Skip to main content
SpringerLink
Log in

Time-Resolved FRET Strategy to Screen GPCR Ligand Library

  • Protocol
  • First Online:
G Protein-Coupled Receptor Screening Assays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1272))

Abstract

Screening chemical libraries to find specific drugs for G protein-coupled receptors is still of major interest. Indeed, because of their major roles in all physiological functions, G protein-coupled receptors remain major targets for drug development programs. Currently, interest in GPCRs as drug targets has been boosted by the discovery of biased ligands, thus allowing the development of drugs not only specific for one target but also for the specific signaling cascade expected to have the therapeutic effect. Such molecules are then expected to display fewer side effects. To reach such a goal, there is much interest in novel, efficient, simple, and direct screening assays that may help identify any drugs interacting with the target, these being then analyzed for their biased activity. Here, we present an efficient strategy to screen ligands on their binding properties. The method described is based on time-resolved FRET between a receptor and a ligand. This method has already been used to develop new assays called Tag-lite® binding assays for numerous G protein-coupled receptors, proving its broad application and its power.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
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

References

  1. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996

    Article  CAS  Google Scholar 

  2. Vogel SS, Thaler C, Koushik SV (2006) Fanciful FRET. Sci STKE 2006:re2

    PubMed  Google Scholar 

  3. Pietraszewska-Bogiel A, Gadella TW (2011) FRET microscopy: from principle to routine technology in cell biology. J Microsc 241:111–118

    Article  CAS  Google Scholar 

  4. Selvin PR (2002) Principles and biophysical applications of lanthanide-based probes. Annu Rev Biophys Biomol Struct 31:275–302

    Article  CAS  Google Scholar 

  5. Mathis G, Bazin H (2011) Stable luminescent chelates and macrocyclic compounds. In: Härmä HD, Hänninen P (eds) Lanthanide luminescence. Springer, Berlin, pp 47–88

    Google Scholar 

  6. Durroux T, Peter M, Turcatti G, Chollet A, Balestre MN, Barberis C, Seyer R (1999) Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applications. J Med Chem 42:1312–1319

    Article  CAS  Google Scholar 

  7. Albizu L, Cottet M, Kralikova M, Stoev S, Seyer R, Brabet I, Roux T, Bazin H, Bourrier E, Lamarque L, Breton C, Rives ML, Newman A, Javitch J, Trinquet E, Manning M, Pin JP, Mouillac B, Durroux T (2010) Time-resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat Chem Biol 6:587–594

    Article  CAS  Google Scholar 

  8. Albizu L, Teppaz G, Seyer R, Bazin H, Ansanay H, Manning M, Mouillac B, Durroux T (2007) Toward efficient drug screening by homogeneous assays based on the development of new fluorescent vasopressin and oxytocin receptor ligands. J Med Chem 50:4976–4985

    Article  CAS  Google Scholar 

  9. Daniels DS, Mol CD, Arvai AS, Kanugula S, Pegg AE, Tainer JA (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. EMBO J 19:1719–1730

    Article  CAS  Google Scholar 

  10. Pegg AE, Dolan ME (1987) Properties and assay of mammalian O6-alkylguanine-DNA alkyltransferase. Pharmacol Ther 34:167–179

    Article  CAS  Google Scholar 

  11. Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K (2003) Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo. Chem Biol 10:313–317

    Article  CAS  Google Scholar 

  12. Juillerat A, Heinis C, Sielaff I, Barnikow J, Jaccard H, Kunz B, Terskikh A, Johnsson K (2005) Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for specific protein labeling in living cells. Chembiochem 6:1263–1269

    Article  CAS  Google Scholar 

  13. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol 21:86–89

    Article  CAS  Google Scholar 

  14. 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 101:9955–9959

    Article  CAS  Google Scholar 

  15. Gronemeyer T, Chidley C, Juillerat A, Heinis C, Johnsson K (2006) Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein Eng Des Sel 19:309–316

