Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

cAMP detection methods in HTS: selecting the best from the rest

Nature Reviews Drug Discovery volume 3, pages 125–135 (2004)Cite this article

Key Points

  • G-protein-coupled receptors (GPCRs) are one of the most important areas of research in the pharmaceutical industry.

  • Recent years have seen an expansion in assay technologies that measure the downstream effects of activation of GPCRs. The key advantage of these functional assays is that they facilitate early and direct pharmacological characterization of compounds.

  • This article focuses on high-throughput technologies available for the detection of changes in levels of a key intracellular signalling molecule that are modulated by GPCR activation — 3′,5′-cyclic adenosine monophosphate (cAMP).

  • Two main types of technology for detecting changes in cAMP levels are discussed: accumulation assays and reporter-gene assays.

  • In accumulation assays, changes in intracellular cAMP are detected by the competition between cellular cAMP and a labelled form of cAMP for binding to an anti-cAMP antibody.

  • In reporter-gene assays, receptor-mediated changes in intracellular cAMP concentrations are detected via changes in the expression level of a particular gene (the reporter), the transcription of which is regulated by the transcription factor cAMP response-element binding protein binding to upstream cAMP response elements.

  • Particular consideration is given to the practical and scientific implications of the methodologies, with the aim of enabling the reader to make an informed choice about their strategy for identifying GPCR modulators.

Abstract

The number of technologies that enable high-throughput functional screening of G-protein-coupled receptors has expanded markedly over the past 5 years. Consequently, choosing the most appropriate technology can be a daunting task, particularly for Gi- or Gs-coupled receptors. The most common systems for cyclic AMP detection are reviewed, highlighting the practical and theoretical aspects that are important in their application to high-throughput screening. Current technologies can do the job, but it is likely that the future may require development of technologies that provide even greater biological information.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Sites on the GPCR signalling cascade commonly employed in in vitro assays of receptor function.
Figure 2: Schematic representation of common technologies available for the detection of cAMP accumulation.
Figure 3: Schematic representation of common technologies available for the detection of cAMP accumulation.
Figure 4: Schematic representation of the luciferase and β-lactamase reporter-gene technologies.

Similar content being viewed by others

References

  1. Langley, J. N. On nerve endings and on special excitable substances in cells. Proc. Roy. Soc. B78, 170–194 (1906).

    Article  Google Scholar 

  2. Ehrlich, P. Chemotherapeutics: scientific principles, methods and results. Lancet 2, 445–451 (1913).

    Google Scholar 

  3. Bleicher, K. H. et al. Hit and lead generation: beyond high-throughput screening. Nature Rev. Drug Disc. 2, 369–378 (2003).

    Article  CAS  Google Scholar 

  4. Hopkins, A. L. & Groom, C. R. The druggable genome. Nature Rev. Drug. Disc. 1, 727–730 (2002).

    Article  CAS  Google Scholar 

  5. Sittampalam, G. S. et al. High-throughput screening: advances in assay technologies. Curr. Opin. Chem. Biol. 13, 384–391 (1997).

    Article  Google Scholar 

  6. Hemmila, I. A. & Hurskainen, P. Novel detection strategies for drug discovery. Drug Disc. Today 7, S150–S156 (2002).

    Article  CAS  Google Scholar 

  7. Walters, W. P. & Namchuk, M. Designing screens: how to make your hits a hit. Nature Rev. Drug. Disc. 2, 259–266 (2003).

    Article  CAS  Google Scholar 

  8. Croston, G. E. Functional cell-based uHTS in chemical genomic drug discovery. Trends Biotech. 20, 110–115 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  10. Johnston, P. A. & Johnston, P. A. Cellular platforms for HTS: three case studies. Drug Disc. Today 7, 353–363 (2002). Highlights some of the benefits and challenges of using cell-based assays in screening campaigns by providing data from three functional assays that have been used in HTS. Specifically, it discusses a luciferase reporter-gene assay and a FLIPR Ca2+ assay for GPCR targets as well as a radioligand-uptake assay for a transporter target.

