SpringerProtocols: Abstract: Screening Technologies for G Protein-Coupled Receptors: From HTS to uHTS

2. Screening Technologies for G Protein-Coupled Receptors: From HTS to uHTS

By: Maite de los Frailes², Emilio Diez²

Abstract

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The discovery of drugs for G protein-coupled receptors (GPCRs) has traditionally been very successful, even before the structural nature of these molecular targets was elucidated. Over the years, this family of proteins has become more important in the understanding and treatment of different human pathologies, representing today close to 30% of the molecular targets of all marketed drugs. The sequencing of the human genome unveiled the existence of many new GPCRs and this has increased even more the interest of this family of proteins as potential drug targets. Today the search for compounds that interfere or modulate the function of GPCRs is one of the major focuses of pharmaceutical companies. The understanding of the molecular events that take place upon receptor activation, together with the need of testing large chemical libraries, has resulted in the development of a variety of methods and technologies to measure the activity of these receptors. In this chapter we will review most of the assay technologies currently in use for “in vitro” pharmacological screening, their evolution, their capabilities, and their limitations.

Affiliation(s): (2) Department of Screening and Compound Profiling, Molecular Discovery Research, GlaxoSmithKline, Madrid, Spain

Book Title: G Protein-Coupled Receptors in Drug Discovery

Series: Methods in Molecular Biology | Volume: 552 | Pub. Date: Aug-01-2009 | Page Range: 15-37 | DOI: 10.1007/978-1-60327-317-6_2

Subject: Pharmacology/Toxicology

Key Words: GPCR (G protein-coupled receptor) - HTS (high-throughput screening) - uHTS (ultra-high-throughput screening)

References

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1.	Venter, J.C., Adams, M.D., Myers, E.W., et al. (2001) The sequence of the human genome. Science 291, 1304–1351.

2.	Drews, J. (2000) Drug discovery: a historical perspective. Science 287, 1960–1964.

3.	Ma, P., and Zemmel, R. (2002) Value of novelty? Nat. Rev. Drug Discov. 1, 571–572.

4.	Overington, J.P., Al-Lazikani, B., and Hopkins, A.L. (2006) How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996.

5.	Foord, S.M., Bonner, T.I., Neubig, R.R., Rosser, E.M., Pin, J.P., Davenport, A.P., Spedding, M., and Harmar, A.J. (2005) International union of pharmacology. XLVI G protein-coupled receptor list. Pharmacol. Rev. 57, 279–288.

6.	Neubig, R.R., Spedding, M., Kenakin, T., and Christopoulos, A. (2003) International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. XXXVIII Update on terms and symbols in quantitative pharmacology Pharmacol. Rev. 55, 597–606.

7.	Jacoby, E., Bouhelal, R., Gerspacher, M., and Seuwen, K. (2006) The 7TM G Protein-Coupled Receptor Target Family. Chem. Med. Chem. 1, 760–782.

8.	Wieland, T., and Michel, M. (2005) Can a GDP-liganded G protein be active? Mol. Pharmacol. 68, 559–562.

9.	Milligan, G., and Kostenis, E. (2006) Heterotrimeric G proteins: a short history. Br. J. Pharmacol. 147(Suppl 1), S46–S55.

10.	Lefkowitz, R.J., and Shenoy, S.K. (2005) Transduction of receptor signals by beta-arrestins. Science 308, 512–517.

11.	Hertzberg, R., and Pope, A.J. (2000) High-throughput screening: new technology for the 21st century. Curr. Opin. Chem. Biol. 4, 445–451.

12.	Ramm, P. (1999) Imaging systems in assay screening. Drug Discov. Today 4,401–410.

13.	Rose, D. (1999) Micro-dispensing technologies in drug discovery. Drug Discov. Today 4, 411–419.

14.	Labcyte home page: http://www.labcyte.com/Index.aspx?MenuID=1000143, Echo R Liquid Handling System.

15.	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 and Drug Develop Technol. 5, 425–451.

16.	Moore, K., and Rees, S. (2001). Cell-based versus isolated target screening: how lucky do you feel? J. Biomol. Screen. 6, 69–74.

17.	Cook, N.D. (1996). Scintillation proximity assay: a versatile high-throughput screening technology. Drug Discov. Today 1, 287–294.

18.	Kahl, S., Hubbard, F.R., Sittampalam, G.S., and Zock, J. (1997) Validation of a High Throughput Scintillation Proximity Assay for 5-Hydroxytryptamine 1E Receptor Binding Activity. J. Biomol. Screen. 2, 33–40.

19.	Bossé, R., Garlick, R., Brown, B., and Menard, L. (1998) Development of Non-separation Binding and Functional Assays for G Protein-Coupled Receptors for High Throughput Screening: Pharmacological Characterization of the Immobilized CCR5 Receptor on FlashPlate. J. Biomol. Screen. 3, 285–292.

20.	Green, A., Walls, S., Wise, A., Green, R.H., Martin, A.M., and Marshall, F.H. (2000) Characterization of [³H]-CGP54626A binding to heterodimeric GABA_B receptors stably expressed in mammalian cells. Br. J. Pharmacol. 131, 1766–1774.

21.	Bylund, D., Deupree, J.D., and Toews, M.L. (2004) Radioligand-binding methods for membrane preparations and intact cells. Methods Mol. Biol. 259, 1–28.

22.	Ahsen, O.V., Schmidt, A., Klotz, M., and Parczyk, K. (2006) Assay Concordance between SPA and TR-FRET in high-throughput screening. J. Biomol. Screen. 11, 606–616.

23.	Zhang, J.H. (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73.

24.	Harrison, C., and Traynor, J.R. (2003) The [(35)S] GTPgammaS binding assay: approaches and applications in pharmacology. Life Sciences 74, 489–508.

25.	Ferrer, M., Kolodin, G.D., Zuck, P., Peltier, R., Berry, K., Mandala, S., Rosen, H., Ota, H., Ozaki, S., Inglese, J., and Strulovice, B. (2003) A Fully Automated [³⁵S-GTPγS] Scintillation proximity sssay for the high-throughput screening of Gi-linked G protein-coupled receptors. Assay and Drug Develop. Technol. 1, 261–273.

26.	Johnson, E.N., Shi, X., Cassaday, J., Ferrer, M., Strulovici, B., and Kunapuli, P. (2008) A 1,536-well [(35)S] GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan Galphai-coupled GPCR. Assay and Drug Develop. Technol. 6, 327–337.

27.	Yan, J.H., Li, Q.Y., Boutin, J.A., Renard, M.P., Ding, Y.X., Hao, X.J., Zhao, W.M., and Wang, M.W. (2008) The high-throughput screening of novel antagonists on melanin-concentrating hormone receptor. Acta Pharmacol. Sinica 29, 752–758.

28.	Windh, R.T., and Manning, D.R. (2002) Analysis of G protein activation in Sf9 and mammalian cells by agonist-promoted [³⁵S-GTPγS] binding. Methods Enzymol. 344, 3–14.

29.	Wang, D., Raehal, K., Bilski, E., and Sadeée, W. (2001) Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signalling in narcotic dependence. J. Neurochem. 77, 1590–1600.

30.	Pagliaro, L., and Praestegaard, M. (2001) Transfected cell lines as tools for high throughput screening: a call for standards. J. Biomol. Screen. 6, 133–136.

31.	Kost, T., Condreay, J.P, Ames, R.S, Rees, S., and Romanos, M.A. (2007) Implementation of BacMam virus gene delivery technology in a drug discovery setting. Drug Discov. Today 12, 396–403.

32.	Soudijn, W., Wijngaarden, I.V., and Ijzerman, A.P. (2004) Allosteric modulation of G protein-coupled receptors: perspectives and recent developments. Drug Discov. Today 9, 752–758.

