Application of Fluorescent Purinoceptor Antagonists for Bioluminescence Resonance Energy Transfer Assays and Fluorescent Microscopy

  • Mark Soave
  • Joëlle Goulding
  • Robert Markus
  • Stephen J. Hill
  • Leigh A. StoddartEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2041)


Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor–ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

Key words

Fluorescence Antagonist Purinergic receptor BRET Confocal microscopy Ligand binding 


  1. 1.
    Abbracchio MP, Burnstock G, Boeynaems J-M, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA (2006) International Union of Pharmacology. LVIII: Update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 58:281–341CrossRefGoogle Scholar
  2. 2.
    Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS (2011) Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev 63:641–683CrossRefGoogle Scholar
  3. 3.
    Fredholm BB, IJzerman AP, Jacobson KA, Linden J, Müller CE (2011) International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors, an update. Pharmacol Rev 63:1–34CrossRefGoogle Scholar
  4. 4.
    Burnstock G (2017) Purinergic signalling: therapeutic developments. Front Pharmacol 8:661CrossRefGoogle Scholar
  5. 5.
    Sarafoff N, Byrne RA, Sibbing D (2012) Clinical use of clopidogrel. Curr Pharm Des 18:5224–5239CrossRefGoogle Scholar
  6. 6.
    Lau OC, Samarawickrama C, Skalicky SE (2014) P2Y2 receptor agonists for the treatment of dry eye disease: a review. Clin Ophthalmol 8:327–334PubMedPubMedCentralGoogle Scholar
  7. 7.
    Garland SL (2013) Are GPCRs still a source of new targets? J Biomol Screen 18:947–966CrossRefGoogle Scholar
  8. 8.
    Stoddart LA, Kilpatrick LE, Briddon SJ, Hill SJ (2015) Probing the pharmacology of G protein-coupled receptors with fluorescent ligands. Neuropharmacology 98:48–57CrossRefGoogle Scholar
  9. 9.
    Stoddart LA, Vernall AJ, Denman JL, Briddon SJ, Kellam B, Hill SJ (2012) Fragment screening at adenosine-A(3) receptors in living cells using a fluorescence-based binding assay. Chem Biol 19:1105–1115CrossRefGoogle Scholar
  10. 10.
    Arruda MA, Stoddart LA, Gherbi K, Briddon SJ, Kellam B, Hill SJ (2017) A non-imaging high throughput approach to chemical library screening at the unmodified adenosine-A3 receptor in living cells. Front Pharmacol 8:908CrossRefGoogle Scholar
  11. 11.
    Vernall AJ, Stoddart LA, Briddon SJ, Ng HW, Laughton CA, Doughty SW, Hill SJ, Kellam B (2013) Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers. Org Biomol Chem 11:5673–5682CrossRefGoogle Scholar
  12. 12.
    Stoddart LA, White CW, Nguyen K, Hill SJ, Pfleger KD (2016) Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding. Br J Pharmacol 173:3028–3037CrossRefGoogle Scholar
  13. 13.
    Stoddart LA, Johnstone EK, Wheal AJ, Goulding J, Robers MB, Machleidt T, Wood KV, Hill SJ, Pfleger KD (2015) Application of BRET to monitor ligand binding to GPCRs. Nat Methods 12:661–663CrossRefGoogle Scholar
  14. 14.
    Stoddart LA, Kilpatrick LE, Hill SJ (2018) NanoBRET approaches to study ligand binding to GPCRs and RTKs. Trends Pharmacol Sci 39:136–147CrossRefGoogle Scholar
  15. 15.
    Soave M, Stoddart LA, Brown A, Woolard J, Hill SJ (2016) Use of a new proximity assay (NanoBRET) to investigate the ligand-binding characteristics of three fluorescent ligands to the human beta1-adrenoceptor expressed in HEK-293 cells. Pharmacol Res 4:e00250Google Scholar
  16. 16.
    Christiansen E, Hudson BD, Hansen AH, Milligan G, Ulven T (2016) Development and characterization of a potent free fatty acid receptor 1 (FFA1) fluorescent tracer. J Med Chem 59:4849–4858CrossRefGoogle Scholar
  17. 17.
    Rose RH, Briddon SJ, Hill SJ (2012) A novel fluorescent histamine H(1) receptor antagonist demonstrates the advantage of using fluorescence correlation spectroscopy to study the binding of lipophilic ligands. Br J Pharmacol 165:1789–1800CrossRefGoogle Scholar
  18. 18.
    Stoddart LA, Vernall AJ, Bouzo-Lorenzo M, Bosma R, Kooistra AJ, de Graaf C, Vischer HF, Leurs R, Briddon SJ, Kellam B, Hill SJ (2018) Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells. Sci Rep 8:1572CrossRefGoogle Scholar
  19. 19.
    Conroy S, Kindon ND, Glenn J, Stoddart LA, Lewis RJ, Hill SJ, Kellam B, Stocks MJ (2018) Synthesis and evaluation of the first fluorescent antagonists of the human P2Y2 receptor based on AR-C118925. J Med Chem 61:3089–3113CrossRefGoogle Scholar
  20. 20.
    Vernall AJ, Stoddart LA, Briddon SJ, Hill SJ, Kellam B (2012) Highly potent and selective fluorescent antagonists of the human adenosine A3 receptor based on the 1,2,4-triazolo(4,3-a)quinoxalin-1-one scaffold. J Med Chem 55:1771–1782CrossRefGoogle Scholar
  21. 21.
    Dong C, Filipeanu CM, Duvernay MT, Wu G (2007) Regulation of G protein-coupled receptor export trafficking. Biochim Biophys Acta 1768:853–870CrossRefGoogle Scholar
  22. 22.
    Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K (2004) Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101:9955–9959CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Mark Soave
    • 1
    • 2
  • Joëlle Goulding
    • 1
    • 2
  • Robert Markus
    • 3
  • Stephen J. Hill
    • 1
    • 2
  • Leigh A. Stoddart
    • 1
    • 2
    Email author
  1. 1.Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life SciencesUniversity of NottinghamNottinghamUK
  2. 2.Centre of Membrane Proteins and Receptors (COMPARE)University of Birmingham and University of NottinghamMidlandsUK
  3. 3.School of Life Sciences Imaging (SLIM), School of Life SciencesUniversity of NottinghamNottinghamUK

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