Synthesis and evaluation of cyclopeptide,a GluA2 endocytosis blocker based on amide methylation strategy
Objective To design methylated cyclopeptides by cyclization and N-methylation strategies based on the GluA2-3Y structure to improve the affinity,neuroprotective activity and stability.Methods The target peptides were synthesized based on Fmoc solid-phase method,and then analyzed and purified by RP-HPLC.The affinity of peptides to BRAG2 protein was tested by surface plasmon resonance.The toxicity of peptides to HT22 cells was detected by CCK8.A model of oxygen-glucose deprivation was established to evaluate the neuroprotective activity of peptides in vitro.The plasma stability of peptides was detected by HPLC.Results Totally 14 peptides were designed and synthesized based on Fmoc solid-phase method,with purity more than 90％by RP-HPLC.The surface plasmon resonance showed that the affinity of cyclopeptide c10c-Y-5 was similar to that of control drug Ac-3Y,the dissociation constant KD=0.68 μmol·L-1,and had a strong affinity with BRAG2 protein.The CCK8 assay showed that 10 μmol·L-1 peptides had no toxicity on HT22 cells.The neuroprotective activity of cyclopeptide on HT22 cells in OGD model showed that:compared with the model group,cyclopeptide c10c-Y-2 had neuroprotective activity at 1 μmol·L-1 and 10 μmol·L-1(P＜0.05),and cyclopeptide c10c-Y-5 had neuroprotective activity at three concentrations(P＜0.05).Cyclopeptide c10c-Y5G6 had neuroprotective activity under 10 μmol·L-1(P＜0.01).The plasma stability test of cyclopeptides showed that the stability of both c10c-Y-2 and c10c-Y-5 was greatly improved as compared with linear peptides after cyclization and N-methylation.Conclusion By surface plasmon resonance technique and evaluation of neuroprotective activity of polypeptides on HT22 cells in OGD model,cyclopeptide c10c-Y-2 and c10c-Y-5 are screened out to show potential neuroprotective activity and may enhance the plasma stability.This study provides ideas and basis for the modification of peptide drugs.
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