TNP-ATP, a derivative of ATP, acts as an antagonist to the purinergic P2Y1, P2X3, and P2X2/3 receptors, demonstrating selectivity by showing lower inhibitory concentrations (IC50 = 6, 0.9, 7 nM, respectively) compared to the P2X2, P2X4, and P2X7 receptors (IC50 = 2, 15.2, >30 µM, respectively) in HEK293 cells expressing these human receptors. It effectively diminishes acetic acid-induced calcium influx in 1321N1 cells targeting P2X3 and P2X2/3 receptors (IC50 = 100 and 62 nM, respectively) and alleviates pain in a mouse model of visceral pain (ED50 = 6.35 µmol/kg). Moreover, TNP-ATP exhibits a notable fluorescence characteristic, with an emission peak at 547 nm after excitation at 403 nm, which enhances four-fold and shifts to 538 nm when bound to the insulin-degrading enzyme (IDE).