天津医药

天津医药 ›› 2015, Vol. 43 ›› Issue (9): 961-964.doi: 10.11958/j.issn.0253-9896.2015.09.001

• 细胞与分子生物学 •    下一篇

靶向肿瘤坏死因子β小分子抑制剂的筛选及其功能鉴定

孙亚薇， 公海艳， 曹善楠， 刘鹏， 朱海燕， 耿广锋， 许元富   

  1. 中国医学科学院 北京协和医学院血液病医院(血液学研究所) 实验血液学国家重点实验室
  • 收稿日期:2015-02-11 修回日期:2015-04-20 出版日期:2015-09-15 发布日期:2015-09-15
  • 通讯作者: 许元富 E-mail:xuyf@ihcams.ac.cn
  • 作者简介: 孙亚薇 （1988）， 女， 硕士在读， 主要从事肿瘤坏死因子的小分子抑制剂的研究
  • 基金资助:
    国家自然科学基金;天津市自然科学基金重点基金;北京协和医学院研究生创新基金

Screening and identification of a novel small-molecule TNFβ inhibitor

SUN Yawei, GONG Haiyan, CAO Shannan, LIU Peng, ZHU Haiyan, GENG Guangfeng, XU Yuanfu   

  1. State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy ofMedical Sciences and Peking Union Medical College, Tianjin 300020, China
  • Received:2015-02-11 Revised:2015-04-20 Published:2015-09-15 Online:2015-09-15
  • Contact: Yuan-Fu XU E-mail:xuyf@ihcams.ac.cn

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摘要： ： 目的 通过计算机虚拟筛选和细胞活性筛选， 获得能靶向抑制肿瘤坏死因子β （TNFβ） 的细胞毒活性的小分子抑制剂。方法 根据 TNFβ与肿瘤坏死因子Ⅰ型受体 （TNFR1） 结合的复合晶体结构， 采用计算机虚拟筛选方法初步选择对接结果较好的 105 个小分子化合物 （C1~C105）， 并对其进行细胞活性筛选； MTT 法检测小分子化合物抑制 TNFβ对 L929 细胞的细胞毒性； 流式细胞术测定小分子化合物对 TNFβ引起的细胞凋亡的抑制作用； 采用碘化丙啶 （PI） 单染和流式分析技术检测小分子化合物对 L929 细胞周期的影响； 采用 Western blot 和双转盘激光共聚焦显微镜分析小分子化合物对 TNFβ引起的下游 Caspase 3 活化的抑制作用。结果 C35 能有效抑制 TNFβ引起的细胞毒作用， 且这种抑制作用呈现剂量依赖性 （半数抑制浓度=8.19 μmol/L)； C35 具有较低的细胞毒性， 且对 L929 细胞周期无影响； C35 能阻断 TNFβ与其受体结合后引起的细胞凋亡通路， 显著抑制 TNFβ引起的 L929 细胞凋亡。结论 成功筛选到一个 TNFβ小分子抑制剂 C35， 其能阻断 TNFβ引起的细胞凋亡通路， 高效抑制 TNFβ的细胞毒活性。

关键词: 关健词：肿瘤坏死因子β, 小分子抑制剂, 细胞凋亡, 信号通路, 细胞周期

Abstract: Abstract: Objective To explore a novel and highly specific small-molecule TNFβ inhibitor by the use of computer-aided virtual screening and cell-based assays in vitro. Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that target TNFβ based on the crystal structure of the TNFβ-TNFR1 complex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cell was detected by MTT assay, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cell was determined by flow cytometry assay. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western Blot and Ultra VIEW VOX 3D Live Cell Imaging System. Results 965 compounds were identified to closely mimic the spatial structure of the docking template, and 105 compounds among them were selected as leading compounds based on their binding energy, structural diversity and potential for future drug development. In further study, those compounds were examined their ability to inhibit TNFβ-induced cytotoxicity in L929 cell line with three different doses. Currently available results suggested that No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent way, and the Half-maximum Inhibition Concentration (IC50) was 8.19μM. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further reveal that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduced L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβ small-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and it could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death induced by TNFβ.

Key words: Key words: TNFβ, Small molecular inhibitor, Cell apoptosis, Signal pathway, Cell cycle