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 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.
Highlights
The nucleoside adenosine is a ubiquitous signalling molecule which modulates cellular responses to stress
AV039 (Fig. 1d) is based on the A3AR selective antagonist 1,2,4-triazolo[4,3-a]quinoxalin-1-one linked to BY630 and has been shown to retain A3AR selectivity [34]
It is becoming increasingly clear that a ligand’s binding kinetics are a crucial part of its pharmacology which can play a role in its success in the clinic [16, 18, 20]
Summary
The nucleoside adenosine is a ubiquitous signalling molecule which modulates cellular responses to stress. Many molecules targeting GPCRs, including those acting at adenosine receptors [12], have exhibited a lack of efficacy in clinical trials [13]. Drugs with a long RT are preferred when an extended duration of action is required and can reduce administration to once a day. This has been demonstrated for the M3 muscarinic receptor antagonist tiotropium and the β2 adrenoceptor agonist olodaterol which both have long RTs and are once a day bronchodilators used to treat chronic obstructive pulmonary disease [14, 19, 20]. Short RT ligands are advantageous when the rapid, i.e. seconds to minutes, replacement of the drug by an endogenous ligand is crucial to avoid long-term side effects which has been proposed to be the case for drugs targeting the dopamine D2 receptor [17, 21, 22]
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