Pharmacological modulation of P2X receptors by approved drugs and natural compounds

P2 X家族的ATP门控的非选择性阳离子通道是响应于组织损伤或炎症的神经胶质-神经元通信和疼痛感知的关键调节剂。它们的活性通过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。对包含1040种批准的药物、800种具有注释的生物活性的天然化合物和160种毒素和药理学调节剂的化合物文库的筛选揭示，与其他通道实体相比，P2 X7的活性化合物的数量和调节效力都是独特的高。因此，本项目将重点关注批准药物对P2 X受体调节的生理和病理生理相关性，以及P2 X受体胞外域上的变构结合位点的结构映射。通过对P2 X2、P2 X4和P2 X7的药理学调节的大规模分析，将评估变构调节的特异性。对于选定的调节剂，作用模式，对内源性表达的P2 X受体和细胞生物学后果的活动将制定和药理学的观点将进行评估。利用最近解决的P2 X4胞外域的结构，将应用各种策略构建变构调节位点的图谱。功能筛选的实验结果将与所用化合物在斑马鱼P2 X4的已知结构上以及在人P2 X4、P2 X2和P2 X7的同源模型上的对接分析进行比较。通过定点诱变和生物化学方法对单个结合腔进行靶向封闭，将用于牢固地建立变构调节位点的映射。为了收集关于潜在的内源性调节剂的信息，将分析结合口袋的系统发育保守性，并且结合保守口袋的调节剂的化学结构将用作用于搜索新的内源性P2 X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.