一种磷脂酶A2抑制剂体外高通量筛选方法的建立.doc

一种磷脂酶A2抑制剂体外高通量筛选方法的建立 朱京童 郑智慧 路新华 范玉玲 张华 贺建功 （华北制药集团新药研究开发中心，微生物药物国家工程研究中心 石家庄050015）μM和反应时间30min时，为最适反应条件。阳性药在该模型上表现出好的浓度剂量依赖抑制效果，IC50为0.37μmol/L。利用该模型，本研究从微生物次生代谢产物筛选得到两个活性化合物F02ZA-2396A、F02ZA-2396B，其IC50分别为152μM和72μM。结论：本研究建立了一个高通量的磷脂酶A2抑制剂体外药物筛选方法，该方法灵敏、快捷、可操作强，可用于各种来源的化合物的快速活性筛选。 关键词：磷脂酶A2；抑制剂；体外；荧光法 A fluorescence-based method on the high throughput screening assay for PLA2 inhibitor in vitro Zhu Jing-tong, Zheng Zhi-hui, Lu Xin-hua, Fan Yu-ling, Zhang Hua, He Jian-gong (New Drugs Research Development Center of North China Pharmaceutical Group Corporation, National Microbial Medicine Engineering Research Center, Shijiazhuang 050015) ABSTRACT: OBJECTIVE: To establish a fluorescence-based method in vitro for high throughput screening phosphplipase A2(PLA2) inhibitor. METHODS: using 7-Hydroxycoumarinyl-arachidonate as a fluorescent substrate，to determine the activity of phosphplipase A2 by measuring the 355ex/460em fluorescence value in 96-well microplates, The effect of positive drugs and samples on phosphplipase A2 activity were evaluated. RESULTS: With 2.25U/ml concentration of the enzyme, 30μM concentration of the substrate and 30 min reaction time, there is a dose-dependent manner between them.. Positive control, DTPA showed good inhibitory activity in dose-dependent manner with IC50 of 0.37μmol/L , In the course of screening for PLA2 inhibitors from metabolites of microorganisms, two compounds named F02ZA-2396A and F02ZA-2396B were conformed as the PLA2 inhibitors, their IC50 value are 152μmol/l and 72μmol/l，respectively. CONCLUSION:The method is sensive, rapid, accurate valid for screening the PLA2 inhibitors in vitro. KEY WORDS: phosphplipase A2 ;inhibitor; in vitro; fluorescence 作者简介：朱京童，（1977年出生），女，硕士在读，工程师。研究方向：药物筛选 基金资助：国家科技部创新药物筛选技术平台研究资助项目，项目编号：2002AA2AZ343D 通信地址：E-mail:zhujingtong2003@ 磷脂酶A2(phospholipase A2 PLA2)是一种重要的代谢和调节酶类，已有150个以上的氨基酸序列可在蛋白质序列库中找到(1(。在生物界中分布广泛，大量存在于蛇毒、蜂毒、蝎毒，动物胰脏及植物组织中(2(。所有的PLA2都有一种最基本的作用，就是水解磷脂sn-2位脂肪酸，释放自由脂肪酸和溶血磷脂，直接或间接