enhances current response to saturating agonist (doubling response amplitude at 0.2-0.5 μM), slows the deactivation time course following rapid removal of glutamate, increases open probability, enhances coagonist potency, and reduces single-channel conductance. We also show that EU1622-240 facilitates NMDAR activation when only glutamate or glycine is bound. EU1622-240-bound NMDARs channels activated by a single agonist (glutamate or glycine) open to a unique conductance level with different pore properties and Mg2+ sensitivity, in contrast to channels arising from activation of NMDARs with both coagonists bound. These data demonstrate that previously hypothesized distinct gating steps can be controlled by glutamate and glycine binding and shows that the 1622-series modulators enable glutamate- or glycine-bound NMDARs to generate open conformations with different pore properties. The properties of this class of allosteric modulators present intriguing therapeutic opportunities for the modulation of circuit function. SIGNIFICANCE STATEMENT: NMDA receptors are expressed throughout the central nervous system and are permeable to calcium. EU1622-240 increases open probability and agonist potency while reducing single-channel conductance and prolonging the deactivation time course. EU1622-240 allows NMDA receptor activation by the binding of one coagonist (glycine or glutamate), which produces channels with distinct properties. Evaluation of this modulator provides insight into gating mechanisms and may lead to the development of new therapeutic strategies.

Abstract GRID1 and GRID2 encode the enigmatic GluD1 and GluD2 proteins, which form tetrameric receptors that play important roles in synapse organization and development of the central nervous system. Variation in these genes has been implicated in neurodevelopmental phenotypes. We evaluated GRID1 and GRID2 human variants from the literature, ClinVar, and clinical laboratories and found that many of these variants reside in intolerant domains, including the amino terminal domain of both GRID1 and GRID2. Other conserved regions, such as the M3 transmembrane domain, show different intolerance between GRID1 and GRID2. We introduced these variants into GluD1 and GluD2 cDNA and performed electrophysiological and biochemical assays to investigate the mechanisms of dysfunction of GRID1/2 variants. One variant in the GRID1 distal amino terminal domain resides at a position predicted to interact with Cbln2/Cbln4, and the variant disrupts complex formation between GluD1 and Cbln2, which could perturb its role in synapse organization. We also discovered that, like the lurcher mutation (GluD2-A654T), other rare variants in the GRID2 M3 domain create constitutively active receptors that share similar pathogenic phenotypes. We also found that the SCHEMA schizophrenia M3 variant GluD1-A650T produced constitutively active receptors. We tested a variety of compounds for their ability to inhibit constitutive currents of GluD receptor variants and found that pentamidine potently inhibited GluD2-T649A constitutive channels (IC50 50 nM). These results identify regions of intolerance to variation in the GRID genes, illustrate the functional consequences of GRID1 and GRID2 variants, and suggest how these receptors function normally and in disease.

N-methyl-D-aspartate (NMDA) receptors mediate a slow component of excitatory synaptic transmission, are widely distributed throughout the central nervous system, and regulate synaptic plasticity. NMDA receptor modulators have long been considered as potential treatments for psychiatric disorders including depression and schizophrenia, neurodevelopmental disorders such as Rett Syndrome, and neurodegenerative conditions such as Alzheimer's disease. New interest in NMDA receptors as therapeutic targets has been spurred by the findings that certain inhibitors of NMDA receptors produce surprisingly rapid and robust antidepressant activity by a novel mechanism, the induction of changes in the brain that well outlast the presence of drug in the body. These findings are driving research into an entirely new paradigm for using NMDA receptor antagonists in a host of related conditions. At the same time positive allosteric modulators of NMDA receptors are being pursued for enhancing synaptic function in diseases that feature NMDA receptor hypofunction. While there is great promise, developing the therapeutic potential of NMDA receptor modulators must also navigate the potential significant risks posed by the use of such agents. We review here the emerging pharmacology of agents that target different NMDA receptor subtypes, offering new avenues for capturing the therapeutic potential of targeting this important receptor class.

