Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry

Abstract

The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR 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 Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki 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 [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization.

Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

“…In such way, a new fluorophore-conjugated A 3 R antagonist ( 16 ) (Table 2) was synthesized and used for cell-based applications. This compound clearly labelled the A 3 R at the plasma membrane of CHO cells expressing the receptor and by means of FCM a very high specific-to-nonspecific binding ratio was obtained (Kozma et al, 2012). Interestingly, by using this last approach an equilibrium binding constant comparable to that calculated from kinetic radioligand experiments in membranes was observed (Kozma et al, 2012).…”

“…This compound clearly labelled the A 3 R at the plasma membrane of CHO cells expressing the receptor and by means of FCM a very high specific-to-nonspecific binding ratio was obtained (Kozma et al, 2012). Interestingly, by using this last approach an equilibrium binding constant comparable to that calculated from kinetic radioligand experiments in membranes was observed (Kozma et al, 2012). Another adenosine receptor fluorescent ligand used in FCM was compound 12 (Table 2), which consisted of an Alexa Fluor 488-conjugated A 2A R agonist.…”

Fluorescent approaches for understanding interactions of ligands with G protein coupled receptors

“…However, the approach has also been used for ligands for adenosine [85, 86], adrenergic [87, 88], cannabinoid [89], chemokine [90, 91], galanin [92], long chain fatty acid [93], neuropeptide Y [94], somatostatin [95, 96], urotensin [97], vasopressin [98]and yeast α-factor [82, 99–102] receptors.…”

Fluorescent ligands for adenosine receptors

“…In order to overcome this problem, two differing methods were recently introduced: one on membrane fragments 7 and another on intact cells. 6 Kecskès et al used an Alexa Fluor 488-tagged A 2A AR antagonist MRS5346 ( 53 ) in a newly developed ligand binding assay that uses fluorescence polarization (FP) with A 2A -HEK293 cell membranes. 7,41 The same study also reported a TAMRA derivative MRS5347 ( 55 ) and a BODIPY 650/665-X derivative MRS5418 ( 54 ) synthesized by Kumar et al 41 Conjugates 54 and 55 were designed for FM, and the structure was based on a pyrazolo[4,3- e ][1,2,4]triazolo[1,5- c ]pyrimidine-5-amine (PTP) scaffold that promotes high affinity and selectivity as antagonists for the A 2A AR.…”

“…6,7 Recently, the expense, health risks, and disposal issues that radioligands pose have increased the need to develop more efficient techniques. Fluorescent methods solve many of these issues associated with radioactivity, in addition to providing other advantages.…”