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

被引:50
作者
Abdelrahman, Aliaa [1 ]
Namasivayam, Vigneshwaran [1 ,3 ]
Hinz, Sonja [1 ]
Schiedel, Anke C. [1 ]
Koese, Meryem [1 ]
Burton, Maggi [2 ]
El-Tayeb, Ali [1 ]
Gillard, Michel [2 ]
Bajorath, Juergen [3 ]
de Ryck, Marc [2 ]
Mueller, Christa E. [1 ]
机构
[1] Univ Bonn, Inst Pharmaceut, PharmaCtr Bonn, Pharmaceut Chem 1, Immenburg 4, D-53121 Bonn, Germany
[2] UCB Pharma, Chemin Foriest, B-1420 Braine Lalleud, Belgium
[3] Rhein Freidrich Wilhelms Univ Bonn, Dept Life Sci Informat, B IT, LIMES Program,Unit Chem Biol & Med Chem, Dahlmannstr 2, D-53113 Bonn, Germany
关键词
ATP; Frozen cells; Radioligand binding; Species differences; Structure-activity relationships; P2X(4) RECEPTOR; NEUROPATHIC PAIN; PURINERGIC RECEPTORS; INTERNATIONAL UNION; SPINAL MICROGLIA; P2Y RECEPTORS; UP-REGULATION; NERVE INJURY; ION-CHANNEL; BRAIN;
D O I
10.1016/j.bcp.2016.11.016
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
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 [S-35]ATP gamma S 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 [S-35]ATP gamma S 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. (C) 2016 Elsevier Inc. All rights reserved.
引用
收藏
页码:41 / 54
页数:14
相关论文
共 68 条
[1]   International union of pharmacology LVIII: Update on the P2Y G protein-coupled nucleotide receptors: From molecular mechanisms and pathophysiology to therapy [J].
Abbracchio, Maria P. ;
Burnstock, Geoffrey ;
Boeynaems, Jean-Marie ;
Barnard, Eric A. ;
Boyer, Jose L. ;
Kennedy, Charles ;
Knight, Gillian E. ;
Fumagalli, Marta ;
Gachet, Christian ;
Jacobson, Kenneth A. ;
Weisman, Gary A. .
PHARMACOLOGICAL REVIEWS, 2006, 58 (03) :281-341
[2]   Trimeric architecture of homomeric P2X2 and heteromeric P2X1+2 receptor subtypes [J].
Aschrafi, A ;
Sadtler, S ;
Niculescu, C ;
Rettinger, J ;
Schmalzing, G .
JOURNAL OF MOLECULAR BIOLOGY, 2004, 342 (01) :333-343
[3]   Identification and Characterization of a Selective Allosteric Antagonist of Human P2X4 Receptor Channels [J].
Ase, Ariel R. ;
Honson, Nicolette S. ;
Zaghdane, Helmi ;
Pfeifer, Tom A. ;
Seguela, Philippe .
MOLECULAR PHARMACOLOGY, 2015, 87 (04) :606-616
[4]   P2X Receptors, Sensory Neurons and Pain [J].
Bele, Tanja ;
Fabbretti, Elsa .
CURRENT MEDICINAL CHEMISTRY, 2015, 22 (07) :845-850
[5]   Pharmacological characterization of recombinant human and rat P2X receptor subtypes [J].
Bianchi, BR ;
Lynch, KJ ;
Touma, E ;
Niforatos, W ;
Burgard, EC ;
Alexander, KM ;
Park, HS ;
Yu, HX ;
Metzger, R ;
Kowaluk, E ;
Jarvis, MF ;
van Biesen, T .
EUROPEAN JOURNAL OF PHARMACOLOGY, 1999, 376 (1-2) :127-138
[6]   P2 receptors activated by uracil nucleotides -: An update [J].
Brunschweiger, A ;
Müller, CE .
CURRENT MEDICINAL CHEMISTRY, 2006, 13 (03) :289-312
[7]   An antagonist-insensitive P-2X receptor expressed in epithelia and brain [J].
Buell, G ;
Lewis, C ;
Collo, G ;
North, RA ;
Surprenant, A .
EMBO JOURNAL, 1996, 15 (01) :55-62
[8]   PURINERGIC RECEPTORS [J].
BURNSTOCK, G .
JOURNAL OF THEORETICAL BIOLOGY, 1976, 62 (02) :491-503
[9]  
Burnstock G., 1978, CELL MEMBRAIN RECEPT, P107, DOI DOI 10.1016/0014-5793(79)81367-8
[10]  
Burnstock G, 2016, ADV PHARMACOL, V75, P91, DOI 10.1016/bs.apha.2015.09.001