33.	Pinkerton, A.B., Cube, R.V., Hutchinson, J.H., James, J.K, Gardner, M.F., Rowe, B.A., Schaffhauser, H., Rodriguez, D.E., Campbell, U.C., Daggett, L.P., and Vernier, J.-M. (2005) Allosteric potentiators of the metabotropic glutamate receptor 2 (mGlu2). Part 3: Identification and biological activity of indanone containing mGluR2 receptor potentiators. Bioorganic Med. Chem. Lett. 15, 1565–1571.

34.	Chambers, C., Smith, F., Williams, C., Marcos, S., Liu, S.M., Hayter, P., Ciaramella, G., Keighley, W., Gribbon, P., and Sewing, A. (2003) Measuring intracellular calcium fluxes in high throughput mode. Comb. Chem. High Throughput Screen. 6, 355–362.

35.	Offermanns, S., and Simon, M.I. (1995) G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175–15180.

36.	Kostensis, E.J. (2002) Potentiation of GPCRs-signalling via membrane targeting of G protein alpha subunits. Receptors Signal Transduct. 22, 267–281.

37.	Ungrin, M.D., Singh, L.M., Stocco, R., Sas, D.E., and Abramovitz, M. (1999) An Automated aequorin luminescence-based functional calcium assay for G protein-coupled receptors. Anal. Biochem. 272, 34–42.

38.	Le Poul, E., Hisada, S., Mizuguchi, 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.

39.	Kariv, I., Stevens, M.E., Behrens, D.L., and Oldenburg, K.R. (1999) High throughput quantification of cAMP production mediated by activation of seven transmembrane domain receptors. J. Biomol. Screen. 4, 27–32.

40.	Kemp, D.M., George, S.E., Kent, T.C., Bungay, P.J., and Naylor, L.H. (2002) The effect of ICRE on screening methods involving CRE-mediated reporter gene expression. J. Biomol. Screen. 7, 141–148.

41.	Gabriel, D., Vernier, M., Pfeifer, M.J., Dasen, B., Tenaillon, L., and Bouhelal, R. (2003) High throughput screening technologies for direct cyclic AMP measurement. Assay Drug Develop. Technol. 1, 291–303.

42.	Golla, R., and Seethala, R. (2002) A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonist. J. Biomol. Screen. 7, 515–525.

43.	Weber, M., Muthusubramaniam, L., Murray, J., Hudak, E., Kornienko, O., Johnson, E.N., Strulovici, B., and Kunapuli, P. (2007) Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-µl 3456-well plate format cAMP Assay. Assay Drug Dev. Technol. 5, 117–125.

44.	Ullman, E.F., Kirakossiona, H., and Singh, S. (1994) Luminescent oxygen channelling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc. Natl. Acad. Sci. USA 91, 5426–5430.

45.	Elster, L., Elling, C., and Heding, A. (2007) Bioluminescence resonance energy transfer as a screening assay: focus on partial and inverse agonism. J. Biomol. Screen. 12, 41–49.

46.	Kasia, P., and Xie, H. (2006) A comparison of PerkinElmer’s LANCE cAMP assay performance to that of state of the art competitive technologies. PE- Application Notes. 1–8, http://www.perkinelmer.com.

47.	Claing, A., Laporte, S.A, Caron, M.G., and Lefkowitz, R.J. (2002). Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and β-arrestin proteins. Prog. Neurobiol. 66, 61–79.

48.	Reiter, E., and Lefkowitz, R.J. (2006). GRKs and beta-arrestins: roles in receptor silencing, trafficking and signalling. Trends Endocrinol. Metab. 17, 159–165.

49.	Ahn, S., Nelson, C., Garrison, T.R., Miller, W.E., and Lefkowitz, R.L. (2002) Desensitization, internalization, and signalling functions of β-arrestins demonstrated by RNA interference. Proc. Natl. Acad. Sci. USA 18, 1740–1744.

50.	Ren, X-R., Reiter E., Ahn, S., Kim, J., Chen, W., and Lefkowitz, R.J. (2004) Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signalling of V2 vasopressin receptor. Proc. Natl. Acad. Sci. USA 102, 1448–1453.

