IngentaConnect A robust homogeneous binding assay for alpha4beta2 nicotinic acet...

Authors: HUI, Xin¹; GAO, Jie¹; XIE, Xin¹; SUTO, Naoki²; OGIKU, Tsuyoshi²; WANG, Ming-Wei¹

Source: Acta Pharmacologica Sinica, Volume 26, Number 10, October 2005 , pp. 1175-1180(6)

Abstract:

Aim:

To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha

2 nicotinic acetylcholine receptor (nAChR) modulators. Methods:

Membrane preparation of HEK293 cells expressing alpha

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 alpha

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 alpha

2 nAChR (K_i<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 alpha

2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.

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

Document Type: Research article

DOI: 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

The full text electronic article is available for purchase. You will be able to download the full text electronic article after payment.

$50.16 plus tax Refund Policy

A robust homogeneous binding assay for 42 nicotinic acetylcholine receptor