The physiological role of the A(3) adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A(3)AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding K(i) value of 6.4±2.5nM in hA(3)AR-expressing CHO cell membranes. MRS5449 antagonized hA(3)AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K(B)=4.8nM). Using flow cytometry (FCM), MRS5449 saturated hA(3)ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15nM, comparable to the K(d) value of 6.65nM calculated from kinetic experiments. K(i) values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5-20 fold weaker than obtained with agonist radioligand [(125)I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A(3)AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA(3)AR characterization.
Published by Elsevier Inc.