A robust homogeneous binding assay for α42 nicotinic acetylcholine receptor
To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α42 nicotinic acetylcholine receptor (nAChR) modulators. Methods:
Membrane preparation of HEK293 cells expressing α42 nAChR, [3H]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 32 000 synthetic compounds. Intracellular calcium measurement was carried out to verify the bioactivities of the hits found by the SPA assay. Results:
IC50 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 [3H]cytisine binding to α42 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α42 nAChR (Ki<2 mol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity. Conclusion:
This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential α42 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.
Document Type: Research Article
Affiliations: 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
Publication date: October 1, 2005