Taspine is a natural product that suppresses P2X4 receptor activity via phosphoinositide 3-kinase inhibition.

Abstract

BACKGROUND & PURPOSE\nP2X4 is a ligand-gated cation channel activated by extracellular ATP, involved in neuropathic pain, inflammation and arterial tone.\n\n\nEXPERIMENTAL APPROACH\nNatural products were screened against human or mouse P2X4 activity using fura-2 loaded 1321N1 cells for measurement of intracellular Ca2+ responses; whole-cell currents were measured by patch clamp electrophysiological. Human primary macrophage chemokine release was used to assess effect of taspine on inflammatory cell function. An enzymatic assay was performed to assess the effect of taspine on recombinant PI3-kinase.\n\n\nKEY RESULTS\nA natural product screen identified taspine as an inhibitor of human P2X4 activity. Taspine inhibits human and mouse P2X4-mediated Ca2+ influx in 1321N1 cells expressing receptors (IC50 1.6±0.4 μM and 1.6±0.3 μM, respectively), but lacked activity at human P2X2, P2X3, P2X2/3 and P2X7 receptors. Taspine inhibited the maximal response at human and mouse P2X4 but had no effect on ATP potency. Taspine has a slow onset rate (~15 mins for half-maximal inhibition), irreversible over 30 minutes of washout. Taspine inhibits P2X4-mediated Ca2+ signalling in mouse BV-2 microglia cells and human primary macrophage. Taspine inhibited P2X4-mediated CXCL5 secretion in human primary macrophage. Taspine reversed ivermectin-induced potentiation of P2X4 currents in 1321N1 stably expressing cells. The known PI3-kinase inhibitor LY294002 mimicked the properties of taspine on P2X4-mediated Ca2+ influx and whole-cell currents. Taspine directly inhibited the enzymatic activity of recombinant PI3-kinase in a competitive manner.\n\n\nCONCLUSIONS AND IMPLICATIONS\nTaspine is a novel natural product P2X4 inhibitor, mediating its effect through PI3-kinase inhibitor rather than receptor antagonism. Taspine can inhibit the pro-inflammatory signalling by P2X4 in human primary macrophage.