The novel heteromeric bivalent ligand SB9 potently antagonizes P2Y1 receptor-mediated responses

Abstract

Effects of 6-[(4,6,8-trisulfo-1-naphthyl)iminocarbonyl-1,3-(4-methylphenylene)iminocarbonyl-1,3-phenylene-azo]-pyridoxal-5′-phosphate (SB9), a heterodimeric bivalent ligand consisting of pyridoxal-5′-phosphate and the suramin monomer, were studied on contractions of the rat vas deferens elicited by αβ-methylene ATP (αβmeATP; mediated by P2X₁-like receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by adenosine 5′-O-(2-thiodiphosphate) (ADPβS; mediated by P2Y₁-like receptors), and the degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. SB9 (0.1–10 μM) antagonized contractile responses produced by αβmeATP or ADPβS in a concentration-dependent manner. Schild analysis yielded linear regression lines of unit slope, indicating competitive antagonism. From the rightward shifts of the agonist concentration–response curves pA₂ values of 6.05±0.13 (vas deferens) and 6.98±0.07 (ileum) were derived. In both preparations, SB9 behaved as a slow onset, slow offset antagonist. Incubation of three oocytes in the presence of ATP produced an increase in inorganic phosphate (P_i) over a 30-min period, which amounted to 35.1±1.9 μM P_i from 100 μM ATP. SB9 (10–1000 μM) reduced this degradation (pIC₅₀=4.33±0.10). The results illustrate that SB9 is a high-affinity P2Y₁ receptor antagonist with a remarkable selectivity for P2Y₁ vs. P2X₁ receptors (about 10-fold) and ecto-nucleotidases (447-fold). These properties make it unique among the pyridoxal-5′-phosphate and suramin derivatives reported to date.