Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 388, Issue 1, pp 51–65 | Cite as

Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the β2-adrenoceptor

  • Michael T. Reinartz
  • Solveig Kälble
  • Timo Littmann
  • Takeaki Ozawa
  • Stefan Dove
  • Volkhard Kaever
  • Irving W. Wainer
  • Roland Seifert
Original Article


Functional selectivity is well established as an underlying concept of ligand-specific signaling via G protein-coupled receptors (GPCRs). Functionally, selective drugs could show greater therapeutic efficacy and fewer adverse effects. Dual coupling of the β2-adrenoceptor (β2AR) triggers a signal transduction via Gsα and Giα proteins. Here, we examined 12 fenoterol stereoisomers in six molecular and cellular assays. Using β2AR-Gsα and β2AR-Giα fusion proteins, (R,S’)- and (S,S’)-isomers of 4′-methoxy-1-naphthyl-fenoterol were identified as biased ligands with preference for Gs. G protein-independent signaling via β-arrestin-2 was disfavored by these ligands. Isolated human neutrophils constituted an ex vivo model of β2AR signaling and demonstrated functional selectivity through the dissociation of cAMP accumulation and the inhibition of formyl peptide-stimulated production of reactive oxygen species. Ligand bias was calculated using an operational model of agonism and revealed that the fenoterol scaffold constitutes a promising lead structure for the development of Gs-biased β2AR agonists.


Functional selectivity β2-Adrenergic receptor Biased ligand Bias quantification Structure-bias relationships Fenoterol 



Seven-transmembrane domain receptor


Adenylyl cyclase




β-Arrestin type 2


Bovine serum albumin




Dulbecco’s modified Eagle’s medium


Extracellular loop


Ethylenediaminetetraacetic acid


Epinephrine (adrenaline)




Inhibitory Gα protein


Stimulatory Gα protein


G protein-coupled receptor


GTP-hydrolyzing activity




Phosphate-buffered saline




Regulatory protein of G protein signaling, type 4


Reactive oxygen species


Clonal isolate of Spodoptera frugiperda ovary cells


Transmembrane domain



The authors thank Dr. Andreas Bock (University of Würzburg) for the helpful discussion on the aspect of bias quantification. We also thank the reviewers for their constructive critique.


