Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist.

P2X receptors are cation channels gated by extracellular ATP and related nucleotides. Because of the widespread distribution of P2X receptors and the high subtype diversity, potent and selective antagonists are needed to dissect their roles in intact tissues. Based on suramin as a lead compound, several derivates have been described that block recombinant P2X receptors with orders of magnitude higher potency than suramin. Here we characterized the suramin analogue 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) with respect to its potency to antagonize ATP or alphabeta-methyleneadenosine 5'-trisphosphate-induced inward currents of homomeric rat P2X(1)-P2X(4) receptors or heteromeric P2X(1 + 5) and P2X(2+3) receptors, respectively. NF449 most potently blocked P2X(1) and P2X(1 + 5) receptors with IC(50) values of 0.3 nM and 0.7 nM, respectively. Three to four orders of magnitude higher NF449 concentrations were required to block homomeric P2X(3) or heteromeric P2X(2 + 3) receptors (IC(50) 1.8 and 0.3 microM, respectively). NF449 was least potent at homomeric P2X(2) receptors (IC(50) 47 microM) and homomeric P2X(4) receptors (IC(50) > 300 microM). Altogether, these results characterize NF449 as the so far most potent and selective antagonist of receptors incorporating the P2X(1) subunit such as the P2X(1) homomer and the P2X(1 + 5) heteromer.

Synthesis and structure-activity relationships of suramin-derived P2Y11 receptor antagonists with nanomolar potency.

Selective and potent P2Y(11) receptor antagonists have yet to be developed, thus impeding an evaluation of this G protein-coupled receptor mainly expressed on immune cells. Taking suramin with moderate inhibitory potency as a template, 18 ureas with variations of the methyl groups of suramin and their precursors were functionally tested at P2Y(11), P2Y(1), and P2Y(2) receptors. Fluorine substitution of the methyl groups of suramin led to the first nanomolar P2Y(11) antagonist (8f, NF157, pK(i): 7.35). For selectivity, 8f was also tested at various P2X receptors. 8f displayed selectivity for P2Y(11) over P2Y(1) (>650-fold), P2Y(2) (>650-fold), P2X(2) (3-fold), P2X(3) (8-fold), P2X(4) (>22-fold), and P2X(7) (>67-fold) but no selectivity over P2X(1). QSAR studies confirm that residues with favored resonance and size parameters in the aromatic linker region can indeed lead to an increased potency as is the case for 8f. A symmetric structure linking two anionic clusters seems to be required for bioactivity. 8f may be helpful for studies evaluating the physiological role of P2Y(11) receptors.

Use-dependent inhibition of the skeletal muscle ryanodine receptor by the suramin analogue NF676.

The skeletal muscle Ca2+ release channel, the ryanodine receptor, is activated by the trypanocidal drug suramin via the calmodulin-binding site. As calmodulin activates and inhibits the ryanodine receptor depending on whether Ca2+ is absent or present, suramin analogues were screened for inhibition of the ryanodine receptor. Up to 300 microM, the novel suramin analogue, 4,4'-(carbonyl-bis(imino-4,1-phenylene-(2,5-benzimidazolylene)carbonylimino))-bis-benzenesulfonic acid disodium salt (NF676) was not able to significantly inhibit the basal [3H]ryanodine binding. However, kinetic analysis of the high affinity [3H]ryanodine binding elucidates a time-dependent increment of inhibition by NF676, which is indicative for an open channel blocker. Moreover, the ryanodine receptor was much more sensitive towards inhibition by NF676 when preactivated with caffeine or the nonhydrolysable ATP analogue, adenylyl-imidodiphosphate. Nonetheless, the suramin activated ryanodine receptor was not susceptible towards high-affinity NF676 inhibition, indicating an allosteric hindrance between the binding sites of suramin and NF676. In the line of this finding, NF676 per se was not capable to elute the purified ryanodine receptor from a calmodulin-Sepharose, but it prevented the elution by suramin. Other than suramin, NF676 did not inhibit the Ca2+ ATPase of the sarcoplasmic reticulum. However, suramin-induced Ca2+ release from sarcoplasmic reticulum was completely abrogated by preincubation with NF676. Taken together, we conclude from these data that NF676 represents a novel lead compound as a potent use-dependent blocker of the skeletal muscle ryanodine receptor via an allosteric interaction with the suramin-binding site.

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range.

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on alphabeta meATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8',8'',8'''-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking alphabeta meATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864 > NF449 > or = NF110 > NF023 = MK-HU1 = suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110 > NF449 > or = NF864 > or = MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

Structure-activity relationships of suramin and pyridoxal-5'-phosphate derivatives as P2 receptor antagonists.

Extracellular adenine and uracil 5'-nucleotides are important signalling molecules that exert a great variety of effects in numerous tissues and cell types through the activation of P2 receptors. In the past eight years, an extended series of P2 receptors (P2X(17), ionotropic subunits; P2Y(1,2,4,6,11,12), metabotropic receptors) has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2 receptor subtypes to the diverse physiological responses mediated by the pharmacological phenotypes of native P2 receptors. Unfortunately, subtype-selective P2 ligands, especially potent and selective antagonists, have been only slowly forthcoming, and this acts as a considerable impediment to progress. However, a number of new P2 receptor antagonists have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2 receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2 receptors and their ligands. It then focuses on structure-activity relationships of PPADS and suramin analogues and will finish with a brief discussion of some related therapeutic possibilities.

Structure-activity relationships of analogues of NF449 confirm NF449 as the most potent and selective known P2X1 receptor antagonist.

NF449 [4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid-octasodiumsalt)] was recently described to inhibit recombinant rP2X(1) receptors (Naunyn Schmiedeberg's Arch. Pharmacol. 364 (2001) 285). The purpose of this study was to examine structure-activity-relationships at P2 receptors of a series of NF449 analogues. Thus, compounds containing various arylaminemono-, di-, or trisulfonic acids and a replacement of the central urea bridge were synthesized. NF449 displayed a pIC(50) at P2X(1) receptors (rat vas deferens) of 6.31 +/- 0.04 being at least 19-fold more potent at P2X(1) than at P2X(3), P2Y(1), P2Y(2), or P2Y(11). Any deletion or change of position of sulfonic acid groups or replacing the central urea bond by the bisamide of terephthalic acid reduced the potency at P2X(1) by at least 90%. All compounds were very weak antagonists at P2Y(2) or P2Y(11) receptors (pIC(50) < 4.5). In conclusion, NF449 remains the most potent and selective P2X(1) antagonist known. Potential lead compounds among the suramin class for P2X(3) (16d) and P2Y(1) (16a) receptors were identified.

Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis-(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt].

Our aim was to determine whether the newly described P2X1 antagonist NF449 [4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X1 receptor and how it affected platelet function. NF449 inhibited alpha,beta-methyleneadenosine 5'-triphosphate-induced shape change (IC50 = 83 +/- 13 nM; n = 3) and calcium influx (pA2 = 7.2 +/- 0.1; n = 3) (pIC50 = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y1 receptor, but with lower potency (IC50 = 5.8 +/- 2.2 microM; n = 3). In contrast, it was a very weak antagonist of the P2Y12-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X1 receptor with NF449 led to reduced collagen-induced aggregation, confirming a role of this receptor in platelet activation induced by collagen. Intravenous injection of 10 mg/kg NF449 into mice resulted in selective inhibition of the P2X1 receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 +/- 4 versus 51 +/- 3%) (P = 0.0061; n = 10) but without prolongation of the bleeding time (106 +/- 16 versus 78 +/- 7 s; n = 10) (N.S.; P = 0.1209). At a higher dose (50 mg/kg), NF449 inhibited the three platelet P2 receptors. This led to a further reduction in platelet consumption compared with mice injected with saline (13 +/- 4 versus 42 +/- 3%) (P = 0.0002; n = 5). NF449 also reduced dose-dependently the size of thrombi formed after laser-induced injury of mesenteric arterioles. Overall, our results indicate that NF449 constitutes a new tool to investigate the functions of the P2X1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.