A robust homogeneous binding assay for α42 nicotinic acetylcholine receptor

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Abstract:

Abstract Aim:

To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel α42 nicotinic acetylcholine receptor (nAChR) modulators. Methods:

Membrane preparation of HEK293 cells expressing α42 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 α42 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to α42 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 α42 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

Keywords: high-throughput screening; nicotinic acetylcholine receptor; scintillation proximity assay

Document Type: Research Article

DOI: http://dx.doi.org/10.1111/j.1745-7254.2005.00202.x

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

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