Kaiser M, Sobottka H, Fischer W, Schaefer M, Nörenberg W
Rudolf-Boehm-Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Leipzig, Germany.
Rudolf-Boehm-Institute of Pharmacology and Toxicology, Medical Faculty, University of Leipzig, Leipzig, Germany
J Pharmacol Exp Ther. 2014 Sep;350(3):531-42. doi: 10.1124/jpet.114.214569. Epub 2014 Jun 26.
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(2+) influx via human P2X7 (hP2X7) with an IC50 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. 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(2+) influx, YO-PRO-1 uptake, and release of the proinflammatory cytokine interleukin-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 pharmacologic properties.
在化合物文库筛选中，丹参酮IIA磺酸盐（TIIAS）被鉴定为嘌呤能受体P2X7的一种强效、选择性阻滞剂。在本研究中，对TIIAS对P2X7的药理作用进行了详细表征。由于TIIAS是丹参酮IIA（TIIA）的衍生物，且这两种化合物一直被交替使用，因此在一些实验中纳入了TIIA。采用荧光法和电生理法来表征TIIAS和TIIA对重组表达的人、大鼠和小鼠P2X7的作用。在表达天然P2X7的人单核细胞衍生巨噬细胞中证实了结果。在所有实验中，使用既定的P2X7拮抗剂验证了P2X7的参与情况。TIIAS而非TIIA可降低通过人P2X7（hP2X7）的Ca(2+)内流，IC50为4.3 μM。TIIAS对小鼠P2X7的效力较低，对大鼠P2X7的抑制作用较差。对YO-PRO-1摄取的监测证实了这些发现，表明hP2X7孔的形成也受到TIIAS的抑制。电生理实验揭示了一种非竞争性作用模式。TIIAS时间依赖性地抑制hP2X7门控，可能是通过与受体的细胞内结构域结合。通过监测Ca(2+)内流、YO-PRO-1摄取和促炎细胞因子白细胞介素-1β的释放，证实了TIIAS对巨噬细胞中天然P2X7的抑制作用。进行了涉及重组表达的大鼠P2X2和人P2X4的荧光实验，并验证了该化合物的选择性。我们的数据表明，hP2X7是TIIAS的分子靶点，而非具有不同药理特性的化合物TIIA的分子靶点。
