Molecular mechanism underlying the subtype-selectivity of competitive inhibitor NF110 and its distinct potencies in human and rat P2X3 receptors

Abstract P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat (rP2X3) and human (hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin (DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF, left flipper (LF) and lower body (LB) to partially, rather than fully, occupy the ATP-binding pocket. Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel’s activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches. Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.