Abstract
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, [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 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 (Ki<2 μmol/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 α4β2 nAChR modulators in an HTS setting. Its application may be expanded to other membrane receptors and ion channels.
Similar content being viewed by others
Article PDF
References
Lukas RJ, Changeux JP, Novere N, Albuquerque EX, Balfour DJ, Berg DK, et al. International Union of Pharmacology. XX. Current status of the nomenclature for nicotinic acetylcholine receptors and their subunits. Pharmacol Rev 1999; 51: 397–401.
Albuquerque EX, Alkondon M, Pereira EF, Castro NG, Schrattenholz A, Barbosa CT, et al. Properties of neuronal nicotinic acetylcholine receptors: pharmacological characterization and modulation of synaptic function. J Pharmacol Exp Ther 1997; 280: 1117–36.
Cordero-Erausquin M, Marubio LM, Klink R, Changeux JP . Nicotinic receptor function: new perspectives from knockout mice. Trends Pharmacol Sci 2000; 21: 211–7.
Kesingland AC, Gentry CT, Panesar MS, Bowes MA, Vernier JM, Cube R, et al. Analgesic profile of the nicotinic acetylcholine receptor agonists, (+)-epibatidine and ABT-594 in models of persistent inflammatory and neuropathic pain. Pain 2000; 86: 113–8.
Boyce S, Webb JK, Shepheard SL, Russell MG, Hill RG, Rupniak NM . Analgesic and toxic effects of ABT-594 resemble epibatidine and nicotine in rats. Pain 2000; 85: 443–50.
Carpenter JW, Laethem C, Hubbard FR, Eckols TK, Baez M, McClure D, et al. Configuring radioligand receptor binding assays for HTS using scintillation proximity assay technology. Methods Mol Biol 2002; 190: 31–49.
Chavez-Noriega LE, Gillespie A, Stauderman KA, Crona JH, Claeps BO, Elliott KJ, et al. Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells. Neuropharmacology 2000; 39: 2543–60.
Donnelly-Roberts DL, Puttfarcken PS, Kuntzweiler TA, Briggs CA, Anderson DJ, Campbell JE, et al. ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective analgesic acting via neuronal nicotinic acetylcholine receptors: In vitro characterization. J Pharmacol Exp Ther 1998; 285: 777–86.
Qian J, Voorbach MJ, Huth JR, Coen ML, Zhang HC, Ng SC, et al. Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms. Anal Biochem 2004; 328: 131–8.
Cheng Y, Prusoff WH . Relationship between the inhibition constant (K i) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099–108.
Khan IM, Yaksh TL, Taylor P . Ligand specificity of nicotinic acetylcholine receptors in rat spinal cord: studies with nicotine and cytisine. J Pharmacol Exp Ther 1994; 270: 159–66.
Wu B, Gao J, Wang M-W . Development of a complex scintillation proximity assay for high-throughput screening of PPAR? modulators. Acta Pharmacol Sin 2005; 26: 339–44.
Crane K, Shih DT . Development of a homogeneous binding assay for histamine receptors. Anal Biochem 2004; 335: 42–9.
Gobel J, Saussy DL, Goetz AS . Development of scintillation-proximity assays for alpha adrenoceptors. J Pharmacol Toxicol Methods 1999; 42: 237–44.
Rodgers G, Hubert C, McKinzie J, Suter T, Statnick M, Emmerson P, et al. Development of displacement binding and GTPγS scintillation proximity assays for the identification of antagonists of the micro-opioid receptor. Assay Drug Dev Technol 2003; 1: 627–36.
Xiao Y, Baydyuk M, Wang HP, Davis HE, Kellar KJ . Pharmacology of the agonist binding sites of rat neuronal nicotinic receptor subtypes expressed in HEK 293 cells. Bioorg Med Chem Lett 2004; 14: 1845–8.
Zhang JH, Chung TD, Oldenburg KR . A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999; 4: 67–73.
Author information
Authors and Affiliations
Corresponding author
Additional information
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.
Rights and permissions
About this article
Cite this article
Hui, X., Gao, J., Xie, X. et al. A robust homogeneous binding assay for α4β2 nicotinic acetylcholine receptor. Acta Pharmacol Sin 26, 1175–1180 (2005). https://doi.org/10.1111/j.1745-7254.2005.00202.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2005.00202.x
Keywords
This article is cited by
-
Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors
Analytical and Bioanalytical Chemistry (2010)