title: Characterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies.
abstract: 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.
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Aliaa
authors.initials: A
authors.lastname: Abdelrahman
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany; Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmannstrasse 2, D-53113 Bonn, Germany.
authors.forename: Vigneshwaran
authors.initials: V
authors.lastname: Namasivayam
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hinz
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Anke C
authors.initials: AC
authors.lastname: Schiedel
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Meryem
authors.initials: M
authors.lastname: Köse
authors.affiliation: UCB Pharma, Chemin du Foriest, B-1420 Braine-L'Alleud, Belgium.
authors.forename: Maggi
authors.initials: M
authors.lastname: Burton
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany.
authors.forename: Ali
authors.initials: A
authors.lastname: El-Tayeb
authors.affiliation: UCB Pharma, Chemin du Foriest, B-1420 Braine-L'Alleud, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Gillard
authors.affiliation: Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmannstrasse 2, D-53113 Bonn, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bajorath
authors.affiliation: UCB Pharma, Chemin du Foriest, B-1420 Braine-L'Alleud, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: de Ryck
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany. Electronic address: christa.mueller@uni-bonn.de.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D017136:Ion Transport; D051379:Mice; D058913:Purinergic Agonists / Q000494:pharmacology*; D058914:Purinergic Antagonists / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D058848:Receptors, Purinergic P2X4 / Q000187:drug effects*
chemical_list: D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D058848:Receptors, Purinergic P2X4; D002118:Calcium
keywords: 2-MeS-ATP (PubChem CID: 16760275); 5-BDBD (PubChem CID: 9841560); 8-Azido-ATP (PubChem CID: 53230101); AR-C67085MX (PubChem CID: 5310954); ATP; ATP (PubChem CID: 16211020); Ap(3)A (PubChem CID: 16219211); Ap(4)A (PubChem CID: 11957521); Ap(5)A (PubChem CID: 44237136); BzATP (PubChem CID: 74764712); Frozen cells; Paroxetine (PubChem CID: 6537147); Radioligand binding; Species differences; Structure–activity relationships; TNP-ATP (PubChem CID: 75358143); α,β-methylene-ATP (PubChem CID: 11957603); β,γ-Imido-ATP (PubChem CID: 16218881); β,γ-methylene-ATP (PubChem CID: 23412783)