  1. Barnes PJ (1999) Effect of beta-agonists on inflammatory cells. J Allergy Clin Immunol 104:S10–S17PubMedCrossRefGoogle Scholar
  2. Black JW, Leff P (1983) Operational models of pharmacological agonism. Proc R Soc Lond B Biol Sci 220:141–162PubMedCrossRefGoogle Scholar
  3. Bock A, Merten N, Schrage R, Dallanoce C, Bätz J, Klöckner J, Schmitz J, Matera C, Simon K, Kebig A, Peters L, Müller A, Schrobang-Ley J, Tränkle C, Hoffmann C, De Amici M, Holzgrabe U, Kostenis E, Mohr K (2012) The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling. Nat Commun 3:1044PubMedCentralPubMedCrossRefGoogle Scholar
  4. Brunskole-Hummel I, Reinartz MT, Kälble S, Burhenne H, Schwede F, Buschauer A, Seifert R (2013) Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity. PLoS One 8:e64556PubMedCentralPubMedCrossRefGoogle Scholar
  5. Casella I, Ambrosio C, Gro MC, Molinari P, Costa T (2011) Divergent agonist selectivity in activating β1- and β2-adrenoceptors for G-protein and arrestin coupling. Biochem J 438:191–202PubMedCrossRefGoogle Scholar
  6. Cazzola M, Page CP, Rogliani P, Matera MG (2013) β2-agonist therapy in lung disease. Am J Respir Crit Care Med 187:690–696PubMedCrossRefGoogle Scholar
  7. Chen X, Sassano MF, Zheng L, Setola V, Chen M, Bai X, Frye SV, Wetsel WC, Roth BL, Jin J (2012) Structure-functional selectivity relationship studies of β-arrestin-biased dopamine D2 receptor agonists. J Med Chem 55:7141–7153PubMedCentralPubMedCrossRefGoogle Scholar
  8. Deshpande DA, Theriot BS, Penn RB, Walker JKL (2008) β-arrestins specifically constrain β2-adrenergic receptor signaling and function in airway smooth muscle. FASEB J 22:2134–2141PubMedCentralPubMedCrossRefGoogle Scholar
  9. Deupi X, Kobilka BK (2010) Energy landscapes as a tool to integrate GPCR structure, dynamics, and function. Physiology (Bethesda) 25:293–303CrossRefGoogle Scholar
  10. Evans BA, Sato M, Sarwar M, Hutchinson DS, Summers RJ (2010) Ligand-directed signalling at β-adrenoceptors. Br J Pharmacol 159:1022–1038PubMedCentralPubMedCrossRefGoogle Scholar
  11. Evans BA, Broxton N, Merlin J, Sato M, Hutchinson DS, Christopoulos A, Summers RJ (2011) Quantification of functional selectivity at the human α1A-adrenoceptor. Mol Pharmacol 79:298–307PubMedCrossRefGoogle Scholar
  12. 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 70:1575–1584PubMedCrossRefGoogle Scholar
  13. Galandrin S, Oligny-Longpré G, Bouvier M (2007) The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol Sci 28:423–430PubMedCrossRefGoogle Scholar
  14. Gether U, Lin S, Kobilka BK (1995) Fluorescent labeling of purified β2 adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem 270:28268–28275PubMedCrossRefGoogle Scholar
  15. Johnson M (2002) Effects of β2-agonists on resident and infiltrating inflammatory cells. J Allergy Clin Immunol 110:S282–S290PubMedCrossRefGoogle Scholar
  16. Jozwiak K, Khalid C, Tanga MJ, Berzetei-Gurske I, Jimenez L, Kozocas JA, Woo A, Zhu W, Xiao RP, Abernethy DR, Wainer IW (2007) Comparative molecular field analysis of the binding of the stereoisomers of fenoterol and fenoterol derivatives to the β2 adrenergic receptor. J Med Chem 50:2903–2915PubMedCrossRefGoogle Scholar
  17. Jozwiak K, Woo AY, Tanga MJ, Toll L, Jimenez L, Kozocas JA, Plazinska A, Xiao RP, Wainer IW (2010) Comparative molecular field analysis of fenoterol derivatives: a platform towards highly selective and effective β2-adrenergic receptor agonists. Bioorg Med Chem 18:728–736PubMedCentralPubMedCrossRefGoogle Scholar
  18. Kenakin T (2012) New concepts in pharmacological efficacy at 7TM receptors: IUPHAR review 2. Br J Pharmacol 168:554–575CrossRefGoogle Scholar
