Characterizationof the Active Enantiomer and Mappingof the Stereospecific Intermolecular Pattern of a Reference P2X7 AllostericAntagonist

The P2X purinergic receptor 7 (P2X7) has an essential role in inflammation, innate immunity, tumor progression, neurodegenerative diseases, and several other diseases, leading subsequently to the development of P2X7 modulators. AZ11645373 is a frequently studied P2X7 antagonist tool compound but always used as a racemic mixture. Racemic AZ11645373 can be separated into its respective enantiomers by chiral chromatography, albeit in small batches, and these were stereochemically intact over two years later, by chiral high-performance liquid chromatography (HPLC) analysis. On a higher scale, significant decomposition is observed during purification. One of the enantiomers was crystallized as a palladium complex, and its (<i>R</i>)-configuration was determined by single-crystal X-ray diffraction, further confirmed, in solution, by vibrational circular dichroism. Biological studies demonstrated that both (<i>S</i>)- and (<i>R</i>)-forms were able to fully inhibit human P2X7, but (<i>R</i>)-AZ11645373 was more potent, with an IC₅₀ of 32.9 nM. Contrary to its effect on human P2X7, (<i>S</i>)-AZ11645373 was ineffective on mouse P2X7, while the (<i>R</i>)-AZ11645373 enantiomer was a full antagonist. These results demonstrated that the antagonistic effects of racemic AZ11645373 are mainly due to its (<i>R</i>)-enantiomer. Site-directed mutagenesis and molecular dynamics simulations indicated that the (<i>R</i>)-enantiomer may form specific interactions with Phe95 and the antagonists bound to other P2X7 monomers. Phe95 is situated in the allosteric binding site at the edge of the upper vestibule and appears to be the pivotal molecular gateway between AZ11645373 allosteric binding and locking of the closed state of the P2X7 channel. All together, these structure–function relationships should be helpful for drug design of P2X7 modulators.