Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.
Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.
复制标题
糖苷作为作用于P2 X7受体的正变构调节剂的结构-活性关系的见解。
DOI：
10.1124/molpharm.120.000129
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发表时间：
2021-03
影响因子：
3.6
通讯作者：
Stokes L
中科院分区：
文献类型：
--
作者：
Piyasirananda W;Beekman A;Ganesan A;Bidula S;Stokes L
P2X7是一个重要的配体门控离子通道，在多种免疫细胞群中表达。本研究旨在探讨三萜苷在一个新的结合袋内的化学需求，以表征其构效关系。在稳定表达野生型人P2X7变异体的HEK-293细胞（HEK-hP2X7细胞）中，采用YO-PRO-1染料摄取法筛选了一组苷对人P2X7的阳性调节活性。阳性调节剂活性最高的是人参皂苷化合物K (CK)，其碳-20位点含有单糖（葡萄糖）。人参皂苷-20(S)-Rg3在碳-3处含有一个双糖基团（葡萄糖-葡萄糖），显示出正向的调节活性，降低了ATP的EC50，增加了人类P2X7的最大反应。外显体20(R)-Rg3无活性。在20(S)-Rh2中观察到类似的立体特异性模式。人参皂苷- f1与人参皂苷- ck高度相似，但含有一个额外的羟基，在P2X7位点也无活性。计算对接表明，口袋中的疏水残基参与了三萜之间的空间区分，而碳水化合物基团的位置和身份对人类P2X7的正调节活性很重要。含单糖的人参皂苷比含二糖或三糖的人参皂苷性能更好。在碳-6位点对三萜支架进行额外修饰是不允许的。除人参外其他植物来源的绞股蓝苷（绞股蓝苷XVII，绞股蓝苷XLIX，绞股蓝苷）也可以作为P2X7的正变构调节剂。我们还研究了阳性变构调节剂对THP-1单核细胞内源性P2X7的影响，并在钙反应实验中证实了我们的发现。细胞活力测定显示，人参皂苷- ck在THP-1和HEK-hP2X7细胞中增强了atp诱导的细胞死亡。人参皂苷作为P2X7的正变构调节剂具有活性，本研究确定了介导这种作用的重要化学特征。糖基的位置和性质对活性很重要，就像一些羟基在三萜支架上的位置一样。人参皂苷- rg3和人参皂苷- rh2的非对映体证明了羟基相对于预测结合袋疏水面位置的重要性。
P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside–compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.