Tanshinone II A Sulfonate, but Not Tanshinone II A, Acts as Potent Negative Allosteric Modulator of the Human Purinergic Receptor P2X7

Abstract

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 pharmacologic effects of TIIAS on P2X7 is provided. Because TIIAS is a derivative of tanshinone II A (TIIA) and both compounds have been used interchangeably, TIIA was included in some assays. Fluorometric and electrophysiologic 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 Ca 21 influx via human P2X7 (hP2X7) with an IC 50 of 4.3 mM. 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. Electrophysiologic experiments revealed a noncompetitive 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 Ca 21 influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-1b. 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 pharmacologic properties.

Critical Evaluation of P2X7 Receptor Antagonists in Selected Seizure Models

“…All fluorometric Ca 2+ assays in cell suspensions were performed in 384-microwell plates (Corning, No. 3655, Lowell, MA, USA) with a fluorescence-imaging microplate reader (POLARstar Omega, BMG Labtech, Offenburg, Germany) (for details, see [ 61 , 68 ]). Briefly, suspensions of HEK293 cells stably expressing human, mouse and rat P2X7R, respectively, were incubated with the fluorescent indicator fluo-4/AM (4 μM, Invitrogen, Darmstadt, Germany), in the dark for 30–45 min at 37°C, centrifuged (100xg for 3 min), and re-suspended in HEPES-buffered solution (HBS), containing 133 mM NaCl, 4.8 mM KCl, 1.2 mM KH 2 PO 4 , 1.3 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES and 10 mM d -glucose adjusted to pH 7.4 with NaOH, or in a similar solution without MgCl 2 (as indicated).…”

“…On the other hand, direct anticonvulsant properties of P2X7R antagonists in traditional screening models or their potential usefulness in preventing epileptogenesis have not been examined in detail. To address this issue, we assessed possible anticonvulsant effects of a series of P2X7R antagonists: three CNS-permeable P2X7R antagonists; Brilliant Blue G (BBG) [ 58 ], JNJ-47965567 (JNJ) [ 59 ] and AFC-5128 (AFC) [ 60 ] as well as a natural compound-based derivative, tanshinone IIA sulfonate (TIIAS), as a negative allosteric modulator of the P2X7R [ 61 ]. Initially, we tested and compared the potency of the compounds using a fluorometric Ca 2+ assay and different HEK293 cell lines stably expressing mouse, rat and human P2X7R.…”

Tanshinone IIA inhibits gastric cancer cell stemness through inducing ferroptosis

“…STS was modified from Tanshinone IIA, thus it has an increased water‐solubility than Tanshinone IIA 16 . Previous researches often interchange the applications of Tanshinone IIA and STS, 17 hence, we used STS instead of Tanshinone IIA to conduct the experiment.…”

Targeting Oxidative Stress and Endothelial Dysfunction Using Tanshinone IIA for the Treatment of Tissue Inflammation and Fibrosis

“…STS suppresses atorvastatin-driven cerebral hemorrhage in zebrafish embryos but not TanIIA [ 32 ]. Human Purinergic Receptor P2X7 is blocked by STS but not TanIIA [ 36 ]. On the other hand, TanIIA inhibits the phosphorylation of Akt when used with anticancer drug epirubicin and increase the apoptosis of breast cancer cell line BT-20.…”