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

Abstract

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 A 3 receptor (A 3 AR) 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 A 3 AR 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 A 3 AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A 3 AR 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 A 3 AR. 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 ([ 3 H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [ 3 H]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.

Fluorescent ligands: Bringing light to emerging GPCR paradigms

“…The Motulsky and Mahan method requires the kinetic ( k on and k off ) parameters of a labelled ligand (radiolabelled or fluorescent‐labelled) to be predetermined, and then by measuring the effect of an unlabelled ligand on these rates, the binding kinetics of the unlabelled ligand can be determined (Figure ; see Bosma et al, , for the Motulsky–Mahan equation used to simulate these data). Due to the ease of collecting larger datasets with fluorescence techniques, there have been several recent papers comparing the binding kinetics of unlabelled ligands measured using radioligand‐ and fluorescence‐based methods (Bosma et al, ; Bouzo‐Lorenzo et al, ; Nederpelt et al, ). Using the gonadotropin‐releasing hormone receptor, Nederpelt et al () compared the binding kinetics of a number of agonists measured in radioligand and TR‐FRET assays.…”

“…In this study, measured dissociation rates ( k off ) at the gonadotropin‐releasing hormone receptor were compared well across both assays, whereas the correlation in association rates ( k on ) was poor. For the adenosine A 3 receptor, a comparison of radioligand and NanoBRET (BRET with the small bright luciferease Nanoluc as the donor) assays using labelled ligands with markedly different binding kinetics suggested that it is difficult to determine the kinetics of fast binding unlabelled ligands when using a probe with a very slow off rate (Bouzo‐Lorenzo et al, ). Using the histamine H 1 receptor as a model system, Bosma et al () compared the binding kinetics of a number of unlabelled antagonists using two radioligands and both NanoBRET and TR‐FRET fluorescence‐based assays.…”

“…Fluorescent probes differ much more widely in their chemical structure as a result of using chemically diverse fluorophores and linkers. Consequently, marked differences have been observed in the binding kinetics of fluorescent ligands with the same pharmacophore but differing fluorescent labels and linker compositions (Bouzo‐Lorenzo et al, ). With increasing knowledge of the structural and chemical factors influencing ligand‐binding kinetics, rationale design of fluorescent ligands with the required binding kinetics for specific compound sets using the Motulsky and Mahan method may become possible.…”

“…ing kinetics of the unlabelled ligand can be determined (Figure 3; seeBosma et al, 2019, for the Motulsky-Mahan equation used to simulate these data). Due to the ease of collecting larger datasets with fluorescence techniques, there have been several recent papers comparing the binding kinetics of unlabelled ligands measured using radioligand-and fluorescence-based methods(Bosma et al, 2019;Bouzo-Lorenzo et al, 2019;Nederpelt et al, 2016). Using the gonadotropin-releasing hormone receptor,Nederpelt et al (2016) T A B L E 1 Summary of GPCRs at which binding kinetics have been measured using fluorescent ligands…”

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

“…In order to overcome these experimental limitations, we established and validated a live-cell, nanoluciferase bioluminescence resonance energy transfer (NanoBRET)based binding assay to assess the binding properties of BODIPY-cyclopamine and unlabeled SMO ligands to an N-terminally Nanoluciferase (Nluc)-tagged SMO and CRD SMO in HEK293 cells devoid of endogenous SMO. This proximity-based ligand-binding assay has recently been developed to assess ligand binding to Class A GPCRs and receptor tyrosine kinases (Bosma et al, 2019;Bouzo-Lorenzo et al, 2019;Mocking, Verweij, Vischer & Leurs, 2018;Stoddart et al, 2015;Stoddart, Kilpatrick & Hill, 2018;Sykes, Stoddart, Kilpatrick & Hill, 2019).…”