NMDA receptor ligands have therapeutic potential in neurological and psychiatric disorders. We designed (R)-3-(5-thienyl)carboxamido-2-aminopropanoic acid derivatives with nanomolar agonist potencies at NMDA receptor subtypes (GluN12/A-D). These compounds are superagonists at GluN1/2C compared to glycine and partial to full agonists at GluN1/2A and GluN1/2D but display functional antagonism at GluN1/2B due to low agonist efficacy. Notably, 8d display 864% agonist efficacy at GluN1/2C relative to glycine, and 8j has high potency at GluN1/2A (0.018 μM), GluN1/2C (0.0029 μM), and GluN1/2D (0.016 μM). We evaluated the binding mode in the glycine site using molecular modeling and mutagenesis. In vitro absorption, distribution, metabolism, and excretion (ADME) assays predict high metabolic stability but poor blood–brain barrier permeability. However, an ester prodrug for the carboxylate group of 7j display moderately high blood–brain barrier permeability. The thiophenecarboxamide agonists expand the synthetic pharmacology of NMDA receptors and provide structural insights that facilitate the design of GluN1 agonists with GluN2 subunit-specific activity.

Recent investigations into the rapid antidepressant effects of ketamine, along with studies on schizophrenia-related susceptibility genes, have highlighted the GluN2A subunit as a critical regulator of both emotion and cognition. However, the specific impacts of acute pharmacological inhibition of GluN2A-containing NMDA receptors on brain microcircuits and the subsequent behavioral consequences remain poorly understood. In this study, we first examined the effects of MPX-004, a selective GluN2A NMDA receptor inhibitor, on behavior within the dorsomedial prefrontal cortex (dmPFC). Local administration of MPX-004 in the dmPFC led to a reduced immobility duration in the forced swim test, an acute antidepressant-like effect, impairments in sensorimotor gating, and a schizophrenia-like phenotype. In vivo multiple-channel recordings and c-Fos staining revealed that MPX-004 decreases the activity of parvalbumin-expressing interneurons (PV-INs) and increases the activity of pyramidal neurons (PYNs). In vivo patch-clamp recordings further confirmed that PV-IN inactivation leads to an elevated PYN firing rate in the PFC. In vitro whole-cell recordings demonstrated that PV-INs receive stronger excitatory synaptic input and respond more robustly to presynaptic stimulation than do somatostatin-expressing interneurons (SST-INs) and PYNs, rendering them susceptible to GluN2A inhibition. Finally, the specific knockdown of GluN2A in prefrontal PV-INs abolished the behavioral effects of MPX-004, underscoring a critical role of the GluN2A-mediated modulation of PV-INs in these phenotypes. Together, these findings reveal that PV-INs are particularly vulnerable to GluN2A inhibition, leading to disinhibition of prefrontal circuits and resulting in both antidepressant-like and schizophrenia-like behaviors.

NMDA-type ionotropic glutamate receptors mediate excitatory neurotransmission and synaptic plasticity, but aberrant signaling by these receptors is also implicated in brain disorders. Here, we present the binding site and the mechanism of action for UCM-101, a novel negative NMDA receptor modulator that produces full inhibition of NMDA receptor-mediated excitatory postsynaptic currents in hippocampal CA pyramidal neurons from juvenile mouse brain slices. UCM-101 has a 59-fold higher binding affinity at GluN1/2A compared with GluN1/2B receptors and inhibits diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors with IC50 values of 110 and 240 nM, respectively, in the presence of 1 µM glycine. The novel binding mode for UCM-101 is revealed in a high-resolution crystal structure of the GluN1/2A agonist binding domain heterodimer. UCM-101 and its analog TCN-213 inhibit NMDA receptors by negatively modulating co-agonist binding to the GluN1 subunit via an allosteric mechanism that is conserved with previously described GluN2A-selective antagonists, TCN-201 and MPX-004. Despite the shared mechanism of action, the structural determinants that mediate subunit selectivity for UCM-101 are distinct from those of TCN-201 and MPX-004. These findings provide detailed insights into the binding site and mechanism of action of a novel NMDA receptor modulator and open new avenues for the development of NMDA receptor ligands with therapeutic potential.

