Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

Abstract P2X receptors are ATP-gated cation channels, and the P2X3 subtype plays crucial roles in peripheral sensory neurons, including in chronic pain and chronic cough. Accordingly, P2X3 has attracted substantial interest as a therapeutic target. Gefapixant, a negative allosteric modulator (NAM) of P2X3, has been approved in some countries for the treatment of chronic cough; however, its limited selectivity for P2X3 homomers over P2X2/P2X3 heteromers is associated with taste disturbance as a prominent adverse effect. These limitations have motivated the development of next-generation NAMs with improved subtype selectivity, but their subtype-specific allosteric inhibition mechanisms are unclear. Here, we report the cryo-EM structure of the human P2X3 receptor in complex with ATP and the P2X3-selective next-generation NAM sivopixant, an investigational drug. Sivopixant binds to an allosteric site at the portal of the central pocket in the extracellular domain, and structure-based mutational analysis by electrophysiology identifies key residues required for sivopixant-dependent inhibition of human P2X3. Comparisons with P2X structures from other subtypes, together with gain-of-function mutants, define a structural basis for subtype-selective allosteric inhibition of the P2X3 receptor. Furthermore, structural comparisons with apo and ATP-bound open states of P2X3 receptors, together with molecular dynamics simulations, revealed that sivopixant expands the upper body domain to suppress the lower-body movements required for channel activation, thereby preventing channel opening even in the presence of ATP.