Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
G Protein-Coupled Receptors in Drug Discovery

Part of the book series: Methods in Molecular Biology ((MIMB,volume 552))

Summary

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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 GABAB receptors stably expressed in mammalian cells. Br. J. Pharmacol. 131, 1766–1774.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

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

    Article  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Google Scholar 

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

    Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

Download references

Acknowledgments

We thank many of our colleagues at GSK that continuously work in the generation of reagent, the development of assays , and the performance of HTS for GPCRs. The generation of the HTS data, the hits, the leads, and the candidates identified would have been impossible without the coordinated effort of all those scientists in many different GSK locations throughout the world. Specially, we thank Robert Hertzberg and Thomas Meek for their continuous support over the years.

Author information

Authors and Affiliations

  1. Department of Screening and Compound Profiling, Molecular Discovery Research, GlaxoSmithKline, Madrid, Spain

    Maite de los Frailes & Emilio Diez

Authors
  1. Maite de los Frailes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emilio Diez
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. CSIRO Human Nutrition, Kintore Ave., Adelaide, 5000, Australia

    Wayne R. Leifert

Rights and permissions

Reprints and permissions

Copyright information

© 2009 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

de los Frailes, M., Diez, E. (2009). Screening Technologies for G Protein-Coupled Receptors: From HTS to uHTS. In: Leifert, W. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 552. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-317-6_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-317-6_2

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-316-9

  • Online ISBN: 978-1-60327-317-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation