人源可溶性环氧化物水解酶抑制剂的高通量筛选 - 药学学报.docVIP

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 ) 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。重组hsEH, 合成其特异性的底物PHOME, 建立了的以384孔微板为的高通量筛选模型 ( = 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。 可溶性环氧化物水解酶; 抑制剂; 高通量筛选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 (环氧化物水解酶 (epoxide hydrolase, EH, EC.3.3.2.3) 是脊椎动物体内广泛存在的可以水解外源性以及内源性环氧化物生成二醇衍生物的一种酶, 由554个氨基酸残基组成, 分子质量约为60 kDa。在哺乳动物EH可以分为微粒体型、胆固醇型、白三烯A4型以及胞质型 (即可溶性EH, sEH) 等类型。这几种EH序列同源性很低 (氨基酸同源性(5%), 底物特异性明显不同?3]。环氧二十碳三烯酸 (EETs) 是参与调节血管稳态、血压、缺血性损伤以及炎症等过程的一种旁分泌和自分泌信号分子。它们广泛分布于肺、心脏