A novel fluorescence-based live cell binding assay for screening of adenosine-A3 receptor ligands

Leigh Stoddart, Andrea Vernall, Stephen Briddon, Barrie Kellam, Stephen Hill. University of Nottingham, NG7 2UH, United Kingdom.

Traditional competitive binding assays measure the ability of increasing concentrations of an unlabelled compound to compete with a single concentration of radiolabelled ligand at equilibrium. However, the use and disposal of radiolabelled ligands is increasingly expensive. Using the human adenosine-A3 receptor as model system, we set out to determine if we could replace the radiolabelled ligand with a fluorescently labelled antagonist and subsequently develop a live cell competition binding assay using a high content screening system.

Chinese Hamster Ovary cells stably expressing the adenosine A3 receptor and a CRE-linked secreted alkaline phosphatase (SPAP) reporter gene were grown to confluency on black walled, clear bottomed 96-well plates in Dulbecco’s Modification of Eagle’s Medium/Ham’sF-12 mix with 10% foetal calf serum and 2mM glutamine. Cells were incubated with varying concentrations of unlabelled antagonist (30 min, 37°C) before addition of 25 nM CA200645, a fluorescent A3 antagonist (CellAura Technologies, Nottingham). Cells were incubated for a further 30 min at 37°C before washing twice in HEPES-buffered saline solution. Automated image capture (four central images per well) was carried out immediately using an ImageXpress Ultra confocal plate reader (Molecular Devices). Each data point was determined in duplicate from the total integrated fluorescence intensity values of the four images per well using MetaExpress software. Functional data were obtained using SPAP reporter gene assays as previously described (Baker at al., 2010). Ki values were calculated using non-linear curve-fitting (GraphPad Prism) and Cheng-Prusoff correction using a ligand Kd of 3.1 nM, calculated from SPAP assays in the same cells.

The high affinity selective A3 receptor antagonist, compound 6, (Lenzi et al., 2006) caused a clear concentration-dependent displacement of CA200645 binding (Ki = 4.5±2.1 nM, mean±s.e.mean, n = 3) which was in close agreement with a Kd value obtained in the same cells when measuring CRE-mediated SPAP production (1.9±0.8 nM, n = 3). Two further antagonists, XAC and MRS1220 also showed affinities at the A3 receptor consistent with values obtained in functional assays (Ki = 15.5±4.1 and 1.3±0.5 nM, n = 3). In contrast, the A1 receptor selective antagonist, DPCPX, was only able to displace CA200645 binding at concentrations >1 μM. Regression analysis of 5 compounds comparing the pKd (SPAP) vs pKi (binding) showed a good correlation (R2 = 0.94).

These data indicate that a live cell competition binding assay using a fluorescently labelled A3-antagonist in conjunction with automated image capture and analysis gives accurate and precise affinity values for known receptor ligands. This assay could operate as a powerful alternative medium-throughput screen for novel A3-receptor ligands, with the potential to incorporate single cell and spatial analyses in future developments.