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 EC50 values for glycine (GluN1 subunit mutations) while not correlating with the measured EC50 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 EC50 values for glycine, as well as with the calculated ΔGbinding 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 EC50 values for l-glutamate or calculated ΔGbinding 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.

• MeSH
• Glycine metabolism   MeSH
• HEK293 Cells MeSH
• Hippocampus cytology   metabolism   MeSH
• Rats MeSH
• Glutamic Acid metabolism   MeSH
• Humans MeSH
• Ligands MeSH
• Mutation genetics   MeSH
• Protein Domains MeSH
• Nerve Tissue Proteins MeSH
• Receptors, N-Methyl-D-Aspartate * metabolism   genetics   chemistry   MeSH
• Protein Transport physiology   genetics   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

We aimed to prepare novel dibenzo [a,d][7]annulen 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 dibenzo [a,d][7]annulen derivatives are promising candidates for developing NMDA receptor-targeted therapies with minimal psychotomimetic side effects.

• MeSH
• Dizocilpine Maleate * pharmacology   MeSH
• Blood-Brain Barrier metabolism   drug effects   MeSH
• Rats MeSH
• Humans MeSH
• Molecular Structure MeSH
• Neuroprotective Agents * pharmacology   chemistry   chemical synthesis   MeSH
• Rats, Sprague-Dawley MeSH
• Receptors, N-Methyl-D-Aspartate * antagonists & inhibitors   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH