β-内酰胺酶抑制剂高通量筛选模型的建立.pdfVIP

.102. 中国抗生素杂志2013年2月第38卷第2期 文章编号: 1001-8689(2013)02-0102-04 应逆翌回 p- 内自先肢酶抑制剂高通量筛选模型的建立 王斯文陈向东·汪辉石功名郭欣张序磊 (中国药科大学，生命科学与技术学院，微生物教研室，南京 210009) 摘要:目的 建立存内酸肢酶抑制剂的高通量筛选模型。方法 从产β内航肢酶菌株中提取酶液，以硝唾吩为底物，三唾 巴坦作为酶抑制剂，通过检测反应体系吸光度的变化，来判断酶促反应的进行情况以及酶抑制剂的效果，建立384微孔板的高 通量筛选模型，优化反应条件并对此模型进行评价。用此模型对7360个化合物组成的随机库进行筛选。结果 确定反应体系条 0 件为:最佳测定波长为482nm，反应温度为30 C ，选取15min时间点检测，底物硝喔吩终浓度为O.lmmollL，评价指标z 因子在 0.8-0.9范围内，表明此模型可以用于高通量筛选。基于此模型初筛出的个有抑制活性的化合物。结论 建立的。-内联胶酶抑制 剂高通量筛选模型具有快速、稳定和微量的特点，是寻找和发现有潜在活性的β内酌肢酶抑制剂的有效手段。 关键词:高通量筛选(HT曰:β内酿肢酶;抑制剂:半数抑制浓度(IC ) so 中图分类号: R978 文献标识码:A EstabUshment of high-throughput screening model for ß-Iactamase inhibitor Wang Si-wen, Chen Xiang-dong , Wang Hui , Shi Gong-ming, Guo Xin and Zhang Xu-lei (Department ofMicrobiology, Schoo1 ofLife Science and technology , China Pharmaceutical University, Na町ing 210009 , Jiangsu) Abstract Objective To establish a high-throughput model for ß-lactamase inhibitors. Methods Extended spectrum ß-lactamase (ESBL) extracted from ß-lactamase producing strains was used to catalyze the hydrolysis of the substrate , nitrocefin. Tazobactam was used as positive contro l. The absorbance value measured from the reaction system was used to evaluate the efficacy of the inhibitor. The screening conditions on the base of 384-well microplate was optimized and the screening model was evaluated with Z-factor, CV and S/B value. 7360 compounds were screened applying this mode l. Results The optínial screening conditions were: 482nm for detecting wave length , 30C for incubation temperature , 15min