Pharmacological and structural investigations of the ATP-gated ion channel receptor P2X4

Abstract

P2X receptors are trimeric ligand-gated ion channels comprising seven subtypes, P2X1-P2X7, which are all activated by adenosine triphosphate (ATP). These receptors are widely expressed in neuronal and non-neuronal cells. Their roles in pathophysiological processes, particularly in inflammatory diseases, make them attractive drug targets, as evidenced by the recent approval of the P2X3 receptor antagonist gefapixant for the treatment of chronic cough. <br /> The P2X4 receptor subtype is expressed in the central nervous system, particularly on neurons and microglia, as well as on peripheral immune cells, and was shown to play a role in chronic pain, neuroinflammation and associated diseases, and in various types of cancer. The inhibition of the P2X4 receptor showed promising effects in preclinical studies of neuropathic and inflammatory pain. Since the polar nature of the P2X4 receptor ATP-binding site and its similarity to that of other P2X receptor subtypes has impeded the discovery of selective orthosteric drug-like P2X4 receptor antagonists, drug research has focused on the development of allosteric antagonists. To date, only a few potent and selective allosteric P2X4 receptor antagonists have been described, and information on their binding site(s) is sparse. <br /> The objective of the present thesis was to advance the pharmacological and structural characterization of the P2X4 receptor, with a particular focus on identifying allosteric binding sites for P2X4 receptor antagonists. Expression and pharmacological characterization of P2X4 receptors from a variety of different species were performed, shedding light on species differences between the human P2X4 receptor and its orthologs. This study successfully established a radioligand binding assay for an allosteric P2X4 receptor antagonist, which indicated the presence of different allosteric binding sites on the P2X4 receptor. Biological investigation of nucleoside thiophosphates at P2X receptor subtypes led to the identification of novel P2X receptor agonists. These compounds may serve as pharmacological tools and provide a basis for the future development of subtype-selective agonists. The allosteric binding site of the P2X4 receptor antagonist BX430 was investigated by mutagenesis studies, indicating that BX430 may bind to the dorsal fin region of the human P2X4 receptor. A high-resolution structure of the human P2X4 receptor in complex with an allosteric antagonist was resolved by cryo-electron microscopy, which revealed an unprecedented binding site for anthraquinone derivatives. The findings of this thesis expand the understanding of the pharmacological and structural features of the P2X4 receptor, thereby facilitating future drug development and investigations of the receptor’s role in health and disease.