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 P2Y(11) 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 (ATP gamma S), 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'-(carbonyl-bis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonicacid)tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATP gamma S and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATP gamma S, 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 ATP gamma S 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.