Extracellular binding sites of positive and negative allosteric P2X4 receptor modulators.

Aims: P2X receptors are ATP-gated ion channels which play a role in many pathophysiological conditions. They are considered as novel drug targets, particularly in the fields of pain, (neuro) inflammation, and cancer. Due to difficulties in developing drug-like orthosteric ligands that bind to the highly polar ATP binding site, the design of positive and negative allosteric modulators (PAMs and NAMs) is a promising strategy. The P2X4 receptor was proposed as a novel target for neuropathic and inflammatory pain (antagonists), and for the treatment of alcoholism (PAMs). So far, little is known about the allosteric binding site(s) of P2X4 receptors. The aim of this study was to identify the binding site(s) of the macrocyclic natural product ivermectin, the urea derivative BX430, and the antidepressant drug paroxetine that act as allosteric modulators of P2X4 receptors.

Material And Methods: We generated chimeric receptors in which extracellular sequences of the human P2X4 receptor were exchanged for corresponding residues of the human P2X2 receptor, complemented by specific single amino acid residue mutants. Chimeric and mutated receptors were stably expressed in 1321N1 astrocytoma cells, and characterized by fluorimetric measurement of ATP-induced Ca-influx. In addition, docking studies utilizing a homology model of the human P2X4 receptor were performed.

Key Findings: Our results suggest a common binding site for ivermectin and BX430 in an extracellular receptor domain, while paroxetine might bind to the cation pore.

Significance: The obtained results provide a basis for the development of positive and negative allosteric P2X4 modulators with improved properties and will support future drug development efforts.