N-Methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors in the central nervous system (CNS), have garnered attention for their role in brain disorders. Specifically, GluN2A-containing NMDA receptors have emerged as a potential therapeutic target for the treatment of depressive disorders and epilepsy. However, the development of GluN2A-containing NMDA receptor-selective antagonists, represented by N-(4-(2-benzoylhydrazine-1-carbonyl)benzyl)-3-chloro-4-fluorobenzenesulfonamide (TCN-201) and its derivatives, faces a significant challenge due to their limited ability to penetrate the blood-brain barrier (BBB), hampering their in vivo characterization and further advancement. In this study, we reported a series of 2-((5-(phemylamino)-1,3,4-thiadiazol-2-yl)thio)-N-(cyclohexylmethyl)acetamide derivatives, achieved through a structure-guided optimization strategy using free energy perturbation (FEP) and BBB permeability estimation. Through systematic exploration of various phenyl substitutions, compound 1f emerged as a standout compound, demonstrating substantially enhanced inhibitory activity compared with the lead compound TCN-213. Compound 1f not only displayed satisfactory BBB permeability but also showed antidepressant-like potency in the hydrocortisone-induced zebrafish depression-like model. All results position it as a promising candidate for developing innovative therapeutics for NMDA receptor-related disorders.

N-methyl-d-aspartate (NMDA)-type ionotropic glutamate receptors are critically involved in excitatory neurotransmission and their dysfunction is implicated in many brain disorders. Allosteric modulators with selectivity for specific NMDA receptor subtypes are therefore attractive as therapeutic agents, and sustained drug discovery efforts have resulted in a wide range of new allosteric modulators. However, evaluation of allosteric NMDA receptor modulators is limited by the lack of operational ligand-receptor models to describe modulator binding dissociation constants (KB) and effects on agonist binding affinity (α) and efficacy (β). Here, we describe a pharmacological equilibrium model that encapsulates activation and modulation of NMDA receptors, and we apply this model to afford deeper understanding of GluN2A-selective negative allosteric modulators, TCN-201, MPX-004, and MPX-007. We exploit slow negative allosteric modulator unbinding to examine receptors at hemi-equilibrium when fully occupied by agonists and modulators to demonstrate that TCN-201 display weaker binding and negative modulation of glycine binding affinity (KB = 42 nM, α = 0.0032) compared with MPX-004 (KB = 9.3 nM, α = 0.0018) and MPX-007 (KB = 1.1 nM, α = 0.00053). MPX-004 increases agonist efficacy (β = 1.19), whereas TCN-201 (β = 0.76) and MPX-007 (β = 0.82) reduce agonist efficacy. These values describing allosteric modulation of diheteromeric GluN1/2A receptors with 2 modulator binding sites are unchanged in triheteromeric GluN1/2A/2B receptors with a single binding site. This evaluation of NMDA receptor modulation reveals differences between ligand analogs that shape their utility as pharmacological tool compounds and facilitates the design of new modulators with therapeutic potential. SIGNIFICANCE STATEMENT: Detailed understanding of allosteric N-methyl-d-aspartate (NMDA) receptor modulation requires pharmacological methods to quantify modulator binding affinity and the strengths of modulation of agonist binding and efficacy. We describe a generic ligand-receptor model for allosteric NMDA receptor modulation and use this model for the characterization of GluN2A-selective negative allosteric modulators. The model enables quantitative evaluation of a broad range of NMDA receptor modulators and provides opportunities to optimize these modulators by embellishing the interpretation of their structure-activity relationships.