    Article  CAS  Google Scholar 

  11. Christopoulos, A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nature Rev. Drug Disc. 1, 198–210 (2002).

    Article  CAS  Google Scholar 

  12. Kenakin, T. Pharmacological Analysis of Drug–Receptor Interaction 3rd edn (Lippincott–Raven, Philadelphia, 1997).

    Google Scholar 

  13. Hanoune, J. & Defer, N. Regulation and role of adenylyl cyclase isoforms. Annu. Rev. Pharmacol. Toxicol. 41, 145–174 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Patel, T. B. et al. Molecular biological approaches to unravel adenylyl cyclase sugnaling and function. Gene 269, 13–25 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Thompson, W. J. Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function. Pharmacol. Ther. 51, 13–33 (1991).

    Article  CAS  PubMed  Google Scholar 

  16. Amersham Life Science. High throughput screening for cAMP formation by scintillation proximity radioimmunoassay. Proximity News Issue No. 23. (1996).

  17. NEN Life Science Products. A novel adenylyl cyclase activation assay on FlashPlate (Flasplate File #1, Application Note). (NEN Life Science Products Inc., Boston, Massachusetts, 1998).

  18. Kariv, I. I. et al. High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J. Biomol. Screen. 4, 27–32 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Prystay, L. et al. Homogeneous cell-based fluorescence polarisation assay for the direct detection of cAMP. J. Biomol. Screen. 6, 75–82 (2001).

    CAS  PubMed  Google Scholar 

  20. Allen, M. et al. A homogeneous high throughput nonradioactive method for measurement of functional activity of Gs-coupled receptors in membranes. J. Biomol. Screen. 7, 35–44 (2002). A good example of a thorough evaluation of a cAMP accumulation assay. Highlights some key considerations for accumulation technologies in general, in addition to specific aspects of the use of membranes and the fluorescence polarization technology in these assays.

    Article  CAS  PubMed  Google Scholar 

  21. Banks, P. et al. Impact of a red-shifted dye label for high throughput fluorescence polarisation assays of G-protein coupled receptors. J. Biomol. Screen. 5, 329–334 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Packard Bioscience. Whole cell cAMP Functional Assay (Technical Note, AN002–ASc). (Packard Instrument Company, Meriden, Connecticut, 2000).

  23. Bouchard, N. et al. cAMPI alphascreen assay: a method for the pharmacological characterisation and screening of GαI-coupled receptors in whole cells. (Perkin Elmer, Montreal, Canada, 2002).

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

    Article  CAS  PubMed  Google Scholar 

  25. Packard Bioscience. Analysis of potential compound interference of ALPHAScreen Signal (Application Note, ASC–012). (Packard Bioscience Company Inc., Meriden, Connecticut, 2001).

  26. Chiulli, A. C. et al. A novel high throughput chemiluminescent assay for the measurement of cellular cyclic adenosine monophosphate levels. J. Biomol. Screen. 5, 239–247 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Scott, M. G. H. et al. Effects of a range of β2-adrenoceptor agonists on changes in intracellular cyclic AMP and on cyclic AMP driven gene expression in cultured human airway smooth muscle cells. Br. J. Pharmacol. 128, 721–729 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hill, S. J. et al. Reporter-gene systems for the study of G-protein-coupled receptors. Curr. Opin. Pharmacol. 1, 526–532 (2001).

    Article  CAS  PubMed  Google Scholar 

  29. Wood, K. V. Marker proteins for gene expression. Curr. Opin. Biotechnol. 6, 50–58 (1995).

    Article  CAS  PubMed  Google Scholar 

  30. Southward, C. M. & Surett, M. G. The dynamic microbe: green fluorescent protein brings bacteria to light. Mol. Microbiol. 45, 1191–1196 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Greer, L. F. & Szalay, A. A. Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17, 43–74 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Dinger, M. C. & Beck-Sickinger, A. G. The first reporter gene assay on living cells: green fluorescent proteon as reporter gene for the investigation of Gi-protein coupled receptors. Mol. Biotechnol. 21, 9–18 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Chen, W. et al. A colorimetric assay for measuring activation of Gs and Gq coupled signalling pathways. Anal. Biochem. 226, 349–354 (1995).

