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 A2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A2AAR (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 Ki value of 111 ± 16 nM in radioligand binding using [3H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A2AAR antagonist. MRS5346 did not show any effect on A1 and A3 ARs in binding or the A2BAR 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 A2AAR 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 Kd value of MRS5346 calculated from kinetic parameters was 16.5 ± 4.7 nM. In FP competition binding experiments using MRS5346 as a tracer, Ki values of known AR agonists and antagonists consistently agreed with Ki 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.

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