NMDA-type ionotropic glutamate receptors are critical for normal brain function and are implicated in central nervous system disorders. Structure and function of NMDA receptors composed of GluN1 and GluN3 subunits are less understood compared to those composed of GluN1 and GluN2 subunits. GluN1/3 receptors display unusual activation properties in which binding of glycine to GluN1 elicits strong desensitization, while glycine binding to GluN3 alone is sufficient for activation. Here, we explore mechanisms by which GluN1-selective competitive antagonists, CGP-78608 and L-689,560, potentiate GluN1/3A and GluN1/3B receptors by preventing glycine binding to GluN1. We show that both CGP-78608 and L-689,560 prevent desensitization of GluN1/3 receptors, but CGP-78608-bound receptors display higher glycine potency and efficacy at GluN3 subunits compared to L-689,560-bound receptors. Furthermore, we demonstrate that L-689,560 is a potent antagonist of GluN1(FA+TL)/3A receptors, which are mutated to abolish glycine binding to GluN1, and that this inhibition is mediated by a non-competitive mechanism involving binding to the mutated GluN1 agonist binding domain (ABD) to negatively modulate glycine potency at GluN3A. Molecular dynamics simulations reveal that CGP-78608 and L-689,560 binding or mutations in the GluN1 glycine binding site promote distinct conformations of the GluN1 ABD, suggesting that the GluN1 ABD conformation influences agonist potency and efficacy at GluN3 subunits. These results uncover the mechanism that enables activation of native GluN1/3A receptors by application of glycine in the presence of CGP-78608, but not L-689,560, and demonstrate strong intra-subunit allosteric interactions in GluN1/3 receptors that may be relevant to neuronal signaling in brain function and disease.

Abstract N-Methyl-d-aspartate (NMDA) receptor hyperfunction plays a key role in the pathological processes of depression and neurodegenerative diseases, whereas NMDA receptor hypofunction is implicated in schizophrenia. Considerable efforts have been made to target NMDA receptor function for the therapeutic intervention in those brain disorders. In this mini-review, we first discuss ion flux-dependent NMDA receptor signaling and ion flux-independent NMDA receptor signaling that result from structural rearrangement upon binding of endogenous agonists. Then, we review current strategies for exploring druggable targets of the NMDA receptor signaling and promising future directions, which are poised to result in new therapeutic agents for several brain disorders.

N-Methyl-d-aspartate (NMDA) receptors play critical roles in central nervous system function and are involved in variety of brain disorders. We previously developed a series of (R)-3-(5-furanyl)carboxamido-2-aminopropanoic acid glycine site agonists with pronounced variation in activity among NMDA receptor GluN1/2A-D subtypes. Here, a series of (R)-2-amino-3-triazolpropanoic acid analogues with a novel chemical scaffold is designed and their pharmacological properties are evaluated at NMDA receptor subtypes. We found that the triazole can function as a bioisostere for amide to produce glycine site agonists with variation in activity among NMDA receptor subtypes. Compounds 13g and 13i are full and partial agonists, respectively, at GluN1/2C and GluN1/2D with 3- to 7-fold preference in agonist potency for GluN1/2C-D over GluN1/2A-B subtypes. The agonist binding mode of these triazole analogues and the mechanisms by which the triazole ring can serve as a bioisostere for amide were further explored using molecular dynamics simulations. Thus, the novel (R)-2-amino-3-triazolpropanoic acid derivatives reveal insights to agonist binding at the GluN1 subunit of NMDA receptors and provide new opportunities for the design of glycine site agonists.

NMDA receptors mediate glutamatergic neurotransmission and are therapeutic targets due to their involvement in a variety of psychiatric and neurological disorders. Here, we describe the design and synthesis of a series of (R)-3-(5-furanyl)carboxamido-2-aminopropanoic acid analogues 8a-s as agonists at the glycine (Gly) binding site in the GluN1 subunit, but not GluN3 subunits, of NMDA receptors. These novel analogues display highly variable potencies and agonist efficacies among the NMDA receptor subtypes (GluN1/2A-D) in a manner dependent on the GluN2 subunit. Notably, compound 8p is identified as a potent partial agonist at GluN1/2C (EC50 = 0.074 μM) with an agonist efficacy of 28% relative to activation by Gly and virtually no agonist activity at GluN1/2A, GluN1/2B, and GluN1/2D. Thus, these novel agonists can modulate the activity of specific NMDA receptor subtypes by replacing the full endogenous agonists Gly or d-serine (d-Ser), thereby providing new opportunities in the development of novel therapeutic agents.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

