Tanshinone II A sulfonate but not tanshinone II A acts as potent negative allosteric modulator of the human purinergic receptor P2X7

Tanshinone II A sulfonate (TIIAS) was identified as a potent, selective blocker of purinergic receptor P2X7 in a compound-library screen. In this study, a detailed characterization of the pharmacological effects of TIIAS on P2X7 is provided. Since TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiological assays were used to characterize effects of TIIAS and TIIA on recombinantly expressed human, rat and mouse P2X7. Results were confirmed in human monocyte-derived macrophages expressing native P2X7. In all experiments, involvement of P2X7 was verified using established P2X7 antagonists. TIIAS, but not TIIA reduces Ca2+ influx via human P2X7 (hP2X7) with an IC 50 of 4.3 μM. TIIAS was less potent at mouse P2X7 and poorly inhibited rat P2X7. Monitoring of YO PRO 1 uptake confirmed these findings, indicating that formation of the hP2X7 pore is also suppressed by TIIAS. Electrophysiological experiments revealed a non-competitive mode of action. TIIAS time-dependently inhibits hP2X7 gating, possibly by binding to the intracellular domain of the receptor. Inhibition of native P2X7 in macrophages by TIIAS was confirmed by monitoring Ca2+ influx, YO PRO 1 uptake and release of the pro-inflammatory cytokine IL-1β. Fluorometric experiments involving recombinantly expressed rat P2X2 and human P2X4 were conducted and verified the compound's selectivity. Our data suggest that hP2X7 is a molecular target of TIIAS, but not of TIIA, a compound with different pharmacological properties.