靶向肿瘤坏死因子β小分子抑制剂的筛选及其功能鉴定

发布时间：2018-11-23 19:27

【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=8.19μmol/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。
[Abstract]:Objective to obtain small molecular inhibitors targeting tumor necrosis factor 尾 (TNF 尾) by computer virtual screening and cell activity screening. Methods according to the compound crystal structure of TNF 尾 combined with tumor necrosis factor type I receptor (TNFR1), 105 small molecular compounds (C1~C105) with good docking results were selected by computer virtual screening method and their cellular activity was screened. The cytotoxicity of TNF 尾 to L929 cells was detected by MTT assay, and the apoptosis induced by TNF 尾 was detected by flow cytometry. The effects of small molecular compounds on the cell cycle of L929 cells were detected by (PI) single staining and flow cytometry, and the inhibition of Caspase 3 activation induced by TNF 尾 by Western blot and double disc laser confocal microscopy was analyzed. Results C35 could effectively inhibit cytotoxicity induced by TNF 尾 in a dose-dependent manner (half inhibitory concentration = 8.19 渭 mol/L), C35 had lower cytotoxicity and had no effect on cell cycle of L929 cells. C35 could block the apoptotic pathway induced by TNF 尾 binding to its receptor and significantly inhibit the apoptosis of L929 cells induced by TNF 尾. Conclusion C35, a small molecule inhibitor of TNF 尾, can block the apoptosis pathway induced by TNF 尾 and inhibit the cytotoxic activity of TNF 尾.
【作者单位】：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;
【基金】：国家自然科学基金资助项目(31271484,31471116) 天津市自然科学基金重点基金(12JCZDJC24600) 北京协和医学院研究生创新基金(100023-0710-1013)
【分类号】：R730.231

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