Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy

The P2X3 receptor (P2X3R), an ATP-gated cation channel
predominantly
expressed in C- and Aδ-primary afferent neurons, has been proposed
as a drug target for neurological inflammatory diseases, e.g., neuropathic
pain, and chronic cough. Aiming to develop novel, selective P2X3R
antagonists, tetrazolopyrimidine-based hit compound 9 was optimized through structure–activity relationship studies
by modifying the tetrazole core as well as side chain substituents.
The optimized antagonist 26a, featuring a cyclopropane-substituted
triazolopyrimidine core, displayed potent P2X3R-antagonistic activity
(IC50 = 54.9 nM), 20-fold selectivity versus the heteromeric
P2X2/3R, and high selectivity versus other P2XR subtypes. Noncompetitive
P2X3R blockade was experimentally confirmed by calcium influx assays.
Cryo-electron microscopy revealed that 26a stabilizes
the P2X3R in its desensitized state, acting as a molecular barrier
to prevent ions from accessing the central pore. In vivo studies in
a rat neuropathic pain model (spinal nerve ligation) showed dose-dependent
antiallodynic effects of 26a, thus presenting a novel,
promising lead structure.