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Label-Free Functional Selectivity Assays

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G Protein-Coupled Receptor Screening Assays

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

Abstract

G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the β2-adrenergic receptor (β2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of β2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the β2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

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References

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Mailman RB (2007) GPCR functional selectivity has therapeutic impact. Trends Pharmacol Sci 8:390–396

    Article  CAS  Google Scholar 

  4. Zhou L, Bohn LM (2014) Functional selectivity of GPCR signaling in animals. Curr Opin Cell Biol 27:102–108

    Article  CAS  PubMed  Google Scholar 

  5. Kenakin T (2012) The potential for selective pharmacological therapies through biased receptor signaling. BMC Pharmacol Tox 13:3

    Article  CAS  Google Scholar 

  6. Kenakin T, Miller LJ (2010) Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol Rev 62:265–304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat Rev Drug Discov 9:373–386

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M, Piñeyro G (2003) β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein coupled receptors. Proc Natl Acad Sci U S A 100:11406–11411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Galandrin S, Bouvier M (2006) Distinct signaling profiles of β1 and β2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol Pharmacol 70:1575–1584

    Article  CAS  PubMed  Google Scholar 

  10. Wisler JW, DeWire SM, Whalen EJ, Violin JD, Drake MT, Ahn S, Shenoy SK, Lefkowitz RJ (2007) A unique mechanism of β-blocker action: carvedilol stimulates β-arrestin signaling. Proc Natl Acad Sci U S A 104:16657–16662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Drake MT, Violin JD, Whalen EJ, Wisler JW, Shenoy SK, Lefkowitz RJ (2008) β-Arrestin-biased agonism at the β2-adrenergic receptor. J Biol Chem 283:5669–5676

    Article  CAS  PubMed  Google Scholar 

  12. Kahsai A, Xiao K, Rajagopal S, Ahn S, Shukla AK, Sun J, Oas TG, Lefkowitz RJ (2011) Multiple ligand-specific conformations of the β2-adrenergic receptor. Nat Chem Biol 7:692–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zocher M, Fung JJ, Kobilka BK, Müller DJ (2012) Ligand-specific interactions modulate kinetic, energetic, and mechanical properties of the human β2 adrenergic receptor. Structure 20:1391–1402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K (2012) Biased signalling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kolinski M, Plazinska A, Jozwiak K (2012) Recent progress in understanding of structure, ligand interactions and the mechanism of activation of the β2-adrenergic receptor. Curr Med Chem 19:1155–1163

    Article  CAS  PubMed  Google Scholar 

  16. Evans BA, Sato M, Sarwar M, Hutchinson DS, Summers RJ (2010) Ligand-directed signalling at β2-adrenoceptors. Br J Pharmacol 159:1022–1038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Patel CB, Noor N, Rockman HA (2010) Functional selectivity in adrenergic and angiotensin signaling systems. Mol Pharmacol 78:983–992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kenakin T, Christopoulos A (2013) Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 12:205–216

    Article  CAS  PubMed  Google Scholar 

  19. Galandrin S, Oligny-Longpre G, Bouvier M (2007) The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol Sci 8:423–430

    Article  CAS  Google Scholar 

  20. Li B, Wang C, Zhou Z, Zhao J, Pei G (2013) β-Arrestin-1 directly interacts with Gαs and regulates its function. FEBS Lett 587:410–416

    Article  CAS  PubMed  Google Scholar 

  21. Saulière A, Bellot M, Paris H, Denis C, Finana F, Hansen JT, Altié MF, Seguelas MH, Pathak A, Hansen JL, Sénard JM, Galés C (2012) Deciphering biased-agonism complexity reveals a new active AT1 receptor entity. Nat Chem Biol 8:622–630

    Article  PubMed  CAS  Google Scholar 

  22. Fang Y, Ferrie AM, Fontaine NH, Mauro J, Balakrishnan J (2006) Resonant waveguide grating biosensor for living cell sensing. Biophys J 91:1925–1940

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ferrie AM, Wang C, Deng H, Fang Y (2013) Label-free optical biosensor with microfluidics identifies an intracellular signalling wave mediated through the β2-adrerengic receptor. Integr Biol 5:1253–1261

    Article  CAS  Google Scholar 

  24. Fang Y, Li G, Peng J (2005) Optical biosensor provides insights for bradykinin B2 receptor signaling in A431 cells. FEBS Lett 579:6365–6374

    Article  CAS  PubMed  Google Scholar 

  25. Tran E, Fang Y (2008) Duplexed label-free G protein-coupled receptor assays for high-throughput screening. J Biomol Screen 13:975–985

    Article  CAS  PubMed  Google Scholar 

  26. Dodgson K, Gedge L, Murray DC, Coldwell M (2009) A 100K well screen for a muscarinic receptor using the Epic label-free system: a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors. J Recept Signal Transduct Res 29:163–172

    Article  CAS  PubMed  Google Scholar 

  27. Schröder R, Janssen N, Schmidt J, Kebig A, Merten N, Hennen S, Müller A, Blättermann S, Mohr-Andrä M, Zahn S, Wenzel J, Smith NJ, Gomeza J, Drewke C, Milligan G, Mohr K, Kostenis E (2010) Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat Biotechnol 28:943–949

    Article  PubMed  CAS  Google Scholar 

  28. Verrier F, An S, Ferrie AM, Sun H, Kyoung M, Fang Y, Benkovic SJ (2011) G protein-coupled receptor signaling regulates the dynamics of a metabolic multienzyme complex. Nat Chem Biol 7:909–915