    Article  CAS  PubMed  Google Scholar 

  34. Stables, J. et al. Development of a dual glow-signal firefly and Renilla luciferase reagent for the analysis of G-protein coupled receptor signalling. J. Recept. Signal Transduct. Res. 19, 395–410 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Viviani, V. R. The origin, diversity and structure function relationships of luciferases. Cell. Mol. Life Sci. 59, 1833–1850 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Zlokarnik, G. et al. Quantitation of transcription and clonal selection of single living cells with β-lactamase as reporter. Science 279, 84–88 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Kanupoli, P. et al. Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening. Anal. Biochem. 314, 16–29 (2003).

    Article  Google Scholar 

  38. Knapp, T. et al. Detection of β-lactamase reporter gene expression by flow cytometry. Cytometry 51A, 68–78 (2003).

    Article  CAS  Google Scholar 

  39. Whitney, M. et al. A genome-wide functional assay of signal transduction in living mammalian cells. Nature Biotech. 16, 1329–1333 (1998).

    Article  CAS  Google Scholar 

  40. George, S. E. Evaluation of a CRE-directed luciferase reporter gene assay as an alternative to measuring cAMP accumulation. J. Biomol. Screen. 2, 235–240 (1997). An early but important study highlighting some of the benefits and pitfalls in using reporter-gene assays. Specifically, it directly compares accumulation and reporter-gene assays for the study of agonism at G i - and G s -coupled receptors that are expressed endogenously in CHO cells.

    Article  CAS  Google Scholar 

  41. Goetz, A. S. et al. Development of a facile method for high throughput screening with reporter gene assays. J. Biomol. Screen. 5, 377–384 (2000).

    Article  CAS  PubMed  Google Scholar 

  42. Kemp, D. M. et al. The effect of ICER on screening methods involving CRE-mediated reporter gene expression. J. Biomol. Screen. 7, 141–148 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Kemp, D. M. et al. Partial agonism at serotonin 5-HT1B and dopamine D2L receptors using a luciferase reporter gene assay. Eur. J. Pharmacol. 373, 215–222 (1999).

    Article  CAS  PubMed  Google Scholar 

  44. Terstappen, G. C. et al. Development of a functional reporter gene HTS assay for the identification of mGluR7 modulators. J. Biomol. Screen. 5, 255–261 (2000).

    Article  CAS  PubMed  Google Scholar 

  45. Baker, J. et al. Influence of agonist efficacy and receptor phosphorylation on antagonist affinity measurements: differences between second messenger and reporter gene responses. Mol. Pharmacol. 64, 679–688 (2003). A recent paper comparing antagonist data generated via accumulation and reporter-gene technologies with a recombinantly expressed G s -coupled receptor. It highlights that desensitization might be an important factor influencing the data observed.

    Article  CAS  PubMed  Google Scholar 

  46. Nagmani, R. Evaluation of beta-adrenergic receptor subtypes in the human prostate cancer cell line-LNCaP. Biochem. Pharmacol. 65, 1489–1494 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Zhang, J. et al. A simple statistical parameter for use in evaluation and validation of high thoughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    Article  CAS  PubMed  Google Scholar 

  48. Offermans, S. & Simon, M. I. Gα15 and Gα16 couple a wide variety of receptors to phospholipase C. J. Biol. Chem. 270, 15175–15180 (1995).

    Article  Google Scholar 

  49. Conklin, B. R. et al. Carboxy-terminal mutations of Gqα and Gsα that alter the fidelity of receptor activation. Mol. Pharmacol. 50, 885–890 (1996).

    CAS  PubMed  Google Scholar 

  50. Milligan, G. & Rees, S. Chimeric Gα proteins: their potential use in drug discovery. Trends Pharmacol. Sci. 20, 118–124 (1999).

    Article  CAS  PubMed  Google Scholar 

  51. Kostenis, E. Is Gα16 the optimal tool for fishing ligands of orphan G-protein-coupled receptors? Trends Pharmacol. Sci. 22, 560–564 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Craig, D. The Cheng–Prussoff relationship:something lost in the translation. Trends Pharmacol. Sci. 14, 89–91 (1993).

