title: Molecular determinants of potent P2X2 antagonism identified by functional analysis, mutagenesis, and homology docking.
abstract: P2X2 receptors are members of the ATP-gated P2X family of cation channels, and they participate in neurotransmission in sympathetic ganglia and interneurons. Here, we identified 7,7'-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770) as a nanomolar-potent competitive P2X2 receptor antagonist within a series of 139 suramin derivatives. Three structural determinants contributed to the inhibition of P2X2 receptors by NF770: 1) a "large urea" structure with two symmetric phenylenecarbonylimino groups; 2) attachment of the naphthalene moiety in position 7,7'; and 3) the specific position of two sulfonic acid groups (3,3'; 6,6') and of one methoxy group (1,1') at the naphthalene moiety. This structure-activity relationship was interpreted using a rat P2X2 homology model based on the crystal structure of the closed zebrafish P2X4 receptor. Docking of the suramin derivatives into the modeled ATP-binding pocket provides a uniform explanation for the observed differences in inhibitory potencies. Changes in the chemical structure that increase the inhibitory potency of the suramin derivatives improved the spatial orientation within the ATP-binding pocket to allow for stronger polar interactions of functional groups with Gly72, Glu167, or Arg290. Gly72 is responsible for the orientation of the methoxy group close to Arg290 or Glu167. Combined mutational and functional analysis confirmed that residues Gly72 and Glu167 are as important for ATP binding as Arg290, the ATP-binding role of which has been shown in previous studies. The in silico prediction of Gly72 and Glu167 as ATP-binding residues strongly supports the validity of our homology docking.
authors.affiliation: Department of Molecular Pharmacology, RWTH Aachen University, Aachen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wolf
authors.forename: Christiane
authors.initials: C
authors.lastname: Rosefort
authors.forename: Ghada
authors.initials: G
authors.lastname: Fallah
authors.forename: Matthias U
authors.initials: MU
authors.lastname: Kassack
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hamacher
authors.forename: Mandy
authors.initials: M
authors.lastname: Bodnar
authors.forename: Haihong
authors.initials: H
authors.lastname: Wang
authors.forename: Peter
authors.initials: P
authors.lastname: Illes
authors.forename: Achim
authors.initials: A
authors.lastname: Kless
authors.forename: Gregor
authors.initials: G
authors.lastname: Bahrenberg
authors.forename: Günther
authors.initials: G
authors.lastname: Schmalzing
authors.forename: Ralf
authors.initials: R
authors.lastname: Hausmann
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D005260:Female; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011237:Predictive Value of Tests; D011485:Protein Binding / Q000235:genetics; D058919:Purinergic P2 Receptor Antagonists / Q000378:metabolism*; D051381:Rats; D058476:Receptors, Purinergic P2X2 / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D013498:Suramin / Q000378:metabolism
chemical_list: D008024:Ligands; D058919:Purinergic P2 Receptor Antagonists; D058476:Receptors, Purinergic P2X2; D018698:Glutamic Acid; D013498:Suramin; D005998:Glycine