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A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor

Bouzo-Lorenzo, Monica; Stoddart, Leigh A.; Xia, Lizi; Jerzman, Adriaan P.I.; Heitman, Laura H.; Briddon, Stephen J.; Hill, Stephen J.


Monica Bouzo-Lorenzo

Leigh A. Stoddart

Lizi Xia

Adriaan P.I. Jerzman

Laura H. Heitman

Professor of Molecular Pharmacology


There is a growing interest in understanding the binding kinetics of compounds that bind to G proteincoupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A3 receptor (A3AR) has been implicated in a range of diseases including immune conditions and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A3AR have been performed using a radiolabelled antagonist but due to the kinetics of this probe they have been carried out at 10oC in membrane preparations. In this study we have developed a live cell NanoBRET ligand binding assay using fluorescent A3AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A3AR at physiological temperatures.

The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A3AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5 and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([3H]PSB- 11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [3H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.


Bouzo-Lorenzo, M., Stoddart, L. A., Xia, L., Jerzman, A. P., Heitman, L. H., Briddon, S. J., & Hill, S. J. (2019). A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor. Purinergic Signalling, 15(2), 139–153.

Journal Article Type Article
Acceptance Date Feb 15, 2019
Online Publication Date Mar 27, 2019
Publication Date Jun 30, 2019
Deposit Date Feb 28, 2019
Publicly Available Date Mar 28, 2020
Journal Purinergic Signalling
Print ISSN 1573-9538
Electronic ISSN 1573-9546
Publisher Springer Verlag
Peer Reviewed Peer Reviewed
Volume 15
Issue 2
Pages 139–153
Public URL
Publisher URL


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