51.	Kang, J., Shi, Y., Xiang, B., Qu, B., Su, W., Zhu, M., Zhang, M., Bao, G., Wang, F., and Zhang, X. (2005). A nuclear function of β-arrestin 1 in GPCR signalling regulation of histone acetylation and gene transcription. Cell 123, 833–847.

52.	Violin, J.D., and Lefkowith, R.J. (2007) β-arrestin-biased ligands at seven-transmembrane receptors. Trends Pharmacol. Sci. 28, 417–422.

53.	Wei, H., Ahn, S., Shenoy, S.K., Hunyady, L., Luttrell, L.M., and Lefkowitz, R. J. (2003) Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl. Acad. Sci. USA 100, 10782–10787.

54.	Kohout, T.A., Nicholas, S.L., Perry, S.J., Reinhart, G., Junger, S., and Struthers R.S. (2004) Differential desensitization, receptor phosphorylation, beta-arrestin recruitment, and ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7. J. Biol. Chem. 279, 23214–23222.

55.	Bruns, R., Chhum, S., Dinh, A.T., Doerr, H., Dunn, N.R., Ly, Y.T., Mitman, C.L., Rickards, H.D., Sol, C., Wan, E.W., and Raffa, R.B. (2006) A potential novel strategy to separate therapeutic- and side-effects that are mediated via the same receptor: β-arrestin 2/G protein coupling antagonists. J. Clin. Pharm. Ther. 31, 119–128.

56.	Conway, B.R., Minor, L.M., Xu, J.Z., Gunnet, J.W., DeBiasio, R., Dándrea, M.R., Rubin, R., Debiasio, R., Giuliano, K., Zhou, L., and Demarest, K.T. (1999) Quantification of G protein receptor internalization using G protein-coupled receptor-green fluorescent protein conjugates with the ArrayScan™ high-content screening systems. J. Biomol. Screen. 4, 75–86.

57.	Bertrand, L., Parent, S., Caron, M., Legault, M., Joly, E.N., Angers, S., Bouvier, M., Brown, M., Benoit, H., Houle, B., and Ménard, L. (2002) The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRs). J. Receptor Signal Transduct. Res. 22, 533–541.

58.	Wehrman, T.S., Casipit, C.L., Gewertz, N.M., and Blau, H.M. (2005) Enzymatic detection of protein translocation. Nat. Methods 2, 521–527.

59.	de Wet, J.R., Wood, K.V., deLuca, M., Helinski, D.R., and Subramini, S. (1987) Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell. Biol. 7, 725–737.

60.	Wilson, T., and Hastings, J.W. (1998). Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14, 197–230.

61.	Athwal, G.K., El-Kholy, E.O., and Cass, A.E.G. (1991) Amplified Bioluminescence Assays. In: Bioluminescence and Chemiluminescence Current Status. Stanley, P.E., and Kricka, L.J. (eds.), pp. 67–70.

62.	Wood, K.V. (1998) The chemistry of bioluminescence reporter assays. Promega Notes 65, 14–20.

63.	Kent, T.C., Thompson, K.S., and Naylor, L.H. (2005) Development of a generic dual-reporter gene assay for screening G protein-coupled receptors. J. Biomol. Screen. 10, 437–447.

64.	Hancock, M., Medina, M.N., Smith, B.M., and Orth, A.P. (2007) Microplate orbital mixing improves high-throughput cell-based assay readouts. J. Biomol. Screen. 12, 140–144.

65.	Chen, D., Liao, J., Li, N., Zhou, C., Wang, G., Zhang, R., Zhang, S., Lin, L., Chen, K., Xie, X., Nan, F., Young, A., and Wang, M.W. (2007) A nonpeptidic agonist of glucagon-like peptide 1 receptors with efficacy in diabetic db/db mice. Proc. Natl. Acad. Sci. USA 104, 943–948.

66.	Terstapen, G.S., Giacometti, A., Ballini, E., and Aldegheri, L. (2000) Development of a functional reporter gene HTS assay for the identification of mGluR7 modulators. J. Biomol. Screen. 5, 255–261.

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