Novel Alexa Fluor-488 labeled antagonist of the A2A adenosine receptor: Application to a fluorescence polarization-based receptor binding assay

Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A_2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A_2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K_i value of 111 ± 16 nM in radioligand binding using [³H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A_2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A_2AAR antagonist. MRS5346 did not show any effect on A₁ and A₃ ARs in binding or the A_2BAR in a cyclic AMP assay at 10 μM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A_2AAR binding. The FP signal was optimal with 20 nM MRS5346 and 150 μg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K_d value of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, K_i values of known AR agonists and antagonists consistently agreed with K_i values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs.