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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; IJzerman, Adriaan P; Heitman, Laura H; Briddon, Stephen J; Hill, Stephen J.
Afiliación
  • Bouzo-Lorenzo M; Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.
  • Stoddart LA; Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK.
  • Xia L; Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.
  • IJzerman AP; Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK.
  • Heitman LH; Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, 9502, 2300 RA, Leiden, The Netherlands.
  • Briddon SJ; Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, 9502, 2300 RA, Leiden, The Netherlands.
  • Hill SJ; Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, 9502, 2300 RA, Leiden, The Netherlands.
Purinergic Signal ; 15(2): 139-153, 2019 06.
Article en En | MEDLINE | ID: mdl-30919204
There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled 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 10 °C 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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Receptor de Adenosina A3 / Antagonistas del Receptor de Adenosina A3 / Mediciones Luminiscentes Límite: Humans Idioma: En Revista: Purinergic Signal Año: 2019 Tipo del documento: Article Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transferencia Resonante de Energía de Fluorescencia / Receptor de Adenosina A3 / Antagonistas del Receptor de Adenosina A3 / Mediciones Luminiscentes Límite: Humans Idioma: En Revista: Purinergic Signal Año: 2019 Tipo del documento: Article Pais de publicación: Países Bajos