基于酰胺甲基化策略的GluA2内吞阻滞剂环肽的合成及评价

Synthesis and evaluation of cyclopeptide,a GluA2 endocytosis blocker based on amide methylation strategy

目的在GluA2-3Y结构基础上通过环化和N-甲基化策略设计甲基化环肽,提高化合物与靶标的亲和力、神经保护活性及稳定性。方法基于Fmoc固相合成法合成目标多肽,经RP-HPLC对多肽进行分析和纯化。采用表面等离子共振技术测试多肽与BRAG2蛋白的亲和力。通过CCK8法检测多肽对HT22细胞的毒性。构建氧糖剥夺(OGD)模型评价多肽的体外神经保护活性。通过HPLC检测多肽的血浆稳定性。结果基于Fmoc固相合成法共设计合成14条多肽,经RP-HPLC分析纯度均在90%以上。表面等离子共振结果显示:环肽c10c-Y-5表现出与对照药Ac-3Y相近的亲和力,解离常数KD为0.68μmol·L^(-1),与BRAG2蛋白有较强的亲和力。CCK8法测试多肽的毒性结果显示:10μmol·L^(-1)多肽在HT22细胞上基本没有毒性。环肽在OGD模型上对HT22细胞的神经保护活性结果显示:与模型组相比,环肽c10c-Y-2在1μmol·L^(-1)和10μmol·L^(-1)下有神经保护活性(P<0.05);环肽c10c-Y-5在3个浓度下有神经保护活性(P<0.05);环肽c10c-Y5G6在10μmol·L^(-1)下有神经保护活性(P<0.01)。环肽的血浆稳定性实验结果显示:c10c-Y-2和c10c-Y-5经环化和N-甲基化修饰后,与线性肽相比稳定性有大幅度提高。结论本研究应用SPR技术和多肽在OGD模型上对HT22细胞的神经保护活性评价手段,筛选出了具有潜在神经保护活性且增强了血浆稳定性的环肽c10c-Y-2和c10c-Y-5,可为多肽药物的修饰改造提供思路和依据。

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=0.68μmol·L^(-1),and had a strong affinity with BRAG2 protein.The CCK8 assay showed that 10μmol·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 1μmol·L^(-1)and 10μmol·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 10μmol·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.