摘要
目的:寻找MDM2的多肽抑制剂,从多角度探讨多肽药物在靶向肿瘤治疗中的潜在价值。方法:通过分子模拟对接技术对已有多肽库中的102条多肽进行结合测试;使用Alpha-lisa技术,分析多肽对MDM2与p53相互作用的影响;BIAcore技术研究多肽与MDM2的相互作用,及对MDM2和p53之间结合的影响;Western Blotting法检测多肽对MCF-7细胞中MDM2、p53及p21表达的影响。结果:Alpha-lisa对分子对接中得分较高的多肽进行验证,发现含苯环结构的Peptide 58:Ser-Tyr-Val-Trp及Peptide 95:Ala-Ser-Asp-Phe两条多肽对MDM2与p53结合破坏较大,分别在50.0和80.0 nmol/L即可产生抑制(Peptide 58:t=5.36,P<0.05,Peptide 95:t=31.26,P<0.05);BIAcore实验发现,Peptide 58与Peptide 95可特异性的与MDM2结合,较p53相比。它们与MDM2的结合常数分别为(389.6±13.4)nmol/L和(1.6±0.2)μmol/L,而与p53的结合常数分别为(19.5±2.4)μmol/L(t=6.85,P<0.01)和(12.8±1.4)μmol/L(t=8.32,P<0.01);Western Blotting结果表明,Peptide 58与Peptide 95能上调p53在MCF-7细胞内的表达水平,并引起p21表达的增加,相对p53基因,两肽对MDM2本身的表达水平影响较小。结论:Peptide 58与Peptide 95在体外对MDM2有较强的亲和抑制效应,但体内作用效果欠佳,原因可能是由于多肽进入细胞的比率较低或在细胞内由于蛋白酶的作用降解失效。
Objective:To seek the pepdide inhibitors of MDM2,then approach the potential value of polypeptide drugs in treatment of tumors from different angles.Methods:An existing peptides library consists of 102 peptides were first utilized to dock with MDM2 crystal structure by molecular simulation technology,then,the hits were followly reanalyzed by alpha-lisa,in order to ascertain that whether them had the capacity of inhibition to MDM2-p53 interplay or not.BIAcoretechnology was employed to comfirm the bingding of peptides to MDM2,and recheck the destructive effect of peptides on MDM2-p53 interplay.Western Blotting assay was finally introduced to ascertain the impact of peptides on MDM2-p53 pathway in MCF-7 cell lines.Results:After being reanalyzed by alpha-lisa assay,two peptides with phenyl structure(Peptide 58:Ser-Tyr-Val-Trp,Peptide 95:Ala-Ser-Asp-Phe)which based on virtual screening were distinguished from the potential hits.Destructiveness of these peptides to MDM2-p53 interplay was conspicuous,only 50.0 nmol/L Peptide 58 nmol/L or 80.0 nmol/L Peptide 95 was able to demonstrate a significant preventation(rather than:t=5.36,P<0.05,Peptide 95:t=31.26,P<0.05).BIAcore experiment found that Peptide 58 and Peptide 95 were able to directly bind to MDM2 rather than p53,for the dissociation constants of MDM2-Peptide 58 and MDM2-Peptide 95 were(389.6±13.4)nmol/L and(1.6±0.2)μmol/L,while(19.5±2.4)μmol/L(t=6.85,P<0.01)and(12.8±1.4)μmol/L(t=8.32,P<0.01)for p53-Peptide 58 and p53-Peptide 95 systems.Western Blotting results indicated that Peptide 58 and Peptide 95 could improve the expression of p53 and p21,while kept MDM2 relative stabilization in MCF-7 cell line.Conclusion:Peptide 58 and Peptide 95 demonstrated a reasonable inhibition effect on MDM2 via its strong binding affinity to MDM2,but unexpectedly fail to do well in vivo.The reason of poor efficiency may be there’s a large part of peptides could not enter cells or be degraded by protease.
作者
包传恩
张雅雅
胡蒙
黄晓媚
解百宜
方尧
郭明
Bao Chuanen;Zhang Yaya;Hu Meng;Huang Xiaomei;Xie Baiyi;Fang Yao;Guo Ming(Xiamen University Affiliated Chenggong Hospital,Fujian Xiamen 361001,China)
出处
《现代肿瘤医学》
CAS
2020年第17期2957-2962,共6页
Journal of Modern Oncology
作者简介
包传恩(1976-),男,安徽池州人,主治医师,硕士研究生,主要从事肿瘤临床治疗及肿瘤发生机制研究。E-mail:baochuanan@163.com;通讯作者:郭明(1963-),男,福建泉州人,主任医师,研究方向:胸外科。
