Anales RANF

S11-03 THE MOLECULAR DETERMINANTS OF SMALL-MOLECULE BINDING AT P2X RECEPTORS Mark T. Young School of Biosciences, Cardiff University, Cardiff, UK P2X receptors are ATP-gated trimeric cation channels which are implicated in several diseases including neuropathic pain, cancer and arthritis, making them attractive drug targets. The availability of a series of P2X receptor crystal structures in resting, open and antagonist-bound states has transformed our understanding of the way small molecules bind to P2X receptors, and enabled us to perform molecular modelling studies to reconcile data from previously published structure-function experiments, describing potential binding conformations for the semi-selective synthetic P2X7 agonist 2’-(3’)-O-(4-benzoyl)benzoyl ATP (BzATP), and the P2X4-selective positive allosteric modulator ivermectin. We find that the distal benzoyl group of BzATP lies in close proximity to Lys-127, a residue previously implicated in BzATP binding to P2X7, which may explain the increased potency of BzATP at rat P2X7 receptors. Ivermectin has been shown to bind between transmembrane domains in the crystal structure of the glutamate-gated chloride channel GluCl α , and a wealth of previous mutagenesis studies on P2X4 receptors suggest that it may bind in a similar location in P2X4 receptors. Our docking of ivermectin into a molecular model of rat P2X4 between the first and second transmembrane domains agrees very well with the previous data, and would likely have the effect of stabilizing the open channel structure, consistent with the mode of action of this positive allosteric modulator. From our docking simulations and analysis of sequence homology we propose a series of mutations likely to confer ivermectin sensitivity to human P2X1. While several subtype-selective P2X receptor antagonists are known, no subtype- selective agonists, and few partially selective agonists have been described to date. In order to discover novel P2X receptor agonists, we have developed a screening platform using transgenic Drosophila melanogaster expressing P2X2 receptors in their taste neurons. Wild-type rat P2X2 expressed in Drosophila was fully functional (ATP EC 50 8.7 µM), and we were able to rapidly screen a library of very small volumes (2 µl) of 80 adenosine nucleotide analogues, enabling us to determine the agonist potency and specificity profile for rat P2X2. We found that triphosphate-bearing analogues displayed broad activity, tolerating a number of substitutions, and that diphosphate and monophosphate analogues displayed very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists, ATP γ S and ATP α S, we also identified a novel agonist, 2-fluoro-ATP, and confirmed its agonist activity on rat P2X2 receptors expressed in human cells. Docking of molecules displaying a range of agonist activity into a molecular model of rat P2X2 gave insights into where substituents are more likely to be tolerated, and will inform the future structure-based design of subtype-selective P2X receptor agonists.