N-methyl- d -aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1 , GRIN2A , and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure–function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1–4 identified in patients with various neurological disorders. These variants produce changes in agonist EC 50 , response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients’ clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

The short pre-M1 helix within the S1-M1 linker (also referred to as the pre-M1 linker) between the agonist-binding domain (ABD, S1) and the M1 transmembrane helix of the NMDA receptor (NMDAR) is devoid of missense variants within the healthy population but is a locus for de novo pathogenic variants associated with neurological disorders. Several de novo variants within this helix have been identified in patients presenting early in life with intellectual disability, developmental delay, and/or epilepsy. In this study, we evaluated functional properties for twenty variants within the pre-M1 linker in GRIN1, GRIN2A, and GRIN2B genes, including six novel missense variants. The effects of pre-M1 variants on agonist potency, sensitivity to endogenous allosteric modulators, response time course, channel open probability, and surface expression were assessed. Our data indicated that virtually all of the variants evaluated altered channel function, and multiple variants had profound functional consequences, which may contribute to the neurological conditions in the patients harboring the variants in this region. These data strongly suggest that the residues within the pre-M1 helix play a key role in channel gating and are highly intolerant to genetic variation.

Advances in sequencing technology have generated a large amount of genetic data from patients with neurological conditions. These data have provided diagnosis of many rare diseases, including a number of pathogenic de novo missense variants in GRIN genes encoding N-methyl-d-aspartate receptors (NMDARs). To understand the ramifications for neurons and brain circuits affected by rare patient variants, functional analysis of the variant receptor is necessary in model systems. For NMDARs, this functional analysis needs to assess multiple properties in order to understand how variants could impact receptor function in neurons. One can then use these data to determine whether the overall actions will increase or decrease NMDAR-mediated charge transfer. Here, we describe an analytical and comprehensive framework by which to categorize GRIN variants as either gain-of-function (GoF) or loss-of-function (LoF) and apply this approach to GRIN2B variants identified in patients and the general population. This framework draws on results from six different assays that assess the impact of the variant on NMDAR sensitivity to agonists and endogenous modulators, trafficking to the plasma membrane, response time course and channel open probability. We propose to integrate data from multiple in vitro assays to arrive at a variant classification, and suggest threshold levels that guide confidence. The data supporting GoF and LoF determination are essential to assessing pathogenicity and patient stratification for clinical trials as personalized pharmacological and genetic agents that can enhance or reduce receptor function are advanced. This approach to functional variant classification can generalize to other disorders associated with missense variants.

OBJECTIVE:To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS:Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS:Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE:This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

ID 53705 Poster Board 71 The N-methyl-D-aspartate receptor (NMDAR) is a ligand gated ion channel that is permeable to Na+ and Ca2+ and mediates a slow component of excitatory neurotransmission. NMDARs are implicated in synaptic plasticity and disease states such as in Parkinson’s, Alzheimer’s, Schizophrenia, and stroke. Here we have evaluated the characteristics at the single channel level of three series of allosteric modulators with structural determinants of bindings in regions that participate in NMDAR channel gating. We have generated two series of positive allosteric modulators (PAMs), EU1622 and EU1794 that generate two and three subconductance states respectively, and reduce the Ca2+:Na+ permeability ratio (Perszyk et al., 2018, 2020) – a first in class features of NMDAR modulators. We are exploring the mechanisms underlying these two classes of unique conductance-modifying allosteric modulators. Specifically, we are measuring effects on open probability, conductance, mean open time, deactivation time course, and agonist potency for conductance modifying modulators for comparison to modulators (EU1180 series) that do not alter conductance. In addition, the reduction of calcium permeability holds interesting therapeutic potential due to reduced neurotoxic risk of NMDA receptor potentiation compared to non-conductance modifying modulators. Ongoing efforts by our group are working to identify the mechanisms by which these modulators mediate changes in calcium permeability, which may be related to changes in calcium binding affinity to residues within or near the pore, or changes in pore diameter. S.F.T. is a PI on a research grants from Janssen to Emory, is a member of the SAB for Sage Therapeutics, Eumentis Therapeutics, and CombinedBrain, a senior advisor for GRIN Therapeutics, is on the Medical Advisory Board for the CureGRIN Foundation and the GRIN2B Foundation, is co-founder of NeurOp Inc and Agrithera, is on the Board of Directors for NeurOp Inc. S.F.T and D.C.L are co-inventors on Emory-owned Intellectual property that includes the allosteric modulators discussed in this work. D.C.L is a member of the Board of Directors for NeurOp Inc

