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.

中文翻译：

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洞察糖苷作为正变构调节剂作用于P2X7受体的糖苷的结构-活性关系。

P2X7是在多个免疫细胞群中表达的重要的配体门控离子通道。这项研究旨在调查在一个新的绑定口袋里三萜类糖苷的化学需求，以表征结构-活性关系。在稳定表达野生型人P2X7变体（HEK-hP2X7细胞）的HEK-293细胞中，使用YO-PRO-1染料摄取测定法筛选了一组糖苷对人P2X7的正调节活性。人参皂苷-化合物K（CK）的阳性调节剂活性最高，其碳20上连接有单糖（葡萄糖）。人参皂甙-20（S）-Rg3，在碳3处包含一个二糖基（葡萄糖-葡萄糖），显示出正调节剂活性，EC ₅₀降低ATP和人类P2X7的最大反应增加。差向异构体20（R）-Rg3没有活性。对于20（S）-Rh2观察到类似的立体特异性模式。人参皂苷-F1与人参皂苷-CK非常相似，但含有一个额外的羟基，在P2X7处也没有活性。计算对接表明，口袋中的疏水残基参与了三萜类化合物之间的空间区分，而碳水化合物基团的位置和特性对人类P2X7上的正调节剂活性很重要。含有单糖附着物的人参皂甙比二或三糖甙有更好的表现。不容许对碳六价位的三萜骨架进行其他修饰。来自人参以外的植物来源的绞股蓝皂甙（绞股蓝皂苷XVII，绞股蓝皂苷XLIX，stevenleaf）也可以充当P2X7的正变构调节剂。我们还研究了正构构调节剂对THP-1单核细胞内源性P2X7的影响，并在钙反应测定中证实了我们的发现。细胞活力分析显示人参皂苷-CK在THP-1和HEK-hP2X7细胞中增强了ATP诱导的细胞死亡。