肿瘤坏死因子-α小分子抑制剂的设计、筛选及初步活性测定  

De novo design and screening of TNF-α-antagonistic small molecular inhibitor

作　　者：谷岳[1] 郭红星[1] 纪庆[1] 谢印良[1] 黄蕊[1] 熊冬生[1] 杨纯正[1] 刘汉芝[1] 许元富[1] 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所实验血液学国家重点实验室,天津300020

出　　处：《中国药理学通报》2009年第10期1287-1291,共5页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No30400405);天津市自然科学基金资助项目(No05YFJZJC01200)

摘　　要：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。Aim To de novo design and explore TNF- α-antagonistic small molecular inhibitor based on the crystal structure-based virtual screening and biological activity assays. Methods Docking templete was deduced from the crystal structures of TNF-α and the TNF-β/TNF receptor（p55） complex by computational molecular modeling, and docking procedure against the templete was performed on 3 D database. Fifty candidates were selected from a small molecule compound library containing about 9× 10^4 compounds by virtual screening; TNF-α antagonistic activity of compounds was evaluated by inhibition of TNF-α-mediated cytotoxicity and PCD on routine L929 cells by MTT and FACs assay ;Results A seven-amino acid peptide of loop2/ domain2 of TNF receptor I （ p55 ） was chosen as docking templete ; three of fifty compounds （ C- 12, C-34, C-35 ） were identified as preliminary candidates by virtual screening and cytotoxicity assays, and among them, C- 12 compound showed higher inhibitory activity in TNF- α-induced cytotoxicity （ 10 μmol · L^-1 of IC50） and TNF-α-meditated apoptosis on murine L929 cells with a dose-dependent fashion. Conclusion This study highlights the potential of virtual screening combined with biological assay method for the design of novel small molecular inhibitor with the ability to neutralize TNF-α, and by this method, a promising lead compound （C-12） can be identified to inhibit TNF-α-induced cy- totoxicity and TNF-α-meditated apoptosis on murine L929 cells.

关 键 词：肿瘤坏死因子-Α 虚拟筛选 小分子抑制剂 分子对接 细胞凋亡 受体 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R341.6[医药卫生—基础医学]