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

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• 作者：Eszter Kozma^a ; T. Santhosh Kumar^a ; Stephanie Federico^b ; Khai Phan^a ; Ramachandran Balasubramanian^a ; Zhan-Guo Gao^a ; Silvia Paoletta^c ; Stefano Moro^c ; Giampiero Spalluto^b ; Kenneth A. Jacobson^a ; ^{kajacobs@helix.nih.gov}
• 关键词：AF488 ; Alexa Fluor-488 ; CHO ; Chinese hamster ovary ; CGS15943 ; N-[9-chloro-2-(2-furanyl)[1 ; 2 ; 4]triazolo[1 ; 5-c]quinazolin-5-amine ; CGS21680 ; 2-[p-(2-carboxyethyl)phenylethylamino]-5&prime ; -N-ethylcarboxamido-adenosine ; Cl-IB-MECA ; 1-[2-chloro-6-[[(3-iodophe
• 刊名：Biochemical Pharmacology
• 出版年：2012
• 期刊代码：2_00062952
• 类别：pha
• 出版时间：1 June, 2012
• 卷：83
• 期：11
• 页码：1552-1561
• 文件大小：1344 K

摘要

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 K_i value of 6.4 卤 2.5 nM in hA₃AR-expressing CHO cell membranes. MRS5449 antagonized hA₃AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (K_B = 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 K_d value of 6.65 nM 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 [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.