Development of a novel TR-FRET-based competition kinetic binding assay to assess the kinetics of unlabeled adenosine A2a receptor ligands

S. Chitlangia, J. Resendedealmeida, N. Groenewoud, S. Briddon, M. Arruda, S. J. Charlton, D. A. Sykes. Cell Signalling Group, The University of Nottingham, Nottingham, UNITED KINGDOM.

Introduction

Adenosine A2a receptor (A2aR) agonists are important regulators of myocardial blood flow whilst antagonists regulate dopamine signalling and are used in Parkinson’s disease. Existing data on adenosine A2aR agonists/antagonists show a range of dissociation and association rates reviewed in Guo et al.1 The majority of these studies have been performed with radiolabelled compounds which are both expensive to synthesise and time consuming to characterise. We have developed a novel higher-throughput TR-FRET based competition kinetic method to investigate the kinetics of a series of adenosine A2 agonists/antagonists at the human A2aR under identical assay conditions in order that these parameters can be directly compared.

Method

The kinetic parameters of unlabeled compounds were assessed using a competition kinetic binding assay essentially as previously described by Klein-Herenbrink et al.2. Specifically fluorescent CA200645 (100nM, final assay volume 40 μL in LABMED buffer (CisBio)) was incubated in the presence of unlabeled antagonist, and terbium-labelled CHO-A2a cell membranes (4 μg well-1) in the presence of GppNHp (100μM) at room temperature. Assays were initiated by addition of membranes to a 384-well Optiplate plate and TR-FRET measurements were taken every 30 sec on a PHERAstar FS (BMG) using standard instrument settings. Specific binding, defined by ZM241385 (1 μM) was plotted against time and fitted to a competitive kinetic binding model (GraphPad Prism 7.0).

Results

The association and dissociation rates of fluorescent CA200645 were 1.93x106 M-1 min-1 and 0.22 min-1 respectively, with a corresponding kinetic Kd of 166nM. The kinetics of competitive binding of 7 adenosine A2a ligands were examined and the results are summarised in Table 1.

Table 1. Kinetic parameters of A2aR ligands. Data are expressed as mean ± SEM (n ≥ 3). Agonists are shown in italics.

Conclusions

All the A2aR compounds tested displayed similar dissociation rates (<3-fold difference). The differences in receptor affinity observed appear driven almost entirely by differences in association rates, which varied by as much as 10,000-fold. The kinetic off-rates of the antagonists closely match existing literature values, whereas agonist off-rates are more rapid than previously reported,1 potentially reflecting differences in assay temperature and the receptor states studied. The novel method described significantly increases assay throughput potentially allows the profiling of 100-1000s of unlabeled compounds and chemical fragments allowing the development of structure-kinetic relationships.

References

1. Guo D, et al., (2016). Chemical Reviews [Epub ahead of print].

2. Klein-Herenbrink C, et al (2016). Nature Communications 7, 10842.