Sephin1 protects from glutamate stress, consistent with negative allosteric modulation of GluN2B-containing NMDA receptor

Glutamate excitotoxicity and GluN2B containing N-methyl- d -aspartate receptors (NMDARs) are involved in neurodegenerative diseases such as Amyotrophic Lateral Sclerosis, Parkinson disease and Alzheimer disease. Recently, Sephin1, a first-in-class, multi-functional, brain-penetrant, orally administered small molecule, has been demonstrated in vitro to improve motor neurons survival following glutamate intoxication. In ALS animal models, Sephin1 was able to reduce motor neuron loss, to improve motor function and increase animal survival. To determine how Sephin1 can reduce motor neurons loss following glutamate intoxication, calcium flux was measured in primary cortical neurons and in GluN1A/GluN2B overexpressing cells following NMDA stimulation. Then, to determine the binding site on GluN1 and GluN2B subunits, binding assays were performed with three benzylideneaminoguanidine derivatives, Sephin1, guanabenz and IFB-048. Here, for the first time, we show that Sephin1 reduces calcium flux in NMDA stimulated primary cortical neurons in a dose dependent manner. Sephin1, guanabenz and IFB-048 reduce the rise of calcium flux in GluN1A/GluN2B subunits overexpressing cells stimulated with NMDA. In addition, we demonstrated that Sephin1, guanabenz and IFB-048, displace ifenprodil from the N-terminal domain (NTD) of NMDAR (GluN1/GluN2B) containing the polyamine site on GluN2B subunits. The three benzylideneaminoguanidine derivatives could possibly be Negative Allosteric Modulator (NAM) of GluN2B-containing NMDARs.