  19. Kenakin T, Christopoulos A (2013) Signalling bias in new drug discovery: detection, quantification and therapeutic impact. Nat Rev Drug Discov 12:205–216PubMedCrossRefGoogle Scholar
  20. Kenakin T, Watson C, Muniz-Medina V, Christopoulos A, Novick S (2012) A simple method for quantifying functional selectivity and agonist bias. ACS Chem Neurosci 3:193–203PubMedCentralPubMedCrossRefGoogle Scholar
  21. Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K (2012) Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110PubMedCentralPubMedCrossRefGoogle Scholar
  22. Misawa N, Kafi AKM, Hattori M, Miura K, Masuda K, Ozawa T (2010) Rapid and high-sensitivity cell-based assays of protein-protein interactions using split click beetle luciferase complementation: an approach to the study of G-protein-coupled receptors. Anal Chem 82:2552–2560PubMedCrossRefGoogle Scholar
  23. Moore RH, Millman EE, Godines V, Hanania NA, Tran TM, Peng H, Dickey BF, Knoll BJ, Clark RB (2007) Salmeterol stimulation dissociates β2-adrenergic receptor phosphorylation and internalization. Am J Respir Cell Mol Biol 36:254–261PubMedCentralPubMedCrossRefGoogle Scholar
  24. Nygaard R, Zou Y, Dror RO, Mildorf TJ, Arlow DH, Manglik A, Pan AC, Liu CW, Fung JJ, Bokoch MP, Thian FS, Kobilka TS, Shaw DE, Mueller L, Prosser RS, Kobilka BK (2013) The dynamic process of β2-adrenergic receptor activation. Cell 152:532–542PubMedCentralPubMedCrossRefGoogle Scholar
  25. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996PubMedCrossRefGoogle Scholar
  26. Plazinska A, Pajak K, Rutkowska E, Jimenez L, Kozocas J, Koolpe G, Tanga M, Toll L, Wainer IW, Jozwiak K (2014) Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor. Bioorg Med Chem 22:234–246PubMedCrossRefGoogle Scholar
  27. R-Core-Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing,
  28. Reiner S, Ambrosio M, Hoffmann C, Lohse MJ (2010) Differential signaling of the endogenous agonists at the β2-adrenergic receptor. J Biol Chem 285:36188–36198PubMedCentralPubMedCrossRefGoogle Scholar
  29. Reiter E, Ahn S, Shukla AK, Lefkowitz RJ (2012) Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 52:179–197PubMedCentralPubMedCrossRefGoogle Scholar
  30. Schneider EH, Seifert R (2010) Fusion proteins as model systems for the analysis of constitutive GPCR activity. Methods Enzymol 485:459–480PubMedCrossRefGoogle Scholar
  31. Seifert R (2013) Functional selectivity of G-protein-coupled receptors: from recombinant systems to native human cells. Biochem Pharmacol 86:853–861PubMedCrossRefGoogle Scholar
  32. Seifert R, Schultz G (1991) The superoxide-forming NADPH oxidase of phagocytes. An enzyme system regulated by multiple mechanisms. Rev Physiol Biochem Pharmacol 117:1–338PubMedGoogle Scholar
  33. Seifert R, Gether U, Wenzel-Seifert K, Kobilka BK (1999a) Effects of guanine, inosine, and xanthine nucleotides on β2-adrenergic receptor/Gs interactions: evidence for multiple receptor conformations. Mol Pharmacol 56:348–358PubMedGoogle Scholar
  34. Seifert R, Wenzel-Seifert K, Kobilka BK (1999b) GPCR-Gα fusion proteins: molecular analysis of receptor-G-protein coupling. Trends Pharmacol Sci 20:383–389PubMedCrossRefGoogle Scholar
  35. Stallaert W, Dorn JF, van der Westhuizen E, Audet M, Bouvier M (2012) Impedance responses reveal β2-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles. PLoS One 7:e29420PubMedCentralPubMedCrossRefGoogle Scholar
  36. Takakura M, Hattori H, Takeuchi M, Ozawa T (2012) Visualization and quantitative analysis of G protein-coupled receptor-β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation. ACS Chem Biol 7:901–910PubMedCrossRefGoogle Scholar
  37. Thompson G, Kelly E, Christopoulos A, Canals M (2014) Novel GPCR paradigms at the μ-opioid receptor. Br J Pharmacol. doi:10.1111/bph.12600 PubMedGoogle Scholar
