A robust homogeneous binding assay for alpha4beta2 nicotinic acetylcholine receptor.
OBJECTIVE
To develop a homogeneous high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel alpha4beta2 nicotinic acetylcholine receptor (nAChR) modulators.


METHODS
Membrane preparation of HEK293 cells expressing alpha4beta2 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 32 000 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 alpha4beta2 nAChR, were identified initially following an HTS campaign. Secondary screening confirmed that 17 compounds with novel chemical structures possessed relatively high binding affinity to alpha4beta2 nAChR (K(i)<2 micromol/L). Eight compounds displayed antagonistic effects with >50% inhibition on ABT-594-induced calcium mobilization while none showed any agonist activity.


CONCLUSIONS
This homogeneous binding assay is a highly efficient, amenable to automation and robust tool to screen potential alpha4beta2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.
Hui
Xin
X
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.
Gao
Jie
J
Xie
Xin
X
Suto
Naoki
N
Ogiku
Tsuyoshi
T
Wang
Ming-Wei
MW
D000470:Alkaloids / Q000378:metabolism; D001384:Azetidines / Q000494:pharmacology; D001392:Azocines / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004622:Embryo, Mammalian; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D009538:Nicotine / Q000031:analogs & derivatives / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D011807:Quinolizines / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012588:Scintillation Counting / Q000379:methods*; D000093843:Quinolizidine Alkaloids
D000470:Alkaloids; D001384:Azetidines; D001392:Azocines; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002118:Calcium; D009538:Nicotine; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011725:Pyridines; D011807:Quinolizines; D011978:Receptors, Nicotinic; C109763:5-(2-azetidinylmethoxy)-2-chloropyridine; C403531:nicotinic receptor alpha4beta2; C012419:metanicotine; C004712:cytisine; D000093843:Quinolizidine Alkaloids; C082748:epibatidine