人源可溶性环氧化物水解酶抑制剂的高通量筛选.docVIP

· PAGE 1372· 药学学报 Acta Pharmaceutica Sinica 2010, 45 (11): 1367?1372 王守宝等: 人源可溶性环氧化物水解酶抑制剂的高通量筛选 · PAGE 1371· 药学学报 Acta Pharmaceutica Sinica 2010, 45 (11): 1367?1372 · PAGE 1367· 人源可溶性环氧化物水解酶抑制剂的高通量筛选 王守宝, 郭 晶, 俞晓明, 杜冠华* (中国医学科学院、北京协和医学院药物研究所国家新药筛选中心, 北京 100050) 摘要: 本研究拟建立体外人源可溶性环氧化物水解酶 (hsEH) 抑制剂高通量筛选模型, 筛选hsEH抑制剂。利用大肠杆菌表达重组hsEH, 化学合成其特异性的底物PHOME, 建立了基于荧光测定的以384孔微板为载体的hsEH抑制剂高通量筛选模型 (Z = 0.65)。对47 360种样品 (包括25 040种化合物和22 320种天然产物) 进行初筛, 选择初筛抑制率大于80%的950种样品作为活性样品进行复筛。最终确定2个化合物具有较强的抑制活性, 其IC50值分别为8.56和4.31 μmol?L?1。结果表明, 所建立的hsEH抑制剂高通量筛选模型具有灵敏、稳定、重复性好的特点。 关键词: 可溶性环氧化物水解酶; 抑制剂; 高通量筛选 中图分类号: R965.1 文献标识码: A 文章编号: 0513-4870 (2010) 11-1367-06 High-throughput screening of human soluble epoxide hydrolase inhibitors WANG Shou-bao, GUO Jing, YU Xiao-ming, DU Guan-hua* (National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China) Abstract: To screen potential human soluble epoxide hydrolase (hsEH) inhibitors, a high-throughput screening model in 384-well microplate with total volume of 50 μL was established. Recombinant hsEH was cloned and expressed in E. coli. and its specific substrate PHOME was synthesized. The HTS model was based on fluorescence analysis with enhanced sensitivity and specificity (Z = 0.65). A total of 47 360 samples (including 25 040 compounds and 22 320 natural products) were screened, of which 950 samples with inhibition greater than 80% were selected for further rescreening. Finally, two compounds with high inhibitory activity were identified, whose IC50 value were 8.56 and 4.31 μmol?L?1, separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening. Key words: soluble epoxide hydrolase; inhibitor; high-throughput screening 收稿日期: 2010-06-08.基金项目: