Abstract
The P2X receptor family of ATP-gated cation channels are attractive drug targets for pain and inflammatory disease, but no subtype-selective agonists, and few partially selective agonists have been described to date. As proof-of-concept for the discovery of novel P2X receptor agonists, here we demonstrate the use of Drosophila taste neurons heterologously expressing rat P2X2 receptors as a screening platform. We demonstrate that wild-type rat P2X2 expressed in Drosophila is fully functional (ATP EC50 8.7 µM), and that screening of small (2 µl) volumes of a library of 80 adenosine nucleotide analogues is rapid and straightforward. We have determined agonist potency and specificity profiles for rat P2X2 receptors; triphosphate-bearing analogues display broad activity, tolerating a number of substitutions, and diphosphate and monophosphate analogues display very little activity. While several ATP analogues gave responses of similar magnitude to ATP, including the previously identified agonists ATPγS and ATPαS, we were also able to identify a novel agonist, the synthetic analogue 2-fluoro-ATP, and to confirm its agonist activity on rat P2X2 receptors expressed in human cells. These data validate our Drosophila platform as a useful tool for the analysis of agonist structure-activity relationships, and for the screening and discovery of novel P2X receptor agonists.
Highlights
P2X receptors are ATP-gated ion channels which play diverse roles in physiology and thereby affect a variety of diseases and conditions, including, not exhaustively: pain sensation[1], cancer[2], arthritis[3,4], osteoporosis[5], and hypertension[6]
Substantial early research in P2X receptors on ATP binding has been carried out using rat P2X218, and parallel work focused on human P2X119–22
To determine whether rat P2X2 formed a functional channel in Drosophila taste neurons, we tested the responses to ATP in taste neurons of flies expressing rat P2X2 via the GAL4-UAS system under control of the Gr5a promoter
Summary
P2X receptors are ATP-gated ion channels which play diverse roles in physiology and thereby affect a variety of diseases and conditions, including, not exhaustively: pain sensation[1], cancer[2], arthritis[3,4], osteoporosis[5], and hypertension[6]. Rat P2X2 exhibited a relatively broad activity profile to adenosine nucleotides with three phosphoryl groups, but did not respond robustly either to ADP or AMP and their derivatives This suggested that the binding pocket can accommodate a range of modifications of the primary ligand. We have developed a sensitive functional assay in Drosophila taste neurons for mammalian ligand-gated ion channels, which permits the rapid screening of compound libraries in small (2 μl per application) quantities. We have used this assay to profile the activity of 80 compounds from an adenosine nucleotide library against rat P2X2, discovering a novel agonist and expanding our understanding of the structure-activity relationship of agonist action
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