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 7,7Ј-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methylphenylene)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 7,7Ј; and 3) the specific position of two sulfonic acid groups (3,3Ј; 6,6Ј) and of one methoxy group (1,1Ј) 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.

Salt Bridge Switching from Arg290/Glu167 to Arg290/ATP Promotes the Closed-to-Open Transition of the P2X2 Receptor

“…This model allowed us to identify the molecular determinants of the nanomolar potency interaction of the suramin-derivative NF770 with the P2X2 receptor (Wolf et al, 2011). The model predicted critical roles for Glu167 and Arg290 in ATP and NF770 binding, and these predictions were experimentally verified.…”

“…The detailed procedure for generating the homology models using the molecular modeling program MOE2008.10 (Molecular Operating Environment 2008; CCG, Montreal, Canada) has been previously described elsewhere (Wolf et al, 2011). To generate the closed-and open-state models of the rP2X2 receptor, we used as templates the X-ray structures of the zfP2X4 receptor in the apoclosed state (PDB entry 3H9V) (Kawate et al, 2009) and the ATPbound open state (PDB entry 4DW1) (Hattori and Gouaux, 2012), respectively.…”

“…The oocyte expression plasmid encoding the N-terminal His 6 -tagged rat P2X2 subunit was available from previous studies (Aschrafi et al, 2004;Hausmann et al, 2006;Meis et al, 2010;Wolf et al, 2011;Franklin et al, 2007). The mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).…”

Activation and Regulation of Purinergic P2X Receptor Channels

“…NF279 acts as a competitive antagonist at rP2X2R with an IC 50 value of 0.76 M (Rettinger et al, 2000), whereas NF023 shows low sensitivity at this receptor (IC 50 Ͼ 50 M) (Soto et al, 1999). The suramin derivates 7,7Ј-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,6-disulfonic acid) tetrasodium salt (NF770), 6,6Ј-(carbonylbis(imino-3,1-(4-methylphenylene)carbonylimino))bis(1-methoxynaphthalene-3,5-disulfonic acid) tetrasodium salt (NF776), and 6,6Ј-(carbonylbis(imino-3,1-phenylenecarbonylimino-3,1-(4-methyl-phenylene)carbonylimino))bis(1-methoxy-naphthalene-3,5-disulfonic acid) tetrasodium salt (NF778) have been shown to act as nanomolar P2X2R antagonists (Wolf et al, 2011). RB-2 inhibits P2X2R with an IC 50 value of 0.4 M (Liu et al, 2001a).…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…This domain is in accordance with the spatial location of R126, a residue that is responsible for the species difference in antagonists' effects of the P2X7 receptor [111] . NF770, a suramin derivative that competitively inhibits the P2X2 receptor at nanomolar concentrations, acts on G72, E167 and R290 (rP2X2 numbering), which are also important for ATP binding [112,113] . Interestingly, nearly all of those identified sites are located in or around the ATP binding pocket except for IVM, a P2X4 positive modulator, which has been identified to act on the TM domains [114][115][116][117] .…”