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.

Abstract

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.

Citation

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. https://doi.org/10.1007/s11302-019-09650-9

• 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
• DOI: https://doi.org/10.1007/s11302-019-09650-9
• Public URL: https://nottingham-repository.worktribe.com/output/1591338
• Publisher URL: http://link-springer-com-443.webvpn.jxust.edu.cn/article/10.1007%2Fs11302-019-09650-9
• Contract Date: Feb 28, 2019