N-methyl-D-aspartate receptors (NMDAR), ionotropic glutamate receptors, mediate a slow component of excitatory synaptic transmission in the central nervous system and play a key role in normal brain function and development. Genetic variations in GRIN genes encoding NMDAR subunits that alter the receptor's functional characteristics are associated with a wide range of neurological and neuropsychiatric conditions. Pathological GRIN variants located in the M2 re-entrant loop lining the channel pore cause significant functional changes, the most consequential alteration being a reduction in voltage-dependent Mg2+ inhibition. Voltage-dependent Mg2+ block is a unique feature of NMDAR biology whereby channel activation requires both ligand binding and postsynaptic membrane depolarization. Thus, loss of NMDAR Mg2+ block will have a profound impact on synaptic function and plasticity. Here, we choose 11 missense variants within the GRIN1, GRIN2A, and GRIN2B genes that alter residues located in the M2 loop and significantly reduce Mg2+ inhibition. Each variant was evaluated for tolerance to genetic variation using the 3-dimensional structure and assessed for functional rescue pharmacology via electrophysiological recordings. Three FDA-approved NMDAR drugs-memantine, dextromethorphan, and ketamine-were chosen based on their ability to bind near the M2 re-entrant loop, potentially rectifying dysregulated NMDAR function by supplementing the reduced voltage-dependent Mg2+ block. These results provide insight of structural determinants of FDA-approved NMDAR drugs at their binding sites in the channel pore and may further define conditions necessary for the use of such agents as potential rescue pharmacology.

Subunit-selective inhibition of N-methyl-d-aspartate receptors (NMDARs) is a promising therapeutic strategy for several neurological disorders, including epilepsy, Alzheimer's and Parkinson's disease, depression, and acute brain injury. We previously described the dihydroquinoline-pyrazoline (DQP) analogue 2a (DQP-26) as a potent NMDAR negative allosteric modulator with selectivity for GluN2C/D over GluN2A/B. However, moderate (<100-fold) subunit selectivity, inadequate cell-membrane permeability, and poor brain penetration complicated the use of 2a as an in vivo probe. In an effort to improve selectivity and the pharmacokinetic profile of the series, we performed additional structure-activity relationship studies of the succinate side chain and investigated the use of prodrugs to mask the pendant carboxylic acid. These efforts led to discovery of the analogue (S)-(-)-2i, also referred to as (S)-(-)-DQP-997-74, which exhibits >100- and >300-fold selectivity for GluN2C- and GluN2D-containing NMDARs (IC50 0.069 and 0.035 μM, respectively) compared to GluN2A- and GluN2B-containing receptors (IC50 5.2 and 16 μM, respectively) and has no effects on AMPA, kainate, or GluN1/GluN3 receptors. Compound (S)-(-)-2i is 5-fold more potent than (S)-2a. In addition, compound 2i shows a time-dependent enhancement of inhibitory actions at GluN2C- and GluN2D-containing NMDARs in the presence of the agonist glutamate, which could attenuate hypersynchronous activity driven by high-frequency excitatory synaptic transmission. Consistent with this finding, compound 2i significantly reduced the number of epileptic events in a murine model of tuberous sclerosis complex (TSC)-induced epilepsy that is associated with upregulation of the GluN2C subunit. Thus, 2i represents a robust tool for the GluN2C/D target validation. Esterification of the succinate carboxylate improved brain penetration, suggesting a strategy for therapeutic development of this series for NMDAR-associated neurological conditions.