Pharmacological modulation of P2X receptors by approved drugs and natural compounds

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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.