Abstract Parvalbumin-containing projection neurons of the medial-septum-diagonal band of Broca ( $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB ) are essential for hippocampal rhythms and learning operations yet are poorly understood at cellular and synaptic levels. We combined electrophysiological, optogenetic, and modeling approaches to investigate $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB neuronal properties. $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB neurons had intrinsic membrane properties distinct from acetylcholine- and somatostatin-containing MS-DBB subtypes. Viral expression of the fast-kinetic channelrhodopsin ChETA-YFP elicited action potentials to brief (1–2 ms) 470 nm light pulses. To investigate $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB transmission, light pulses at 5–50 Hz frequencies generated trains of inhibitory postsynaptic currents (IPSCs) in CA1 stratum oriens interneurons. Using a similar approach, optogenetic activation of local hippocampal PV ( $$\hbox {PV}_{\text{HC}}$$ PV HC ) neurons generated trains of $$\hbox {PV}_{\text{HC}}$$ PV HC -mediated IPSCs in CA1 pyramidal neurons. Both synapse types exhibited short-term depression (STD) of IPSCs. However, relative to $$\hbox {PV}_{\text{HC}}$$ PV HC synapses, $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB synapses possessed lower initial release probability, transiently resisted STD at gamma (20–50 Hz) frequencies, and recovered more rapidly from synaptic depression. Experimentally-constrained mathematical synapse models explored mechanistic differences. Relative to the $$\hbox {PV}_{\text{HC}}$$ PV HC model, the $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB model exhibited higher sensitivity to calcium accumulation, permitting a faster rate of calcium-dependent recovery from STD. In conclusion, resistance of $$\hbox {PV}_{\text{MS-DBB}}$$ PV MS-DBB synapses to STD during short gamma bursts enables robust long-range GABAergic transmission from MS-DBB to hippocampus.

Competitive antagonists for ionotropic glutamate receptors (iGluRs) are highly valuable tool compounds for studying health and disease states in the central nervous system. However, only few subtype selective tool compounds are available and the discovery of antagonists with novel iGluR subtype selectivity profiles remains a profound challenge. In this paper, we report an elaborate structure-activity relationship (SAR) study of the parental scaffold 2,3-trans-3-carboxy-3-phenyl-proline by the synthesis of 40 new analogues. Three synthetic strategies were employed with two new strategies of which one being a highly efficient and fully enantioselective strategy based on C(sp(3))-H activation methodology. The SAR study led to the conclusion that selectivity for the NMDA receptors was a general trend when adding substituents in the 5'-position. Selective NMDA receptor antagonists were obtained with high potency (IC50 values as low as 200 nM) and 3-34-fold preference for GluN1/GluN2A over GluN1/GluN2B-D NMDA receptors.

NMDA receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. The diverse functions of these receptors are tuned by deploying different combinations of GluN1 and GluN2 subunits (GluN2A-D) to form either diheteromeric NMDA receptors, which contain two GluN1 and two identical GluN2 subunits, or triheteromeric NMDA receptors, which contain two GluN1 and two distinct GluN2 subunits. Here, we characterize PTC-174, a novel positive allosteric modulator (PAM) of receptors containing GluN2C or GluN2D subunits. PTC-174 potentiates maximal current amplitudes by 1.8-fold for diheteromeric GluN1/2B receptors and by > 10-fold for GluN1/2C and GluN1/2D receptors. PTC-174 also potentiates responses from triheteromeric GluN1/2B/2D and GluN1/2A/2C receptors by 4.5-fold and 1.7-fold, respectively. By contrast, PTC-174 produces partial inhibition of responses from diheteromeric GluN1/2A and triheteromeric GluN1/2A/ 2B receptors. PTC-174 increases potencies of co-agonists glutamate and glycine by 2- to 5-fold at GluN1/2C and GluN1/2D receptors, and NMDA receptor activation facilitates allosteric modulation by PTC-174. At native NMDA receptors in GluN2D-expressing subthalamic nucleus neurons, PTC-174 increases the amplitude of responses to NMDA application and slows the decay of excitatory postsynaptic currents (EPSCs) evoked by internal capsule stimulation. Furthermore, PTC-174 increases the amplitude and slows the decay of EPSCs in hippocampal interneurons, but has not effect on the amplitudes of NMDA receptor-mediated EPSCs in hippocampal CA1 pyramidal neurons. Thus, PTC-174 provides a useful new pharmacological tool to investigate the molecular pharmacology and physiology of GluN2C- and GluN2D-containing NMDA receptors.

