Characterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies

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• 作者：Aliaa Abdelrahman^a ; Vigneshwaran Namasivayam^a ; ^c ; Sonja Hinz^a ; Anke C. Schiedel^a ; Meryem Kö ; se^a ; Maggi Burton^b ; Ali El-Tayeb^a ; ¹ ; Michel Gillard^b ; Jü ; rgen Bajorath^c ; Marc de Ryck^b ; Christa E. Mü ; ller^a ; ^{christa.mueller@uni-bonn.de}
• 关键词：Ap3A ; diadenosine triphosphate ; Ap4A ; diadenosine tetraphosphate ; Ap5A ; diadenosine pentaphosphate ; AR-C67085MX ; 2-propylthio-β ; γ-dichloromethylene-ATP ; PSB0412 ; 2-propargylthio-β ; γ-dichloromethylene-ATP ; 5-BDBD ; 5-(3-bromophenyl)-1 ; 3-dihydro-2H-benzofuro[3 ; 2-e]-1 ; 4-diazepin-2-one ; BzATP ; 2&prime ; (3&prime ; )-O-(4-benzoylbenzoyl)adenosine 5&prime ; -triphosphate ; CHO-K1 cells ; Chinese hamster ovary cells ; HBSS ; Hank&rsquo ; s balanced salt solution ; HEPES ; 4-(2-hydroxyethyl)-1-piperazine ethanesulfoni
• 刊名：Biochemical Pharmacology
• 出版年：2017
• 出版时间：1 February 2017
• 年：2017
• 卷：125
• 期：Complete
• 页码：41-54
• 全文大小：2287 K
• 卷排序：125

文摘

Antagonists for ATP-activated P2X4 ion channel receptors are currently in the focus as novel drug targets, in particular for the treatment of neuropathic and inflammatory pain. We stably expressed the human, rat and mouse P2X4 receptors in 1321N1 astrocytoma cells, which is devoid of functional nucleotide receptors, by retroviral transfection, and established monoclonal cell lines. Calcium flux assay conditions were optimized for high-throughput screening resulting in a Z′-factor of >0.8. The application of ready-to-use frozen cells did not negatively affect the results of the calcium assays, which is of great advantage for the screening of compound libraries. Species differences were observed, the rat P2X4 receptor being particularly insensitive to many ATP derivatives. Membrane preparations of the cell lines showed high levels of specific [35S]ATPγS binding with low nonspecific binding (<5% of total binding), while non-transfected cells were devoid of specific binding sites for the radioligand. Conditions were employed which allow binding studies to be performed at room temperature. While a variety of nucleotide-derived agonists and the antagonist TNP-ATP displaced [35S]ATPγS from its binding site at human P2X4 receptors, the non-nucleotidic antagonists paroxetine and 5-BDBD did not compete with radioligand binding and were therefore characterized as allosteric antagonists. Homology modeling was applied to find an explanation for the observed species differences.