Abstract
G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Gαq-coupled GPCRs are activated. Although Gαi- and Gαs-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Gαprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.
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Eglen, R. M., Bosse, R., and Reisine, T. (2007) Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening. Assay Drug Dev. Technol. 5, 425–451.
Coward, P., Chan, S. D., Wada, H. G., Humphries, G. M., and Conklin, B. R. (1999) Chimeric G proteins allow a high-throughput signaling assay of Gi-coupled receptors. Anal. Biochem. 270, 242–248.
Yokoyama, T., Kato, N., and Yamada, N. (2003) Development of a high-throughput bioassay to screen melatonin receptor agonists using human melatonin receptor expressing CHO cells. Neurosci. Lett. 344, 45–48.
Kostenis, E., Waelbroeck, M., and Milligan, G. (2005) Techniques: promiscuous Gα proteins in basic research and drug discovery. Trends Pharmacol. Sci. 26, 595–602.
Schroeder, K. S. and Neagle, B. D. (1996) FLIPR: a new instrument for accurate, high throughput optical screening. J. Biomol. Screen. 1, 75–80.
Zhang, J. Y., Nawoschik, S., Kowal, D., Smith, D., Spangler, T., Ochalski, R., Schechter, L., and Dunlop, J. (2003) Characterization of the 5-HT6 receptor coupled to Ca2+ signaling using an enabling chimeric G-protein. Eur. J. Pharmacol. 472, 33–38.
Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Milligan, G., Groarke, D. A., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) Receptor for the pain modulatory neuropeptides FF and AF is an orphan G protein-coupled receptor. J. Biol. Chem. 275, 25965–25971.
Sawyer, N., Cauchon, E., Chateauneuf, A., Cruz, R. P., Nicholson, D. W., Metters, K. M., O'Neill, G. P., and Gervais, F. G. (2002) Molecular pharmacology of the human prostaglandin D2 receptor, CRTH2. Br. J. Pharmacol. 137, 1163–1172.
Kowal, D., Zhang, J., Nawoschik, S., Ochalski, R., Vlattas, A., Shan, Q., Schechter, L., and Dunlop, J. (2002) The C-terminus of Gi family G-proteins as a determinant of 5-HT(1A) receptor coupling. Biochem. Biophys. Res. Commun. 294, 655–659.
Lubin, M. L., Reitz, T. L., Todd, M. J., Flores, C. M., Qin, N., and Xin, H. (2006) A nonadherent cell-based HTS assay for N-type calcium channel using calcium 3 dye. Assay Drug Dev. Technol. 4, 689–694.
Ott, T. R., Pahuja, A., Lio, F. M., Mistry, M. S., Gross, M., Hudson, S. C., Wade, W. S., Simpson, P. B., Struthers, R. S., and Alleva, D. G. (2005) A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: utilization of U937 monocytic cells. J. Pharmacol. Toxicol. Methods 51, 105–114.
Kunapuli, P., Zheng, W., Weber, M., Solly, K., Mull, R., Platchek, M., Cong, M., Zhong, Z., and Strulovici, B. (2005) Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells. Assay Drug Dev. Technol. 3, 17–26.
Chen, J., Lake, M. R., Sabet, R. S., Niforatos, W., Pratt, S. D., Cassar, S. C., Xu, J., Gopalakrishnan, S., Pereda-Lopez, A., Gopalakrishnan, M., Holzman, T. F., Moreland, R. B., Walter, K. A., Faltynek, C. R., Warrior, U., and Scott, V. E. (2007) Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists. J. Biomol. Screen. 12, 61–69.
Lundholt, B. K., Scudder, K. M., and Pagliaro, L. (2003) A simple technique for reducing edge effect in cell-based assays. J. Biomol. Screen. 8, 566–570.
Xin, H., Wang, Y., Todd, M. J., Qi, J., and Minor, L. K. (2007) Evaluation of no-wash calcium assay kits: enabling tools for calcium mobilization. J. Biomol. Screen. 12, 705–714.
Dupriez, V., Maes, K., Le Poul, E., Burgeon, E., and Detheux, M. (2002) Aequorin-based functional assays for G-protein-coupled receptors, ion channels, and tyrosine kinase receptors. Receptors Channels 8, 319–330.
Le Poul, E., Hisada, S., Miziguchi, Y., Dupriez, V. J., Burgeon, E., and Detheux, M. (2002) Adaptation of aequorin functional assay to high throughput screening. J. Biomol. Screen. 7, 57–65.
Bovolenta, S., Foti, M., Lohmer, S., and Corazza, S. (2007) Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening. J. Biomol. Screen. 12, 694–704.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Emkey, R., Rankl, N.B. (2009). Screening G Protein-Coupled Receptors: Measurement of Intracellular Calcium Using the Fluorometric Imaging Plate Reader. In: Janzen, W., Bernasconi, P. (eds) High Throughput Screening. Methods in Molecular Biology, vol 565. Humana Press. https://doi.org/10.1007/978-1-60327-258-2_7
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DOI: https://doi.org/10.1007/978-1-60327-258-2_7
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