NF449: a subnanomolar potency antagonist at recombinant rat P2X₁ receptors

Abstract.

Antagonistic effects of the novel suramin analogue 4,4′,4′′,4′′′-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by α,β-methylene ATP (αβmeATP; mediated by P2X₁ receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by αβmeATP (mediated by P2X₃ receptors) or adenosine 5′-O-(2-thiodiphosphate) (ADPβS; mediated by P2Y₁ receptors), ATP-induced increases of [Ca²⁺]_i in human embryonic kidney (HEK) 293 cells (mediated by P2Y₂ receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X₁ or rP2X₃ receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (α_1A-adrenoceptors) and guinea-pig ileum (histamine H₁ and muscarinic M₃ receptors).

At native (pIC₅₀=7.15) and recombinant (pIC₅₀=9.54) P2X₁ receptors, NF449 was a highly potent antagonist. The P2X₃ receptors present in guinea-pig ileum (pIC₅₀=5.04) or expressed in oocytes (pIC₅₀≈5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y₁ receptors in guinea-pig ileum (pIC₅₀=4.85) and P2Y₂ receptors in HEK 293 cells (pIC₅₀=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC₅₀<3.5). NF449 (100 µM) did not interact with α_1A-adrenoceptors or histamine H₁ and muscarinic M₃ receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors.

In conclusion, the subnanomolar potency at rP2X₁ receptors and the rank order of potency, P2X₁ >> P2X₃ > P2Y₁ > P2Y₂ > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X₁-selective radioligand.