NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocyte-derived dendritic cells.

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor.

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.

New iantherans from the marine sponge Ianthella quadrangulata: novel agonists of the P2Y(11) receptor.

Three new iantherans, iso-iantheran A (1), 8-carboxy-iso-iantheran A (2), and iso-iantheran B (4) were isolated together with two further new brominated tyrosine-derived metabolites 5 and 6 from the polar extract of the Australian marine sponge Ianthella quadrangulata. Structures were elucidated on the basis of extensive spectral analysis. The dimeric benzofuran skeleton including a 2,3-dihydroxy-1,3-butadiene disulfate moiety found in 1, 2, and 4 is a unique feature of iantherans and to date only described for iantherans A and B. Iso-iantheran A (1) and 8-carboxy-iso-iantheran A (2) exhibited agonist activity at P2Y11 receptors with EC50 values of 1.29 muM and 0.48 muM, respectively. Compound 2 showed some selectivity for P2Y11 over P2Y1 and P2Y2 receptors. Compounds 1 and 2 represent a new structural type for the development of P2Y11 receptor agonists.

Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes.

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.

The suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype selectivity is determined by location of sulfonic acid groups.

We have previously identified the suramin analog 4,4',4'',4'''-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) as a low nanomolar potency antagonist of recombinant P2X(1) receptors. Here, we characterize, by two-electrode voltage-clamp electrophysiology, three isomeric suramin analogs designated para-4,4',4'',4''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetrakis-benzenesulfonic acid (NF110), meta-(3,3',3'',3''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (NF448), and ortho-(2,2',2'',2''''-(carbonylbis(imino-5,1,3-benzenetriylbis (carbonylimino)))tetra-kis-benzenesulfonic acid (MK3) with respect to their potency in antagonizing rat P2X receptor-mediated inward currents in Xenopus laevis oocytes. Meta, para, and ortho refer to the position of the single sulfonic acid group relative to the amide bond linking the four symmetrically oriented benzenesulfonic acid moieties to the central, invariant suramin core. NF448, NF110, and MK3 were >200-fold less potent in blocking P2X(1) receptors than NF449, from which they differ structurally only by having one instead of two sulfonic acid residues per benzene ring. Although the meta- and ortho-isomers retained P2X(1) receptor selectivity, the para-isomer NF110 exhibited a significantly increased activity at P2X(3) receptors (K(i) approximately 36 nM) and displayed the following unique selectivity profile among suramin derivatives: P2X(2+3) = P2X(3) > P2X(1) > P2X(2) >> P2X(4) > P2X(7). The usefulness of NF110 as a P2X(3) receptor antagonist in native tissues could be demonstrated by showing that NF110 blocks alphabeta-methylene-ATP-induced currents in rat dorsal root ganglia neurons with similar potency as recombinant rat P2X(3) receptors. Together, these data highlight the importance of both the number and exact location of negatively charged groups for P2X subtype potency and selectivity.

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.