Discovery of an allosteric binding site for anthraquinones at the human P2X4 receptor.

P2X receptors are trimeric ATP-gated ion channels. The P2X4 receptor subtype is a promising drug target for the treatment of inflammatory diseases, neuropathic pain, and cancer. Here, the water-soluble anthraquinone derivative Cibacron Blue, previously described as a P2X4 receptor modulator, is selected as a lead structure, and structure-activity relationships are investigated. A chimeric receptor approach, combined with mutagenesis and docking studies, is applied to identify the allosteric binding site and the interacting amino acid residues. We discover that Glu307, located in the upper body of the receptor, is prone to form an intermolecular "ionic lock" with basic amino acid residues, thereby preventing high-affinity binding of anthraquinone derivatives. Exchange of Glu307 for threonine leads to a dramatic potency increase for anthraquinones in blocking P2X4 receptor function. The structure of the human P2X4-E307T receptor in complex with the anthraquinone derivative PSB-0704 is determined by cryo-electron microscopy at a resolution of 3.35 Å. This reveals an allosteric binding site in the upper body at the interface of the receptor trimer subunits, which differs from previously described allosteric sites on P2X receptors. Our results provide a rational basis for structure-based drug design towards potent and selective P2X4 receptor antagonists.