A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

查看全文 作　　者：Xin [1]HUI;Jie [1]GAO;Xin [1]XIE;Naoki [2]SUTO;Tsuyoshi [2]OGIKU;Ming-Wei [1]WANG

机构地区：[1]The National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Shanghai 201203, China;[2]Discovery and Pharmacology Research Laboratories, Tanabe Seiyaku Co, 2-50, Kawagishi 2-Chome, Toda, Saitama 335-8505, Japan

出　　处：《Acta Pharmacologica Sinica》索引2005年第26卷第10期,共6页

基　　金：Project supported in part by grants from the Ministry of Science and Technology of China (2002AA2Z343A and 2004CB518902);Chinese Academy of Sciences (KSCX1-SW-11-2) and Shanghai Pharmaceutical (Group) Co 摘　　要：<正> Aim: To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α4β2 nicotinic acetylcholine receptor (nAChR) modulators. Methods: Membrane preparation of HEK293 cells expressing α4β2 nAChR, [3~H]cytisine and wheat germ agglutinin (WGA)-coupled microbeads were used to develop an HTS assay based on SPA technology. This method was validated against a conventional filter binding approach and applied to large-scale screening of a library containing 32000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results: IC_(50) values of 2 reference compounds (epibatidine and RJR 2403) determined by SPA and filter binding methods were comparable and consistent with those reported elsewhere. A total of 54 compounds, showing more than 60% competitive inhibition on [3~H]cytisine binding to α4β2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α4β2 nAChR (K_i<2 μmol/L). Eight compounds displayed antagonistic effects with>50% inhibition on ABT-594-induced calcium mobilization while none showed any agonistactivity. Conclusions: This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels. 关键词：α4β2 烟碱酸乙酰胆碱受体微生物凝集素实验室检查

相关文献

参考文献（17）
引证文献（4）
耦合文献（11）

参考文献(17)

引证文献(4)

耦合文献(11)

互联网出版许可证新出网证(渝)字10号全国400电话 - 免长途话费

A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor

参考文献(17)

引证文献(4)

耦合文献(11)

用户提示