Determining biophysical details of spatially extended neurons is a challenge that needs to be overcome if we are to understand the dynamics of brain function from cellular perspectives. Moreover, we now know that we should not average across recordings from many cells of a given cell type to obtain quantitative measures such as conductance since measures can vary multiple-fold for a given cell type. In this work we examine whether a tight combination of experimental and computational work can address this challenge. The oriens-lacunosum/moleculare (OLM) interneuron operates as a “gate” that controls incoming sensory and ongoing contextual information in the CA1 of the hippocampus, making it essential to understand how its biophysical properties contribute to memory function. OLM cells fire phase-locked to the prominent hippocampal theta rhythms, and we previously used computational models to show that OLM cells exhibit high or low theta spiking resonance frequencies that depend respectively on whether their dendrites have hyperpolarization-activated cation channels (h-channels) or not. However, whether OLM cells actually possess dendritic h-channels is unknown at present. We performed a set of whole-cell recordings of OLM cells from mouse hippocampus and constructed three multi-compartment models using morphological and electrophysiological parameters extracted from the same OLM cell, including per-cell pharmacologically isolated h-channel currents. We found that the models best matched experiments when h-channels were present in the dendrites of each of the three model cells created. This strongly suggests that h-channels must be present in OLM cell dendrites and are not localized to their somata. Importantly, this work shows that a tight integration of model and experiment can help tackle the challenge of characterizing biophysical details and distributions in spatially extended neurons. Full spiking models were built for two of the OLM cells, matching their current clamp cell-specific electrophysiological recordings. Overall, our work presents a technical advancement in modeling OLM cells. Our models are available to the community to use to gain insight into cellular dynamics underlying hippocampal function.

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission. Most native NMDA receptors are tetrameric assemblies of two glycine-binding GluN1 and two glutamate-binding GluN2 subunits. Co-assembly of the glycine-binding GluN1 with glycine-binding GluN3 subunits (GluN3A-B) creates glycine activated receptors that possess strikingly different functional and pharmacological properties compared to GluN1/GluN2 NMDA receptors. The role of GluN1/GluN3 receptors in neuronal function remains unknown, in part due to lack of pharmacological tools with which to explore their physiological roles. We have identified the negative allosteric modulator EU1180-438, which is selective for GluN1/GluN3 receptors over GluN1/GluN2 NMDA receptors, AMPA, and kainate receptors. EU1180-438 is also inactive at GABA, glycine, and P2X receptors, but displays inhibition of some nicotinic acetylcholine receptors. Furthermore, we demonstrate that EU1180-438 produces robust inhibition of glycine-activated current responses mediated by native GluN1/GluN3A receptors in hippocampal CA1 pyramidal neurons. EU1180-438 is a non-competitive antagonist with activity that is independent of membrane potential (i.e. voltage-independent), glycine concentration, and extracellular pH. Non-stationary fluctuation analysis of neuronal current responses provided an estimated weighted mean unitary conductance of 6.1 pS for GluN1/GluN3A channels, and showed that EU1180-438 has no effect on conductance. Site-directed mutagenesis suggests that structural determinants of EU1180-438 activity reside near a short pre-M1 helix that lies parallel to the plane of the membrane below the agonist binding domain. These findings demonstrate that structural differences between GluN3 and other glutamate receptor subunits can be exploited to generate subunit-selective ligands with utility in exploring the roles GluN3 in neuronal function.

Determining details of spatially extended neurons is a challenge that needs to be overcome. The oriens-lacunosum/moleculare (OLM) interneuron has been implicated as a critical controller of hippocampal memory making it essential to understand how its biophysical properties contribute to function. We previously used computational models to show that OLM cells exhibit theta spiking resonance frequencies that depend on their dendrites having hyperpolarization-activated cation channels (h-channels). However, whether OLM cells have dendritic h-channels is unknown. We performed a set of whole-cell recordings of OLM cells from mouse hippocampus and constructed multi-compartment models using morphological and electrophysiological parameters extracted from the same cell. The models matched experiments only when dendritic h-channels were present. Immunohistochemical localization of the HCN2 subunit confirmed dendritic expression. These models can be used to obtain insight into hippocampal function. Our work shows that a tight integration of model and experiment tackles the challenge of characterizing spatially extended neurons.

We report a series of glutamate and aspartate analogues designed using the hydroxy-1,2,3-triazole moiety as a bioisostere for the distal carboxylic acid. Compound 6b showed unprecedented selectivity among ( S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtypes, confirmed also by an unusual binding mode observed for the crystal structures in complex with the AMPA receptor GluA2 agonist-binding domain. Here, a methionine (Met729) was highly disordered compared to previous agonist-bound structures. This observation provides a possible explanation for the pharmacological profile. In the structure with 7a, an unusual organization of water molecules around the bioisostere arises compared to previous structures of ligands with other bioisosteres. Aspartate analogue 8 with the hydroxy-1,2,3-triazole moiety directly attached to glycine was unexpectedly able to activate both the glutamate and glycine agonist-binding sites of the N-methyl-d-aspartic acid receptor. These observations demonstrate novel features that arise when employing a hydroxytriazole moiety as a bioisostere for the distal carboxylic acid in glutamate receptor agonists.

Development of pharmacological tools for the ionotropic glutamate receptors (iGluRs) is imperative for the study and understanding of the role and function of these receptors in the central nervous system. We report the synthesis of 18 analogues of (2 S,3 R)-2-carboxy-3-pyrrolidine acetic acid (3a), which explores the effect of introducing a substituent on the ε-carbon (3c-q). A new synthetic method was developed for the efficient synthesis of racemic 3a and applied to give expedited access to 13 racemic analogues of 3a. Pharmacological characterization was carried out at native iGluRs, cloned homomeric kainate receptors (GluK1-3), NMDA receptors (GluN1/GluN2A-D), and excitatory amino acid transporters (EAAT1-3). From the structure-activity relationship studies, several new ligands emerged, exemplified by triazole 3p-d1, GluK3-preferring (GluK1/GluK3 Ki ratio of 15), and the structurally closely related tetrazole 3q-s3-4 that displayed 4.4-100-fold preference as an antagonist for the GluN1/GluN2A receptor ( Ki = 0.61 μM) over GluN1/GluN2B-D ( Ki = 2.7-62 μM).

Triheteromeric NMDA receptors contain two GluN1 and two distinct GluN2 subunits and mediate excitatory neurotransmission in the CNS. Triheteromeric GluN1/2B/2D receptors have functional properties intermediate to those of diheteromeric GluN1/2B and GluN1/2D receptors. GluN1/2B/2D receptors are more sensitive to channel blockade by ketamine and memantine compared to GluN1/2B receptors in the presence of physiological Mg2+. GluN2B‐selective antagonists produce robust inhibition of GluN1/2B/2D receptors, and the GluN2B‐selective positive allosteric modulator spermine enhances responses from GluN1/2B/2D but not GluN1/2A/2B receptors. These insights into the properties of triheteromeric GluN1/2B/2D receptors are necessary to appreciate their physiological roles in neural circuit function and the actions of therapeutic agents targeting NMDA receptors.