  38. Tikhonova IG, Selvam B, Ivetac A, Wereszczynski J, McCammon JA (2013) Simulations of biased agonists in the β2 adrenergic receptor with accelerated molecular dynamics. Biochemistry 52:5593–5603PubMedCentralPubMedCrossRefGoogle Scholar
  39. Uzkeser H, Cadirci E, Halici Z, Odabasoglu F, Polat B, Yuksel TN, Ozaltin S, Atalay F (2012) Anti-inflammatory and antinociceptive effects of salbutamol on acute and chronic models of inflammation in rats: involvement of an antioxidant mechanism. Mediat Inflamm 2012:438912Google Scholar
  40. van der Westhuizen ET, Breton B, Christopoulos A, Bouvier M (2014) Quantification of ligand bias for clinically relevant β2-adrenergic receptor ligands: implications for drug taxonomy. Mol Pharmacol 85:492–509PubMedCrossRefGoogle Scholar
  41. Warne T, Moukhametzianov R, Baker JG, Nehme R, Edwards PC, Leslie AG, Schertler GF, Tate CG (2011) The structural basis for agonist and partial agonist action on a β1-adrenergic receptor. Nature 469:241–244PubMedCentralPubMedCrossRefGoogle Scholar
  42. Weitl N, Seifert R (2008) Distinct interactions of human β1- and β2-adrenoceptors with isoproterenol, epinephrine, norepinephrine, and dopamine. J Pharmacol Exp Ther 327:760–769PubMedCrossRefGoogle Scholar
  43. Wenzel-Seifert K, Seifert R (2000) Molecular analysis of β2-adrenoceptor coupling to Gs-, Gi-, and Gq-proteins. Mol Pharmacol 58:954–966PubMedGoogle Scholar
  44. Whalen EJ, Rajagopal S, Lefkowitz RJ (2011) Therapeutic potential of β-arrestin- and G protein-biased agonists. Trends Mol Med 17:126–139PubMedCentralPubMedCrossRefGoogle Scholar
  45. White KL, Scopton AP, Rives M-L, Bikbulatov RV, Polepally PR, Brown PJ, Kenakin T, Javitch JA, Zjawiony JK, Roth BL (2014) Identification of novel functionally selective κ-opioid receptor scaffolds. Mol Pharmacol 85:83–90PubMedCrossRefGoogle Scholar
  46. 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 104:16657–16662PubMedCentralPubMedCrossRefGoogle Scholar
  47. Woo AYH, Xiao RP (2012) β-Adrenergic receptor subtype signaling in heart: from bench to bedside. Acta Pharmacol Sin 33:335–341PubMedCentralPubMedCrossRefGoogle Scholar
  48. Woo AY, Wang TB, Zeng X, Zhu W, Abernethy DR, Wainer IW, Xiao RP (2009) Stereochemistry of an agonist determines coupling preference of β2-adrenoceptor to different G proteins in cardiomyocytes. Mol Pharmacol 75:158–165PubMedCentralPubMedCrossRefGoogle Scholar
  49. Xiao RP, Zhang SJ, Chakir K, Avdonin P, Zhu W, Bond RA, Balke CW, Lakatta EG, Cheng H (2003) Enhanced Gi signaling selectively negates β2-adrenergic receptor (AR)—but not β1-AR–mediated positive inotropic effect in myocytes from failing rat hearts. Circulation 108:1633–1639PubMedCrossRefGoogle Scholar
  50. Zhu W, Zeng X, Zheng M, Xiao R-P (2005) The enigma of β2-adrenergic receptor Gi signaling in the heart: the good, the bad, and the ugly. Circ Res 97:507–509Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Michael T. Reinartz
    • 1
  • Solveig Kälble
    • 1
  • Timo Littmann
    • 1
  • Takeaki Ozawa
    • 2
  • Stefan Dove
    • 3
  • Volkhard Kaever
    • 1
    • 4
  • Irving W. Wainer
    • 5
  • Roland Seifert
    • 1
  1. 1.Institute for PharmacologyHannover Medical SchoolHannoverGermany
  2. 2.Department of Chemistry, School of ScienceThe University of TokyoTokyoJapan
  3. 3.Department of Pharmaceutical and Medicinal Chemistry IIUniversity of RegensburgRegensburgGermany
  4. 4.Core Unit MetabolomicsHannover Medical SchoolHannoverGermany
  5. 5.Laboratory of Clinical Investigation, Biomedical Research Center, National Institute on AgingNational Institutes of HealthBaltimoreUSA

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