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Long receptor residence time of C26 contributes to super agonist activity at the human ?2 adrenoceptor

Rosethorne, Elizabeth M.; Bradley, Michelle E.; Gherbi, Karolina; Sykes, David A.; Sattikar, Afrah; Wright, John D.; Renard, Emilie; Trifilieff, A.; Fairhurst, Robin A.; Charlton, Steven J.

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Elizabeth M. Rosethorne

Michelle E. Bradley

Karolina Gherbi

David A. Sykes

Afrah Sattikar

John D. Wright

Emilie Renard

A. Trifilieff

Robin A. Fairhurst

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Professor of Molecular Pharmacology and Drug Discovery


Super agonists produce greater functional responses than endogenous agonists in the same assay, and their unique pharmacology is the subject of increasing interest and debate. We propose that receptor residence time and the duration of receptor signaling contribute to the pharmacology of super agonism. We have further characterized the novel β2 adrenoceptor agonist C26 (7-[(R)-2-((1R,2R)-2-benzyloxycyclopentylamino)-1-hydroxyethyl]-4-hydroxybenzothiazolone), which displays higher intrinsic activity than the endogenous ligand adrenaline in cAMP accumulation, β-arrestin-2 recruitment, and receptor internalization assays. C26 recruited β-arrestin-2, and internalized the Green Fluorescent Protein (GFP)-taggedβ2 adrenoceptor at a slow rate, with half-life (t1/2) values of 0.78 ± 0.1 and 0.78 ± 0.04 hours, respectively. This was compared with 0.31 ± 0.04 and 0.34 ± 0.01 hours for adrenaline-mediated β-arrestin-2 recruitment and GFP-β2 internalization, respectively. The slower rate for C26 resulted in levels of β-arrestin-2 recruitment increasing up to 4-hour agonist incubation, at which point the intrinsic activity was determined to be 124.3 ± 0.77% of the adrenaline response. In addition to slow functional kinetics, C26 displayed high affinity with extremely slow receptor dissociation kinetics, giving a receptor residence half-life of 32.7 minutes at 37°C, which represents the slowest dissociation rate we have observed for any β2 adrenoceptor agonist tested to date. In conclusion, we propose that the gradual accumulation of long-lived active receptor complexes contributes to the increased intrinsic activity of C26 over time. This highlights the need to consider the temporal aspects of agonist binding and signaling when characterizing ligands as super agonists.

Journal Article Type Article
Acceptance Date Jan 13, 2016
Online Publication Date Mar 15, 2016
Publication Date Apr 1, 2016
Deposit Date Mar 22, 2017
Publicly Available Date Mar 22, 2017
Journal Molecular Pharmacology
Print ISSN 0026-895X
Electronic ISSN 1521-0111
Publisher American Society for Pharmacology and Experimental Therapeutics
Peer Reviewed Peer Reviewed
Volume 89
Issue 4
Pages 467-475
Public URL
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