    Article  CAS  PubMed  Google Scholar 

  53. Leff, P. & Dougall, G. Further concerns over Cheng–Prusoff analysis. Trends Pharmacol. Sci. 14, 110–112 (1993).

    Article  CAS  PubMed  Google Scholar 

  54. Giuliano, K. A. High-content profiling of drug–drug interactions: cellular targets involved in modulation of microtubule drug action by the antifungal ketoconazole. J. Biomol. Screen. 8, 125–135 (2003).

    Article  CAS  PubMed  Google Scholar 

  55. Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Conway, B. R. et al. Quantification of G-protein coupled receptor internalisation using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan TM High-content screening system. J. Biomol. Screen. 4, 75–86 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Barak, L. S. et al. A β-arrestin/green fluorescent protein biosensor for detecting G-protein coupled receptor activation. J. Biol. Chem. 272, 27497–27500 (1997).

    Article  CAS  PubMed  Google Scholar 

  58. Hermans, E. Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors. Pharmacol. Ther. 99, 25–44 (2003). An excellent review of the evidence supporting agonist trafficking at GPCRs and its relevance in both the physiological and drug discovery contexts.

    Article  CAS  PubMed  Google Scholar 

  59. Goetz, A. S. et al. A combination assay for simultaneous assessment of multiple signalling pathways. J. Pharmacol. Toxicol. Methods 42, 225–235 (1999).

    Article  CAS  PubMed  Google Scholar 

  60. Meyer, T. & Teruel, M. N. Fluorescence imaging of signalling networks. Trends Cell Biol. 13, 101–106 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Hurskainen, P. et al. Time-resolved fluoremetry in bioanalytical assays. Chem. Today 5, 22–24 (2001).

    Google Scholar 

  62. Milligan, G. Principles: extending the utility of [35S]GTPγS binding assays. Trends Pharmacol. Sci. 24, 87–90 (2003).

    Article  CAS  PubMed  Google Scholar 

  63. Horstman, D. A. et al. Formation and metabolism of [3H]inositolphophates in AR42J pancreatoma cells. Substance P-induced Ca2+ mobilization in the apparent absence of inositol 1,4,5-triphosphate 3-kinase activity. J. Biol. Chem. 263, 15297–15303 (1988).

    CAS  PubMed  Google Scholar 

  64. Chambers, C. et al. Measuring intracellular calcium fluxes in high throughput mode. Comb. Chem. & High Throughput Screen. 6, 355–362 (2003).

    Article  CAS  Google Scholar 

  65. Schroeder, K. S. & Neagle, B. D. FLIPR: a new instrument for accurate, high throughput optical screening. J. Biomol. Screen. 1, 75–80 (1996).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The author wishes to thank A. Sewing, C. Perros-Huguet, F. Smith, S. Patrick, F. Bertelli and G. Ciaramella for reviewing the document and providing helpful comments.

Author information

Authors and Affiliations

  1. Hit Discovery Group (HDG), Pfizer Global Research and Development, Ramsgate Road, Sandwich, CT13 9NJ, Kent, UK

    Christine Williams

Authors
  1. Christine Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Ethics declarations

Competing interests

The author declares no competing financial interests.

Related links

Related links

DATABASES

LocusLink

calmodulin

CREB

β-galactosidase

β-lactamase

ICER

β2-adrenoceptor

FURTHER INFORMATION

Amersham Biosciences

Atto Biosciences

CIS Bio International HTRF web site

DiscoveRx

Meso Scale Discovery

Perkin Elmer

The Automation Partnership

Glossary

ALLOSTERIC MODULATOR

A compound that acts on a modulatory binding site on a receptor that is topographically distinct from the agonist binding site.

RED-SHIFTED PLATES

Shifting the assay to the red range ensures the most sensitive imaging cameras can be used and reduces interference from yellow/brown compounds.

COLOUR QUENCH

An inappropriate decrease in an assay signal due to the presence of a coloured entity.

PARTIAL AGONIST

An agonist that is unable to induce maximal activation of a receptor population, regardless of the amount of compound applied.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Williams, C. cAMP detection methods in HTS: selecting the best from the rest. Nat Rev Drug Discov 3, 125–135 (2004). https://doi.org/10.1038/nrd1306

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrd1306

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing