Discovery of natural product antagonists for the P2X7 receptor through virtual screening

The P2X7 receptor is a purinergic ionotropic receptor widely expressed in hematopoietic and glial cells. P2X7 receptor activation triggers a cascade of proinflammatory responses and cell death, thereby contributing to the pathogenesis of inflammatory disorders, neurodegenerative diseases, and pain. Although in vivo studies of P2X7 receptor antagonists have shown significant anti-inflammatory and antinociceptive effects, no antagonist has yet been approved for clinical use, highlighting the need for novel inhibitors. In this study, virtual screening was employed to identify natural product-derived candidates with potential P2X7 receptor antagonistic activity. Approximately 21,000 compounds from the ZINC15 database were first evaluated by docking, and the top 100 were ranked based on binding affinity scores ranging from -14.109 to -11.346 kcal/mol. Notably, these values surpassed those of most reference P2X7 receptor ligands used as controls. Five compounds (C28, C58, C63, C68, and C71) were selected for further evaluation based on drug-likeness criteria, including agreement with Lipinski's Rule of Five. A visual inspection analysis of the best-scored docking poses revealed that all five compounds interact with key residues at the P2X7 receptor allosteric site, particularly L95, P96, L97, and Y295, through hydrophobic and hydrogen-bonding interactions. In vitro assays revealed that some compounds were noncytotoxic; however, only compound C71 significantly inhibited P2X7 receptor-mediated dye uptake and ATP-induced intracellular reactive oxygen species (ROS) formation. These findings suggest that compound C71 acts as a promising P2X7 receptor inhibitor capable of blocking ATP-induced pore formation and downstream oxidative signaling, supporting further investigation as a potential therapeutic agent.