黄嘌呤氧化酶抑制剂_超氧阴离子清除剂双靶点高通量筛选模型的建立_谢涛

黄嘌呤氧化酶抑制剂/超氧阴离子清除剂双靶点高通量筛选模型的建立

谢涛†, 秦至臻†, 周睿, 赵赢, 杜冠华*

(中国医学科学院、北京协和医学院药物研究所, 北京市药物靶点研究与新药筛选重点实验室, 北京 100050)

摘要: 本文建立了适用于同时筛选黄嘌呤氧化酶抑制剂和超氧阴离子清除剂的双靶点高通量复合筛选模型。在黄嘌呤氧化酶超氧阴离子生成体系中,加入WST-1作为超氧阴离子生成量的探针,以反应体系中标识性产

物尿酸为黄嘌呤氧化酶活性指示剂, 采用SpectraMax M5酶标测试仪, 同时检测两种指示剂的浓度变化, 通过

对反应体系中的影响因素进行优化建立双靶点HTS筛选模型, 并利用阳性药物对该模型进行评价。在反应体系

中, 反应终体积50 μL, 黄嘌呤氧化酶4 mU·mL−1、黄嘌呤250 μmol·L−1、WST-1浓度为100 μmol·L−1, 黄嘌呤氧

化酶抑制剂筛选模型的Z'-因子为0.5374, S/N为47.5199; 超氧阴离子清除剂筛选模型的Z'-因子为0.5074, S/N

为5.3889。结果表明, 本文建立的黄嘌呤氧化酶抑制剂/氧自由基清除剂高通量筛选模型具有稳定性好、成本较

低和重复性高等特点, 可以广泛应用于药物筛选。

关键词: 高通量筛选方法; 黄嘌呤氧化酶; 超氧阴离子清除剂; 抗氧化; 药物评价

中图分类号: R965 文献标识码:A 文章编号: 0513-4870 (2015) 04-0447-06 Establishment of double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers

XIE Tao†, QIN Zhi-zhen†, ZHOU Rui, ZHAO Ying, DU Guan-hua*

(Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China)

Abstract: A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators’ concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU·mL−1, xanthine 250 μmol·L−1 and WST-1 100 μmol·L−1, separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.5374, S/N is 47.5199; the Z'-factor of model for superoxide anion scavengers is 0.5074, S/N is 5.3889. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.

Key words: high throughput screening method; xanthine oxidase; superoxide anion scavenger; antioxidant;

drug evaluation

收稿日期: 2014-10-20; 修回日期: 2014-12-22.

基金项目: 重大新药创制科技重大专项 (2012ZX09101); 国际合作项目 (2012DFH30070).

†并列第一作者.

*通讯作者 Tel / Fax: 86-10-63165184, E-mail: dugh@