基于p53- mdm2复合物结构的mdm2阻断剂的初步探讨

Initial Evaluation of mdm2 Inhibitors Based on p53- mdm2 Complex Structure

目的：寻找基于 p53- mdm2复合物晶体结构的 mdm2抑制剂，使 p53释放出来，发挥其抗癌活性。方法：根据已知的 p53- mdm2复合物晶体的空间结构，在计算机上模拟设计出 23个非肽小分子化合物。将 p53的全长开放阅读框（ ORF）和 mdm2的部分 ORF克隆进原核表达载体，异丙基硫代－β－ D半乳糖苷（ IPTG）诱导蛋白表达。金属螯和层析柱纯化蛋白。 Western blot检测蛋白的质量，十二烷基磺酸钠－聚丙烯酰胺凝胶电泳 (SDS- PAGE)及标准曲线法进行蛋白定量。用 ELISA方法检测非肽小分子化合物与 mdm2的亲和性及作用的饱和浓度。用 MTT方法进行细胞生长抑制实验。结果： SDS- PAGE和 Western blot检测显示原核表达的 p53和 mdm2蛋白质量和纯度良好，可供后续实验。 ELISA结果显示非肽小分子目标化合物及中间化合物 32个化合物中 13个与对照相比对 mdm2有一定的亲和作用，其中 5个亲和性较高，并有一定的饱和作用量。体外实验表明这 5个化合物对几种表达野生型 p53的肿瘤细胞系有生长抑制作用。结论：基于 p53- mdm2复合物晶体的空间结构设计的非肽小分子化合物，通过抑制 mdm2- p53的结合，在体外显示有一定的抑制肿瘤细胞生长的作用。

Objective： This study was designed to seek mdm2（ murine double minute2） inhibitors that can block p53- mdm2 binding， thus release p53 from the binding complex and recover anti- tumor function of p53. Methods： Twenty- three non- peptidic small- molecule compounds based on the crystal structure of p53- mdm2 complex were obtained by computer aided design and subsequently synthesized by chemical method. p53 full- length open reading frame(ORF) and partial ORF of mdm2 (17－ 125 amino acid) were cloned into a prokaryotic vector pET－ 22b(＋ ) respectively. p53 and mdm2 proteins were expressed in E.coli cells BL21(DE3) and purified by metal affinity resin. These proteins were identified by SDS- PAGE and Western blot analysis and quantified by SDS- PAGE and BCA method. The affinity of these synthetic compounds with mdm2 protein was detected by ELISA. Growth inhibition of tumor cells was observed by MTT method. Results： Western blot analysis showed the quality and purity of p53 and mdm2 proteins were excellent. ELISA results showed that among 23 synthetic small compounds and 9 intermediate products (totally 32 inhibitors), 13 showed affinity with mdm2, and 5 of them showed high affinity compared with the controls. These 5 compounds can inhibit the growth of several tumor cell lines which express wt- p53. Conclusions： Non- peptidic small- molecule compounds based on crystal structure of p53- mdm2 complex can effectively inhibit p53- mdm2 binding, thus arrest in vitro growth of some tumor cell lines. Developing mdm2 inhibitors based on p53- mdm2 complex structure may be a promising way to find new anti- tumor drugs.