CNS4 causes subtype‐specific changes in agonist efficacy and reversal potential of permeant cations in NMDA receptors

The NMDA subtype of glutamate receptor serves as an attractive drug target for the treatment of disorders evolving from hyper‐ or hypoglutamatergic conditions. Compounds that optimize the function of NMDA receptors are of great clinical significance. Here, we present the pharmacological characterization of a biased allosteric modulator, CNS4. Results indicate that CNS4 sensitizes ambient levels of agonists and reduces higher‐concentration glycine & glutamate efficacy in 1/2AB receptors, but minimally alters these parameters in diheteromeric 1/2A or 1/2B receptors. Glycine efficacy is increased in both 1/2C and 1/2D, while glutamate efficacy is decreased in 1/2C and unaltered in 1/2D. CNS4 does not affect the activity of competitive antagonist binding at glycine (DCKA) and glutamate (DL‐AP5) sites; however, it decreases memantine potency in 1/2A receptors but not in 1/2D receptors. Current–voltage (I‐V) relationship studies indicate that CNS4 potentiates 1/2A inward currents, a phenomenon that was reversed in the absence of permeable Na+ ions. In 1/2D receptors, CNS4 blocks inward currents based on extracellular Ca2+ concentration. Further, CNS4 positively modulates glutamate potency on E781A_1/2A mutant receptors, indicating its role at the distal end of the 1/2A agonist binding domain interface. Together, these findings reveal that CNS4 sensitizes ambient agonists and allosterically modulates agonist efficacy by altering Na+ permeability based on the GluN2 subunit composition. Overall, the pharmacology of CNS4 aligns with the need for drug candidates to treat hypoglutamatergic neuropsychiatric conditions such as loss function GRIN disorders and anti‐NMDA receptor encephalitis. Working hypothesis of CNS4 mechanism of action: Changes in glutamate concentration alter agonist binding domain (ABD) intersubunit interaction, that plays a crucial role in desensitization leading to channel constriction. CNS4 (blue) preferentially reverses the low glutamate (broken heart) induced changes in ABD conformation back to normal glutamate‐like ABD conformation facilitating cation influx through the NMDA receptor channel. This will be experimentally evaluated in the future.