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fang Y (2013) Troubleshooting and deconvoluting label-free cell phenotypic assays in drug discovery. J Pharmacol Toxicol Methods 67:69–81

    Article  CAS  PubMed  Google Scholar 

  30. Fang Y (2014) Label-free drug discovery. Front Pharmacol 5:52

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Friend J, Yeo L (2010) Fabrication of microfluidic devices using polydimethylsiloxane. Biomicrofluidics 4:026502

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Goral V, Jin Y, Sun H, Ferrie AM, Wu Q, Fang Y (2011) Agonist-directed desensitization of the β2-adrenergic receptor. PLoS One 6:e19282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Goral V, Wu Q, Sun H, Fang Y (2011) Label-free optical biosensor with microfluidics for sensing ligand-directed functional selectivity on trafficking of thrombin receptor. FEBS Lett 585:1054–1060

    Article  CAS  PubMed  Google Scholar 

  34. Zaytseva N, Miller W, Goral V, Hepburn J, Fang Y (2011) Microfluidic resonant waveguide grating biosensor system for whole cell sensing. Appl Phys Lett 98:163703

    Article  CAS  Google Scholar 

  35. Coller HA, Sang L, Roberts JM (2006) A new description of cellular quiescence. PLoS Biol 4:e83

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Fang Y, Ferrie AM (2008) Label-free optical biosensor for ligand-directed functional selectivity acting on β2-adrenoceptor in living cells. FEBS Lett 582:558–564

    Article  CAS  PubMed  Google Scholar 

  37. Fang Y (2010) Label-free receptor assays. Drug Discov Today Technol 7:e5–e11

    Article  CAS  Google Scholar 

  38. Fang Y, Ferrie AM, Tran E (2009) Resonant waveguide grating biosensor for whole cell GPCR assays. Methods Mol Biol 552:239–252

    Article  CAS  PubMed  Google Scholar 

  39. Guo D, Mulder-Krieger T, Ijzerman AP, Heitman LH (2012) Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time. Br J Pharmacol 166:1846–1959

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Deng H, Sun H, Fang Y (2013) Label-free cell phenotypic assessment of the biased agonism and efficacy of agonists at the endogenous muscarinic M3 receptors. J Pharmacol Toxicol Methods 68:323–333

    Article  CAS  PubMed  Google Scholar 

  41. Kenakin TP, Morgan PH (1989) The theoretical effects of single and multiple transducer receptor coupling proteins on estimates of the relative potency of agonists. Mol Pharmacol 35:214–222

    CAS  PubMed  Google Scholar 

  42. Swaminath G, Deup X, Lee TW, Zhu W, Thian FS, Kobilka TS, Kobilka B (2005) Probing the β2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists. J Biol Chem 280:22165–22171

    Article  CAS  PubMed  Google Scholar 

  43. Calebiro D, Nikolaev VO, Persani L, Lohse MJ (2010) Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 31:221–228

    Article  CAS  PubMed  Google Scholar 

  44. Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495:534–538

    Article  CAS  PubMed  Google Scholar 

  45. Ferrie AM, Sun H, Fang Y (2011) Label-free integrative pharmacology on-target of drugs at the β2-adrenergic receptor. Sci Rep 1:33

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Morse M, Tran E, Levension RL, Fang Y (2011) Ligand-directed functional selectivity at the mu opioid receptor revealed by label-free on-target pharmacology. PLoS One 6:e25643

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Morse M, Sun H, Tran E, Levenson RL, Fang Y (2013) Label-free integrative pharmacology ontarget of opioid ligands at the opioid receptor family. BMC Pharmacol Toxicol 14:17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Ferrie AM, Sun H, Zaytseva N, Fang Y (2014) Divergent label-free cell phenotypic pharmacology of ligands at the overexpressed β2-adrenergic receptors. Sci Rep 4:3828

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Deng H, Wang C, Fang Y (2013) Label-free cell phenotypic assessment of the molecular mechanism of action of epidermal growth factor receptor inhibitors. RSC Adv 3:10370–10378

    Article  CAS  Google Scholar 

  50. Ferrie AM, Wu Q, Fang Y (2010) Resonant waveguide grating imager for live cell sensing. Appl Phys Lett 97:223704

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Ferrie AM, Deichmann OD, Wu Q, Fang Y (2012) High resolution resonant waveguide grating imager for cell cluster analysis under physiological condition. Appl Phys Lett 100:223701

    Article  CAS  Google Scholar 

  52. Tran E, Sun H, Fang Y (2012) Dynamic mass redistribution assays decodes surface influence on signaling of endogenous purinergic receptors. Assay Drug Dev Technol 10:37–45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY, 14831, USA

    Ann M. Ferrie, Vasiliy Goral, Chaoming Wang & Ye Fang Ph.D.

  2. Department of Mechanical, Materials and Aerospace Engineering, NanoScience Technology Center, University of Central Florida, Orlando, FL, USA

    Chaoming Wang

Authors
  1. Ann M. Ferrie
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  2. Vasiliy Goral
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  3. Chaoming Wang
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  4. Ye Fang Ph.D.
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Corresponding author

Correspondence to Ye Fang Ph.D. .

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

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Ferrie, A.M., Goral, V., Wang, C., Fang, Y. (2015). Label-Free Functional Selectivity Assays. 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_16

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  • DOI: https://doi.org/10.1007/978-1-4939-2336-6_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-2335-9

  • Online ISBN: 978-1-4939-2336-6

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