Profiling of a suramin-derived compound library at recombinant human P2Y receptors identifies NF272 as a competitive but non-selective P2Y receptor antagonist.

Extracellular nucleotides mediate multiple physiological effects such as proliferation, differentiation, or induction of apoptosis through G protein-coupled P2Y receptors or P2X ion channels. Evaluation of the complete physiological role of nucleotides has long been hampered by a lack of potent and selective ligands for all P2 subtypes. Meanwhile, for most of the P2 receptors, selective ligands are available, but only a few potent and selective P2Y receptor antagonists are described. This limits the understanding of the role of P2Y receptors. The purpose of this study was to search for P2Y receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y receptors recombinantly expressed in human 1321N1 astrocytoma cells identified NF272 [8-(4-methyl-3-(3-phenoxycarbonylimino-benzamido)benzamido)-naphthalene-1,3,5-trisulfonic acid trisodium salt] as a competitive P2Y receptor antagonist with a K of 19 μM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA value of 5.03 ± 0.22 (mean ± SEM) with a slope not significantly different from unity. Among uracil-nucleotide-preferring P2Y receptors, NF272 shows a moderate selectivity over P2Y (3.6-fold) and P2Y (5.7-fold). However, NF272 is equipotent at P2Y, and even more potent at P2Y and P2Y receptors. Up to 250 μM, NF272 showed no cytotoxicity in MTT cell viability assays in 1321N1, HEK293, and OVCAR-3 cells. Further, NF272 was able to inhibit the ATP-induced calcium signal in OVCAR-3 cells demonstrated to express P2Y receptors. In conclusion, NF272 is a competitive but non-selective P2Y receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y receptor antagonists.