    Article  CAS  Google Scholar 

  16. Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M, Beaufils F, Johnsson K (2008) An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136

    Article  CAS  Google Scholar 

  17. Zhang Y, So MK, Loening AM, Yao H, Gambhir SS, Rao J (2006) HaloTag protein-mediated site-specific conjugation of bioluminescent proteins to quantum dots. Angew Chem Int Ed Engl 45:4936–4940

    Article  CAS  Google Scholar 

  18. Zwier JM, Roux T, Cottet M, Durroux T, Douzon S, Bdioui S, Gregor N, Bourrier E, Oueslati N, Nicolas L, Tinel N, Boisseau C, Yverneau P, Charrier-Savournin F, Fink M, Trinquet E (2010) A fluorescent ligand-binding alternative using Tag-lite(R) technology. J Biomol Screen 15:1248–1259

    Article  CAS  Google Scholar 

  19. Loison S, Cottet M, Orcel H, Adihou H, Rahmeh R, Lamarque L, Trinquet E, Kellenberger E, Hibert M, Durroux T, Mouillac B, Bonnet D (2012) Selective fluorescent nonpeptidic antagonists for vasopressin V(2) GPCR: application to ligand screening and oligomerization assays. J Med Chem 55:8588–8602

    Article  CAS  Google Scholar 

  20. Emami-Nemini A, Roux T, Leblay M, Bourrier E, Lamarque L, Trinquet E, Lohse MJ (2013) Time-resolved fluorescence ligand binding for G protein-coupled receptors. Nat Protoc 8:1307–1320

    Article  Google Scholar 

  21. Middleton RJ, Kellam B (2005) Fluorophore-tagged GPCR ligands. Curr Opin Chem Biol 9:517–525

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The development of the technique has been supported by the Fonds Unique Interministériel and OSEO in a collaborative program named “CELL2Lead.” Thanks are due to the Plateforme ARPÈGE of the Institut de Génomique Fonctionnelle.

Author information

Authors and Affiliations

  1. Institut de Génomique Fonctionnelle, CNRS, UMR 5203, 141 Rue de la Cardonille, 34094, Montpellier Cedex 5, France

    Nadia Oueslati, Candide Hounsou, Abderazak Belhocine, Thieric Rodriguez, Jean-Philippe Pin & Thierry Durroux

  2. INSERM, U. 661, Montpellier, France

    Nadia Oueslati, Candide Hounsou, Abderazak Belhocine, Thieric Rodriguez, Jean-Philippe Pin & Thierry Durroux

  3. Université Montpellier 1,2, Montpellier, France

    Nadia Oueslati, Candide Hounsou, Abderazak Belhocine, Thieric Rodriguez, Jean-Philippe Pin & Thierry Durroux

  4. Cisbio Bioassays, BP 84175, 30200, Codolet, France

    Elodie Dupuis, Jurriaan M. Zwier & Eric Trinquet

Authors
  1. Nadia Oueslati
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Candide Hounsou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abderazak Belhocine
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thieric Rodriguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Elodie Dupuis
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jurriaan M. Zwier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eric Trinquet
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Philippe Pin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Thierry Durroux
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thierry Durroux .

Editor information

Editors and Affiliations

  1. IBB – Institute for Biotechnology and Bioengineering, Lisbon, Portugal

    Duarte Miguel F. Prazeres  & Sofia Aires M. Martins  & 

  2. Department of Bioengineering, Instituto Superior Técnico Universidade de Lisboa, Lisbon, Portugal

    Duarte Miguel F. Prazeres

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Oueslati, N. et al. (2015). Time-Resolved FRET Strategy to Screen GPCR Ligand Library. In: Prazeres, D.M.F., Martins, S.A.M. (eds) G Protein-Coupled Receptor Screening Assays. Methods in Molecular Biology, vol 1272. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-2336-6_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2336-6_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-2335-9

  • Online ISBN: 978-1-4939-2336-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation