Martin Horak

Background and Purpose: Voltage-dependent Mg ²⁺ block is a defining feature of the GluN1/GluN2 subtype of N -methyl-D-aspartate receptors (NMDARs). We tested how impaired Mg ²⁺ block at GluN1/GluN2A receptors mutated at the GluN2A-N615 position modifies inhibition by dizocilpine-derived K2060 compared with clinically used memantine and ketamine. Experimental Approach: Electrophysiological recordings were performed on recombinant wild-type (WT) and mutated GluN1/GluN2A receptors expressed in HEK293T cells. Acute hippocampal slice electrophysiology and behavioural studies were conducted in WT and knock-in GluN2A-N615S mice. In vivo tolerability and efficacy were evaluated using open field testing, prepulse inhibition (PPI), and protection against audiogenic seizures (AGS). Key Results: Mg ²⁺ reduced the apparent potency of all three blockers and slowed onset at the GluN1/GluN2A-WT receptor. These Mg ²⁺ -dependent effects were absent or markedly reduced at the GluN1/GluN2A-N615S and GluN1/GluN2A-N615K receptors. In addition, K2060 showed slower unbinding and greater trapping than memantine. In hippocampal slices from GluN2A-N615S mice, K2060 exhibited enhanced but slower onset inhibition than memantine and ketamine. In vivo , K2060 caused no overt behavioural disruption at 5 mg/kg ( i.p. ) and did not further disrupt PPI. All three blockers protected GluN2A-N615S mice against AGS at 5 mg/kg ( i.p. ); only K2060 remained protective at 1 mg/kg ( i.p. ). Conclusions and Implications: Impaired Mg²⁺ block removes a physiological constraint on the action of NMDAR blockers. K2060 exhibited the most favourable pharmacological profile, supporting its further evaluation for studies with NMDAR hyperfunction models. Additional pharmacokinetic, safety, and disease-model studies are warranted. What is already known?: NMDAR blockers, including memantine, ketamine and the dizocilpine-derived K2060, bind within the ion channel pore, where Mg ²⁺ can influence their potency and kinetics. The GluN2A-N615S variant reduces Mg ²⁺ block and causes NMDAR hyperfunction associated with neuronal hyperexcitability and audiogenic seizure susceptibility in mice. What does this study add?: Impaired Mg ²⁺ block of the mutated NMDARs at the GluN2A-N615 position removes Mg ²⁺ -dependent modulation of blocker potency and kinetics. K2060 produces sustained inhibition of recombinant and endogenous GluN2A-N615S-containing NMDARs and protects GluN2A-N615S mice against AGS at a lower dose than memantine or ketamine. What is the clinical significance?: GRIN2A pore variants that reduce Mg ²⁺ block alter sensitivity to NMDAR blockers. K2060 warrants further preclinical evaluation for NMDAR hyperfunction animal models.

N -Methyl- d -aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (Embryonic Day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared with WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared with the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant.

N -Methyl- d -aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the human embryonic kidney 293 (HEK293) cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC ₅₀ values for glycine (GluN1 subunit mutations) while not correlating with the measured EC ₅₀ values for l -glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC ₅₀ values for glycine, as well as with the calculated Δ G _binding values for glycine obtained from molecular dynamics simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC ₅₀ values for l -glutamate or calculated Δ G _binding values for l -glutamate. Coexpression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.

