Pharmacological modulation of P2X receptors by approved drugs and natural compounds

atp门控的P2X家族非选择性阳离子通道是组织损伤或炎症反应中神经胶质细胞通信和疼痛感知的关键调节因子。它们的活性被许多细胞生物学机制进一步调节，包括磷酸化、糖基化和细胞外配体和脂质的变构调节。筛选的化合物文库包括1040种获批药物、800种具有生物活性的天然化合物和160种毒素和药理学调节剂，结果显示，与其他通道实体相比，P2X7的活性化合物数量和调节效力都非常高。因此，本项目将重点关注经批准的药物对P2X受体调节的生理和病理生理相关性，以及P2X受体外结构域上变构结合位点的结构定位。通过对P2X2， P2X4和P2X7的药理调节的大规模分析，将评估变构调节的特异性。对于选定的调节剂，将研究其作用方式、对内源性表达的P2X受体的活性和细胞生物学后果，并对药理学观点进行评估。利用最近解决的P2X4外畴结构，将采用各种策略构建变构调制位点图。功能筛选的实验结果将与使用的化合物与斑马鱼P2X4已知结构以及人类P2X4、P2X2和P2X7同源模型的对接分析进行比较。通过位点定向诱变和生化方法靶向阻断单结合腔将被应用于牢固地建立变构调节位点的定位。为了收集潜在内源性调节剂的信息，我们将分析结合口袋的系统发育保守性，并将结合到保守口袋的调节剂的化学结构作为寻找新型内源性P2X受体调节剂的模板。

ATP-gated, non-selective cation channels of the P2X family are key regulators of glio-neuronal communication and pain perception in response to tissue damage or inflammation. Their activity is further tuned by numerous cell-biological mechanisms, including phosphorylation, glycosylation and allosteric modulation by extracellular ligands and lipids. Screening of a compound library that comprises 1040 approved drugs, 800 natural compounds with annotated biological activity and 160 toxins and pharmacological modulators revealed that both the number of active compounds and the potency of modulation is uniquely high for P2X7 compared to other channel entities. Therefore, this project will focus on the physiological and pathophysiological relevance of P2X receptor modulation by approved drugs and on structurally mapping allosteric binding sites on the P2X receptor ectodomains. By large-scale analysis of pharmacological modulation of P2X2, P2X4 and P2X7, the specificity of allosteric modulation will be assessed. For selected modulators, the mode of action, activity on endogenously expressed P2X receptors and cell-biological consequences will be worked out and pharmacological perspectives will be evaluated. Taking advantage of the recently solved structure of the P2X4 ectodomain, a map of allosteric modulation sites will be constructed applying various strategies. Experimental results of functional screens will be compared to docking analyses of the used compounds onto the known structure of zebrafish P2X4 and on homology models of human P2X4, P2X2 and P2X7. Targeted occlusion of single binding cavities by site-directed mutagenesis and biochemical approaches will be applied to firmly establish the mapping of allosteric modulatory sites. To gather information about potential endogenous modulators, phylogenetic conservation of binding pockets will be analyzed and the chemical structure of modulators that bind to conserved pockets will serve as template for the search of novel endogenous P2X receptor modulators.