GW201 mediates neuroprotection via allosteric modulation of NMDA receptor activity in the middle cerebral artery occlusion (MCAO) model

BACKGROUND AND PURPOSE: Neuroexcitotoxicity mediated by NMDA receptor is a central contributor to ischemic stroke pathology. Neuroprotective effect and mechanism of the new compound GW201, which targets NMDA receptor, were studied. EXPERIMENTAL APPROACH: We evaluated the neuroprotective effects of GW201 using in vivo and in vitro models of ischemic stroke, including the middle cerebral artery occlusion/reperfusion (MCAOi/r) model, permanent MCAO (pMCAO) model and primary neuronal oxygen-glucose deprivation (OGD) model. Pharmacodynamic studies examined the dose-response relationship and therapeutic time window of GW201 neuroprotective effects. Electrophysiological membrane clamp experiments assessed the NMDA receptor subunit selectivity of GW201 and its influence on neuronal currents under ischemia-mimicking conditions. The mechanism of its action was further analysed using molecular docking technology. Live-cell fluorescence imaging was employed to evaluate the impact of GW201 on intracellular calcium levels in simulated ischemic stroke. Furthermore, bioinformatics analysis of the GEO database identified 16 significant calcium-related genes, with their regulation by GW201 validated in the MCAOi/r model. KEY RESULTS: Across all models tested, GW201 exhibited marked neuroprotective effects. Membrane clamp studies identified GW201 as an allosteric modulator of the NMDA receptor GluN2A subunit reducing current amplitude under high glutamate/NMDA + glycine conditions and subsequently lowering intracellular calcium levels. Bioinformatics analysis revealed 16 calcium-related genes, with GW201 significantly modulating Ccl3, Stat3, Anxa1, Anxa2, Mgp, S100A8, S100A9 and Cacna1a. CONCLUSIONS AND IMPLICATIONS: These results suggest that the neuroprotective effects of GW201 are mediated by its ability to allosterically modulate NMDA receptor activity, reducing calcium overload, influence downstream gene expression and suppress inflammatory responses.