We aimed to prepare novel dibenzosuberane derivatives that act on N-methyl-D-aspartate (NMDA) receptors with potential neuroprotective effects. Our approach involved modifying the tropane moiety of MK-801, a potent open-channel blocker known for its psychomimetic side effects, by introducing a seven-membered ring with substituted base moieties specifically to alleviate these undesirable effects. Our in silico analyses showed that these derivatives should have high gastrointestinal absorption and cross the blood-brain barrier (BBB). Our pharmacokinetic studies in rats supported this conclusion and confirmed the ability of leading compounds 3l and 6f to penetrate the BBB. Electrophysiological experiments showed that all compounds exhibited different inhibitory activity towards the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B. Of the selected compounds intentionally differing in the inhibitory efficacy, 6f showed high relative inhibition (∼90% for GluN1/GluN2A), while 3l showed moderate inhibition (∼50%). An in vivo toxicity study determined that compounds 3l and 6f were safe at 10 mg/kg doses with no adverse effects. Behavioral studies demonstrated that these compounds did not induce hyperlocomotion or impair prepulse inhibition of startle response in rats. Neuroprotective assays using a model of NMDA-induced hippocampal neurodegeneration showed that compound 3l at a concentration of 30 μM significantly reduced hippocampal damage in rats. These results suggest that these novel dibenzosuberane derivatives are promising candidates for developing NMDA receptor-targeted therapies with minimal psychotomimetic side effects.

N -methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer’s disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity “membrane-to-channel inhibition” (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg ²⁺ . K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 µM K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by ∼51%, outperforming 30 µM memantine (∼21% inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 µM, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060’s potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.

The search for novel drugs to address the medical needs of Alzheimer’s disease (AD) is an ongoing process relying on the discovery of disease-modifying agents. Given the complexity of the disease, such an aim can be pursued by developing so-called multi-target directed ligands (MTDLs) that will impact the disease pathophysiology more comprehensively. Herewith, we contemplated the therapeutic efficacy of an amiridine drug acting as a cholinesterase inhibitor by converting it into a novel class of novel MTDLs. Applying the linking approach, we have paired amiridine as a core building block with memantine/adamantylamine, trolox, and substituted benzothiazole moieties to generate novel MTDLs endowed with additional properties like N-methyl-D-aspartate (NMDA) receptor affinity, antioxidant capacity, and anti-amyloid properties, respectively. The top-ranked amiridine-based compound 5d was also inspected by in silico to reveal the butyrylcholinesterase binding differences with its close structural analogue 5b. Our study provides insight into the discovery of novel amiridine-based drugs by broadening their target-engaged profile from cholinesterase inhibitors towards MTDLs with potential implications in AD therapy.

The essential role of N-methyl-D-aspartate receptors (NMDARs) in excitatory neurotransmission is underscored by numerous pathogenic variants in the GluN subunits, including those identified in their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs; however, whether it controls the structure of LBDs or the functionality of NMDARs is unknown. Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A receptors, the primary NMDAR subtype in the adult forebrain, strongly correlates with EC ₅₀ values for glycine and L-glutamate. Interestingly, co-expression of both GluN1 and GluN2A subunits with alanine substitutions led to an additive reduction in the surface number of GluN1/GluN2A receptors, as did co-expression of both GluN1 and GluN2A subunits containing closed cleft conformation of LBDs. The synchronized ER release confirmed the altered regulation of early trafficking of GluN1/GluN2A receptors bearing alanine substitutions in the LBDs. Furthermore, the human versions of GluN1/GluN2A receptors containing pathogenic GluN1-S688Y, GluN1-S688P, GluN1-D732E, GluN2A-S511L, and GluN2A-T690M variants exhibited distinct surface expression compared to the corresponding alanine substitutions. Mutant cycles of GluN1-S688, GluN1-D732, GluN2A-S511, and GluN2A-T690 residues revealed, in most cases, a weak correlation between surface expression of the mutant GluN1/GluN2A receptors and their EC ₅₀ values for glycine or L-glutamate. Consistent with our experimental data, molecular modeling and dynamics showed that the ER quality control machinery likely perceives structural changes of the LBDs but not the functionality of GluN1/GluN2A receptors. Significant statement: Our study showed that structural changes in LBDs independently regulate the early trafficking of GluN1/GluN2A receptors and that the surface numbers of mutant GluN1/GluN2A receptors do not necessarily correlate with their agonist sensitivity. In addition, we validated a novel system of synchronized release of GluN1/GluN2A receptors from the ER. Together, our experimental and in silico findings support the urgency of further detailed research on the regulation of early trafficking of NMDARs, as it may open the avenue to targeted therapeutic intervention of CNS disorders associated with pathogenic variants in GluN subunits.