荧光共振能量转移电压敏感探针在钾通道抑制剂高通量筛选中的应用

中国临床药理学与治疗学 ›› 2006, Vol. 11 ›› Issue (11): 1261-1264.

荧光共振能量转移电压敏感探针在钾通道抑制剂高通量筛选中的应用

孙丽新, 廖红, 严明, 马怡娜, 张陆勇

中国药科大学新药筛选中心, 南京 210038 , 江苏

收稿日期:2006-10-10 修回日期:2006-11-05 出版日期:2006-11-26 发布日期:2020-11-06
通讯作者: 张陆勇,男, 教授, 博士生导师, 研究方向为分子毒理学和药物筛选。Tel:025-83721500 E-mail:drugscreen@126.com
作者简介:孙丽新, 女, 实习助理研究员, 从事新药筛选及分子药理科研工作。Tel:025-85391036 　E-mail:sunlixin78@126.com
基金资助:
国家高技术研究发展计划(863 计划)课题(No2004AA234011)

Screening potassium channel blocker with FRET-bases voltage sensor probes

SUN Li-xin, LIAO Hong, YAN Ming, MA Yi-na, ZHANG Lu-yong

National Drug Screening Laboratory , China Pharmaceutical University , Nanjing 210038 , Jiangsu , China

Received:2006-10-10 Revised:2006-11-05 Online:2006-11-26 Published:2020-11-06

摘要/Abstract

摘要： 目的建立以荧光共振能量转移(FRET)电压敏感探针为染料的钾通道抑制剂高通量筛选模型。方法分别用电压敏感探针的能量供体CC2-DMPE与能量受体DiSBAC₂(3)先后负载RBL-2H3细胞, 测量细胞去极化前后460nm和570nm的荧光变化,根据荧光的变化反映细胞膜电压的变化。结果本筛选方法稳定可靠, 系统Z' 因子达到0.60以上, 日筛选通量能够达到10000 药次。对102个定向合成的化合物进行筛选, 有11个化合物的荧光信号变化率有较好的剂量效应关系。结论此模型具有良好的稳定性和特异性, FRET 电压敏感探针是钾通道抑制剂高通量筛选的理想染料。

关键词: 荧光共振能量转移, 电压敏感探针, 钾通道抑制剂, 高通量筛选

Abstract: AIM: To establish high-throughput potassium channel assay using FRET-based voltage sensor probes (VSPs).METHODS: RBL-2H3 Cells , which endogenously express high level Kir channels , were loaded with CC2-DMPE and DiSBAC2(3).The fluorescence response ratio of 460 and 570 nm before and after addition of high K+ was then detected.The response ratio can be used to evaluate membrane potential changes.RESULTS: The assay is robust as indicated by a Z' factor larger than0.6.The throughput is in the range of 10 000 data points per day , which is significantly higher than many other methods presently available for potassium channel.In 102 compounds screened with VSPs , 11 compounds were found to change the fluorescence ratio dosedependently.CONCLUSION: The assay allows semiautomated screening of compounds for potassium channel activity on 384-well plates and is sufficiently rapid for testing a large number of compounds.

Key words: fluorescence resonance energy transfer, voltage sensor probes, potassium channel blockers, high throughput screening

中图分类号:

R965.1

孙丽新, 廖红, 严明, 马怡娜, 张陆勇. 荧光共振能量转移电压敏感探针在钾通道抑制剂高通量筛选中的应用[J]. 中国临床药理学与治疗学, 2006, 11(11): 1261-1264.

SUN Li-xin, LIAO Hong, YAN Ming, MA Yi-na, ZHANG Lu-yong. Screening potassium channel blocker with FRET-bases voltage sensor probes[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2006, 11(11): 1261-1264.

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