Discovery And Evaluation Of Neuroprotective Activity Of Allosteric Modulators Targeting The N-terminal Domain Of The GluN1/N2A Receptor

Objective:The NMDA receptor is an ionotropic glutamate receptor with high calcium permeability and is a key mediator of excitatory synaptic transmission.Physiological activation of NMDA receptors is involved in important neurobiological processes such as synaptic plasticity,memory formation,brain development,and neuronal survival.However,excessive activation of NMDA receptors under pathological conditions can produce excitotoxicity,leading to neuronal death.The subunit selection doctrine reveals that activation of NMDA receptors containing Glu N2 A promotes neuronal survival and exerts neuroprotective effects,while activation of NMDA receptors containing Glu N2 B is the main cause of excitotoxicity and neuronal apoptosis.The positive allosteric modulator(PAM)of Glu N2 A protects neurons by activating neuronal survival signals and has a longer therapeutic window and fewer adverse effects than conventional NMDAR antagonists.The aim of this study was to identify novel allosteric modulators targeting the N-terminal structural domain(NTD)of the Glu N1/N2 A receptor using a classical drug discovery strategy combined with in vitro activity evaluation,providing new preclinical pharmacological tools for ischemic stroke and new ideas for neuroprotection.Methods:First,this study used a molecular docking-based virtual screening technique to identify potential compounds with a metastable modulatory effect by acting on the N-terminal structural domain of the Glu N1/N2 A receptor.Candidate compounds were identified with reference to simulated small molecule-receptor affinity scoring and by hand selection.Next,a cell model of transient transfection of HEK-293 with different isoforms of NMDA receptors was constructed at the molecular level,and the calcium in-flow fluorescence assay was used to verify the metastable activity and selectivity of the candidate compounds.After ADME prediction of the compounds,excitotoxic pharmacological models of glutamateinduced PC-12 cells and HT-22 cells were constructed at the cellular level to evaluate the in vitro neuroprotective activity and toxicity of the candidate compounds.Selected target compounds were validated for in vivo neuroprotective activity in a mouse t MCAO model to examine their therapeutic effects on neurological deficits in ischemic brain injury and their effects on cerebral infarct size.Finally,molecular simulations and protein immunoblotting were used to preliminarily investigate the mechanisms by which the compounds exert neuroprotection.Results:During the virtual screening process,the fluorescence assay of calcium inward flow showed that compounds 02,03,04,05,06,15,18,19,20,and 23 had variable activation of the Glu N2 A subunit and good selectivity and were identified as potential NMDAR PAMs.Compounds 15 and 23 had the most significant effect on agonist-induced enhancement of calcium inward flow compared to the agonist group(12.5% and 11.8%,P<0.005);in vitro toxicity evaluation showed that compounds 02,03,04,06,15,and 19 had no significant effect on the growth status of PC-12 and HT-22 cells.Compounds 18,20,and 23 showed toxic effects on PC-12 cells and 05,18,and 20 on HT-22 cells.In the in vitro neuroprotective activity evaluation assay,the results of the glutamate-induced cytokinetic toxicity assay showed that compounds 02,03,04,05,19,20,and 23 exhibited significant cytoprotective effects in the PC-12 cell model with 0.1 M,1 M,and 10 M concentration interventions.Compounds 06 and 18 showed significant cytoprotective effects at two concentrations.In the HT-22 cell model,compound 15 and 23 showed significant cytoprotective effects at all three concentrations.The compounds that showed significant cytoprotective effects at two concentrations were 02,04,18,and 19.In the in vivo neuroprotective activity evaluation of compound 15,the medium(5 mg/kg)and high(10 mg/kg)dose groups showed significant improvement in neurological deficit symptoms and a significant reduction in the area of post-ischemic cerebral infarction compared to the model group(30.68% and 42.70%,P<0.005).Molecular docking results showed that compound 15 was stably bound in the pocket through hydrophobic interactions with the receptor residues Leu219,Ile 227,and Ile 239,as well as hydrogen bonding interactions with Ser224.WB results showed that compound 15 enhanced Glu N2A-mediated phosphorylation of downstream CREB(c AMP response element binding protein).Conclusion:This study established a systematic drug discovery strategy of "in silico,in vitro,and in vivo",which provided a new idea for the clinical treatment of ischemic stroke and confirmed the feasibility of a computationally assisted drug discovery method in this system.After physical screening at the molecular level,compounds 02,03,04,05,06,15,18,19,20,and 23 were identified as potential Glu N1/N2 A PAMs;the in vitro evaluation model of excitotoxicity was established in PC-12 and HT-22 double cell lines;the in vitro neuroprotective activity of the above 10 compounds was verified;further,the in vivo anti The neuroprotective effect of compound 15(Aegeline)was also reported for the first time.In summary,the results of this thesis provide a new basis for further screening of the development of lead compounds with therapeutic efficacy against ischemic stroke.