Dizocilpine-derived K2060 exhibits enhanced blockage at NMDA receptors with impaired Mg 2+ block and provides strong protection against audiogenic seizures

Abstract 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.