新型MicroRNA-21小分子抑制剂的研究

MicroRNA-21 is an oncogene. Inhibiting the expression or activity of MicroRNA-21 can inhibit tumor growth. MicroRNA-21 inhibitors could be next hot spots to developmental anti-tumor drugs. In this project, compound 1j, discovered by our group, is used as a lead compounds. We shall adopt rational drug design method to design the novel types of MicroRNA-21 small molecule inhibitors by ring closure of different position compound 1j. The designed new compounds will be synthesized and characterized. The activity of synthesized compounds inhibiting MicroRNA-21 in Hela cell line will be evaluated by real time quantitative PCR technology. The structure activity relationship of tested compounds will be revealed. The active compound target will be elucidated by photoaffinity labeling technology. The antiproliferative activity of synthesized compounds will be determined by MTT method. The antitumor effects of compounds in vivo will be investigated with nude mice transplantation tumor model. The study of project can discover novel structure, high active MicroRNA-21 small molecule inhibitors, and disclose the structure-activity relationship and the target of active compound. Meanwhile, the study of project can promote the research on MicroRNA-21 inhibitor, discover the antitumor drugs with independent intellectual property. There are both theoretical significance and development prospects of a new antitumor drug to carry out the project.

MicroRNA-21是一种致癌基因，抑制MicroRNA-21的表达或活性可抑制肿瘤的生长。MicroRNA-21抑制剂可能是下一个抗肿瘤药物研发的热点。本项目以我们发现的MicroRNA-21小分子抑制剂1j为先导化合物，采用合理药物设计法，将化合物1j的不同位置关环，设计出多种类型的化合物；合成、表征设计的化合物；采用实时定量PCR技术研究化合物抑制MicroRNA-21在Hela细胞中的活性，总结构效关系；采用光亲和标记技术研究高活性化合物的作用靶点和作用位点；采用MTT法评价化合物抑制人肿瘤细胞增值的活性；采用人肿瘤裸鼠移植瘤模型考察化合物的体内抗肿瘤活性。通过本项目的研究，可发现结构新颖、活性高的MicroRNA-21小分子抑制剂，并阐明其构效关系和作用靶点。同时，可推动MicroRNA-21抑制剂的研究，发现具有自主知识产权的抗肿瘤药物。既有理论意义，又有潜在的研发前景。

miRNA-21是一种致癌基因，抑制miRNA-21的表达或活性可抑制肿瘤的生长。本项目以我们发现的miRNA-21小分子抑制剂1j为先导化合物，设计出多种类型的化合物。以诺氟沙星等为原料，合成了氟喹诺酮衍生物（A类化合物）70多个；以2-氨基苯甲酸衍生物为原料，合成了3-取代-4(3H)-喹唑啉酮类化合物(B类)40多个。建立了双荧光素酶报告实验筛选体系和茎环法RT-qPCR实验体系，并对合成化合物抑制miRNA-21的活性进行了筛选和验证。发现诺氟沙星衍生物A36（N-环己基-1-乙基-6-氟-7-（4-苄基-1-哌嗪基）-4-氧-1,4-二氢喹啉-3-羧酰胺）的活性最好。B类化合物对miRNA-21也有一定的抑制活性。随后，对化合物A36进行了体外抗肿瘤活性评价和作用机制研究。结果表明，A36对Hela等六种肿瘤细胞的IC50在1.76-12.98 µmol/L，并且对人正常肺上皮细胞BEAS-2B的增殖没有抑制活性。在浓度为10 µmol/L时，A36可显著促进Hela细胞的凋亡，并将细胞周期阻滞在G0/G1期，也可显著抑制Hela细胞克隆的形成。A36可显著抑制Hela细胞中成熟miRNA-21的表达，促进pri-miRNA-21和pre-miRNA-21的表达；同时，A36上调了靶蛋白PTEN、PDCD4和DUSP5的表达水平。A36可使Hela细胞中171个mRNA发生显著变化，其中MAPK信号通路发生的改变最为显著。综上，A36是新型的miRNA-21小分子抑制剂，其通过抑制miRNA-21的表达，上调其靶蛋白PTEN、PDCD4和DUSP5的表达水平，发挥特异性抗肿瘤作用。本项目发现了结构新颖的miRNA-21小分子抑制剂，初步阐明了其作用机制，丰富了miRNA-21的抑制剂的结构类型。