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

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Abstract

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.

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Abbreviations

7TMR:

Seven-transmembrane domain receptor

AC:

Adenylyl cyclase

β2AR:

β2-Adrenoceptor

β-arr-2:

β-Arrestin type 2

BSA:

Bovine serum albumin

DHA:

Dihydroalprenolol

DMEM:

Dulbecco’s modified Eagle’s medium

ECL:

Extracellular loop

EDTA:

Ethylenediaminetetraacetic acid

EPI:

Epinephrine (adrenaline)

fMLF:

N-formyl-l-methionyl-l-leucyl-l-phenylalanine

Giα:

Inhibitory Gα protein

Gsα:

Stimulatory Gα protein

GPCR:

G protein-coupled receptor

GTPase:

GTP-hydrolyzing activity

ISO:

Isoproterenol

PBS:

Phosphate-buffered saline

PMSF:

Phenylmethanesulfonylfluoride

RGS4:

Regulatory protein of G protein signaling, type 4

ROS:

Reactive oxygen species

Sf9:

Clonal isolate of Spodoptera frugiperda ovary cells

TM:

Transmembrane domain

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Acknowledgments

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.

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Authors and Affiliations

  1. Institute for Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Michael T. Reinartz, Solveig Kälble, Timo Littmann, Volkhard Kaever & Roland Seifert

  2. Department of Chemistry, School of Science, The University of Tokyo, Tokyo, Japan

    Takeaki Ozawa

  3. Department of Pharmaceutical and Medicinal Chemistry II, University of Regensburg, Regensburg, Germany

    Stefan Dove

  4. Core Unit Metabolomics, Hannover Medical School, Hannover, Germany

    Volkhard Kaever

  5. Laboratory of Clinical Investigation, Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA

    Irving W. Wainer

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  1. Michael T. Reinartz
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  2. Solveig Kälble
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  4. Takeaki Ozawa
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  8. Roland Seifert
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Correspondence to Roland Seifert.

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Reinartz, M.T., Kälble, S., Littmann, T. et al. Structure-bias relationships for fenoterol stereoisomers in six molecular and cellular assays at the β2-adrenoceptor. Naunyn-Schmiedeberg's Arch Pharmacol 388, 51–65 (2015). https://doi.org/10.1007/s00210-014-1054-5

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  • DOI: https://doi.org/10.1007/s00210-014-1054-5

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