Drosophila taste neurons as an agonist-screening platform for P2X receptors

Abstract

van der Goes van naters 1 ✉ 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 ec 50 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. P2X receptors are ATP-gated ion channels which play diverse roles in physiology and thereby affect a variety of diseases and conditions, including, although not exhaustively: pain sensation 1 , cancer 2 , arthritis 3,4 , osteoporosis 5 , and hypertension 6. Together with the metabotropic P2Y receptors, P2X receptors mediate cellular responses to extracellular ATP 7. ATP functions as an intercellular signaling molecule through release as a (co)transmitter in many synapses, during muscle contraction in skeletal and heart muscle, and by receptor-mediated release in many non-excitable tissues. ATP is also released from cells following damage or stress to the cell membrane by a variety of conductive mechanisms 8 , and is released constitutively at low flux rates 9. Seven P2X genes in mammals (P2RX1-P2RX7), some of which may be alternatively spliced, encode channel subunits that trimerize to form a complex repertoire of homo-and heterotrimeric P2X receptors. Each subunit has two transmembrane domains connected by a large extracellular loop; N-and C-termini are intracellular. P2X genes have also been identified in many other classes of vertebrates and invertebrates, as well as in lower organisms, such as slime mold, where the P2X receptor has an intracellular function in vesicle fusion 10,11. The P2X2 subunit in mammals functions as a homotrimer, or in association with other subunits, in signaling in both the peripheral nervous system (PNS) and central nervous system (CNS). In the PNS, P2X2 receptors of the carotid sinus nerve endings are crucial to carotid body oxygen signal transduction 6. Functions of P2X2 receptors have also been re...

Identification of a novel P2X7 antagonist using structure-based virtual screening

“…Indeed, the binding pose of ATP in the pocket is strikingly similar in Danio rerio P2X4, human P2X3, gulf coast tick P2X and rat P2X7, adopting a U-shaped conformation where conserved lysine and arginine residues coordinate the phosphates. The presence of the gamma phosphate is crucial for agonist activity, as well as the conformation of the ribose moiety and the localization of the adenine moiety into a relatively hydrophobic pocket ( Dal Ben et al, 2019 ; Gasparri et al, 2019 ; Grimes et al, 2020 ). We chose a model of human P2X4 based on the ATP-bound structure of Danio rerio P2X4 for virtual screening and validated our docking algorithms using ATP (in a model where the bound ATP was removed prior to docking).…”

Delineating Purinergic Signaling in Drosophila

“…Ectopic expression of P2X2 in gustatory sensory neurons, and particularly in sweet-, bitter-, and water-sensing neurons committed to the sense of taste and feeding, has demonstrated the existence of independent processing of appetitive and aversive tastes, aimed at ensuring innate responses to appropriate feeding [ 45 ]. Moreover, heterologous expression of mammalian P2X2 in Drosophila taste neurons has expanded our knowledge of the structure–activity relationship of agonists’ action, through the rapid screening and identification of novel selective P2X2 agonists from an adenosine nucleotide library [ 46 ].…”

Animal Models of Neurodegenerative Disease: Recent Advances in Fly Highlight Innovative Approaches to Drug Discovery

“…Flies have been used extensively in drug discovery, in large part due to their ease of use and relevance to human disease models (Fernández-Hernández et al, 2016 ; Su, 2019 ). Small molecules have been identified in screens with relevance to several human disease conditions including cancer (Willoughby et al, 2013 ; Yadav et al, 2016 ; Bangi, 2019 ; Al Outa et al, 2020 ), pain and inflammatory disease (Grimes et al, 2020 ), sleep and circadian rhythm (Nall and Sehgal, 2013 ; Wang et al, 2020 ), neurodegenerative (ND) disorders including Alzheimer's disease (Hannan et al, 2016 ; Elovsson et al, 2021 ), Parkinson's disease (Sanz et al, 2021 ), Huntington's disease (Sarkar et al, 2007 ; Schulte et al, 2011 ), Lysosomal storage disorders (Rigon et al, 2021 ), Alexander disease (Wang et al, 2016 ), Friedreich's disease (Seguin et al, 2015 ), Fragile X syndrome (Chang et al, 2008 ), Ataxia-telangiectasia (Rimkus and Wassarman, 2018 ), obesity (Gasque et al, 2013 ), atrial fibrillation (van Marion et al, 2019 ), and in human hazard identification (testing potential genotoxins such as food, dietary supplements, cosmetics, drugs, pesticides, herbicides, and nanoparticles) (Barik and Mishra, 2019 ; Pitchakarn et al, 2021 ). Created with BioRender.com .…”