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目的结合计算机虚拟筛选和实物活性筛选,寻找新的TNF-α的小分子抑制剂,并完成对其生物学活性的测定。方法基于TNF-α受体蛋白的晶体结构和计算机辅助设计,用docking方法对含有约9万个小分子三维数据库进行筛选;选择对接结果较好的50个化合物进行初步的抑制TNF-α细胞毒活性实验;MTT法进一步分析筛选出的小分子化合物的细胞毒性,以及其在不同剂量时抑制TNF-α对L929细胞毒性的作用;采用AnnexinV-FITC检测上述小分子对TNF-α诱导的细胞凋亡的抑制作用。结果确定受体p55蛋白第二结构域的第二loop环中7肽(RKEMGQV)作为docking的配体模板;经计算机虚拟筛选后得到965种结构相似的化合物,综合分析后最终选择并购买了50个代表性化合物做进一步生活性测定;活性筛选结果显示有3个小分子化合物(C-12、C-34、C-35)能抑制TNF-α的细胞毒活性;对其中抑制活性较好的C-12进行了深入研究的结果表明:该化合物具有较低的细胞毒性和较强的抑制TNF-α(1μg.L-1)对L929细胞毒性的作用,表现为剂量依赖性,半数抑制浓度(IC50)为10μmol.L-1;该化合物能抑制TNF-α诱导的L929细胞凋亡,并呈剂量依赖性。结论通过计算机虚拟筛选并结合生物活性分析,筛选到一种能直接靶向TNF-α,并能中和TNF-α的细胞毒活性的全新的先导化合物。
OBJECTIVE: To find a new small molecule inhibitor of TNF-α and to determine its biological activity by combining with computer virtual screening and physical activity screening. METHODS: Based on the crystal structure and computer-aided design of the TNF-α receptor protein, about 90,000 small molecule three-dimensional databases were screened by docking method. Fifty compounds with better docking results were selected to preliminarily inhibit TNF-α cytotoxicity The cytotoxicity of the small molecule compounds screened by MTT assay and the effect of TNF-α on the cytotoxicity of L929 cells at different doses were also analyzed. Annexin V-FITC was used to detect the effect of these small molecules on TNF-α-induced apoptosis Inhibition of death. As a result, it was confirmed that RKEMGQV 7 in the second loop of the second domain of the receptor p55 protein was used as a docking ligand template; after the computer virtual screening, 965 structurally similar compounds were finally selected and purchased 50 Three representative compounds were further tested for their viability. Three small molecule compounds (C-12, C-34, C-35) were found to inhibit the cytotoxic activity of TNF-α. The results of in-depth study of C-12 showed that the compound has lower cytotoxicity and stronger inhibitory effect on TNF-α (1μg.L-1) on L929 cell toxicity in a dose-dependent and half-maximal inhibitory concentration (IC50) was 10μmol.L-1. The compound could inhibit TNF-α-induced L929 cell apoptosis in a dose-dependent manner. Conclusions A new lead compound, which can directly target TNF-α and neutralize the cytotoxic activity of TNF-α, was screened by computer virtual screening combined with bioactivity analysis.