Profiling of a suramin-derived compound library at recombinant human P2Y receptors identifies NF272 as a competitive but non-selective P2Y2 receptor antagonist
- 69 Downloads
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 P2Y2 receptor antagonists are described. This limits the understanding of the role of P2Y2 receptors. The purpose of this study was to search for P2Y2 receptor antagonists by a combinatorial screening of a library of around 415 suramin-derived compounds. Calcium fluorescence measurements at P2Y2 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 P2Y2 receptor antagonist with a Ki of 19 μM which is 14-fold more potent than suramin at this receptor subtype. The SCHILD analysis of competitive inhibition resulted in a pA2 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 P2Y4 (3.6-fold) and P2Y6 (5.7-fold). However, NF272 is equipotent at P2Y1, and even more potent at P2Y11 and P2Y12 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 P2Y2 receptors. In conclusion, NF272 is a competitive but non-selective P2Y2 receptor antagonist with 14-fold higher potency than suramin lacking cytotoxic effects. Therefore, NF272 may serve as a lead structure for further development of P2Y2 receptor antagonists.
KeywordsPurinergic receptors P2Y2 receptor Calcium assay Antagonist Suramin
We gratefully acknowledge the device of around 1000 suramin analogues from Prof. Peter Nickel, Bonn.
The Deutsche Forschungsgemeinschaft (DFG) is acknowledged for funds used to purchase the Arrayscan XTI high content imager, ThermoFisher, Langenselbold, Germany, used in this research (INST 208/690-1).
Compliance with ethical standards
Conflict of interest
Nicole Brockmann declares that she has no conflict of interest.
Parichat Sureechatchaiyan declares that she has no conflict of interest.
David Müller declares that he has no conflict of interest.
Tatiana Hennicke declares that she has no conflict of interest.
Ralf Hausmann declares that he has no conflict of interest.
Gerhard Fritz declares that he has no conflict of interest.
Alexandra Hamacher declares that she has no conflict of interest.
Matthias U. Kassack declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 4.Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, Park D, Woodson RI, Ostankovich M, Sharma P, Lysiak JJ, Harden TK, Leitinger N, Ravichandran KS (2009) Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature 461(7261):282–286CrossRefGoogle Scholar
- 15.Eun SY, Park SW, Lee JH, Chang KC, Kim HJ (2014) P2Y(2)R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production. Free Radic Biol Med 69:157–166CrossRefGoogle Scholar
- 16.Stachon P et al (2014) Extracellular ATP induces atherosclerosis and vascular inflammation via purinergic receptor 2 (P2Y2) in mice. Cardiovasc Res 103(suppl 1):S97Google Scholar
- 22.Lau OCF, Samarawickrama C, Skalicky SE (2014) P2Y2 receptor agonists for the treatment of dry eye disease: a review. Clin Ophthalmol 8:327–334Google Scholar
- 38.Meghani P (2002) The design of P2Y2 antagonists for the treatment of inflammatory diseases. Abstr Pap Am Chem Soc 224(1–2):12Google Scholar
- 43.Ullmann H, Meis S, Hongwiset D, Marzian C, Wiese M, Nickel P, Communi D, Boeynaems JM, Wolf C, Hausmann R, Schmalzing G, Kassack MU (2005) Synthesis and structure-activity relationships of suramin-derived P2Y11 receptor antagonists with nanomolar potency. J Med Chem 48(22):7040–7048CrossRefGoogle Scholar
- 45.Meis S, Hamacher A, Hongwiset D, Marzian C, Wiese M, Eckstein N, Royer HD, Communi D, Boeynaems JM, Hausmann R, Schmalzing G, Kassack MU (2010) 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 monoc. J Pharmacol Exp Ther 332(1):238–247CrossRefGoogle Scholar
- 46.Wolf C, Rosefort C, Fallah G, Kassack MU, Hamacher A, Bodnar M, Wang H, Illes P, Kless A, Bahrenberg G, Schmalzing G, Hausmann R (2011) Molecular determinants of potent P2X2 antagonism identified by functional analysis, mutagenesis, and homology docking. Mol Pharmacol 79(4):649–661CrossRefGoogle Scholar
- 47.Kassack MU, Höfgen B, Lehmann J, Eckstein N, Quillan JM, Sadée W (2002) Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader. J Biomol Screen 7(3):233–246Google Scholar
- 65.Voogd TE, Vansterkenburg EL, Wilting J, Janssen LH (1993) Recent research on the biological activity of suramin. Pharmacol Rev 45(2):177–203Google Scholar