PROJECT SUMMARY: P2X purinergic receptors are trimeric, non-selective cation channels activated by extracellular ATP to modulate processes in the cardiovascular and immune systems. The P2X7 receptor, the most structurally and functionally distinct P2X receptor subtype, is involved in signaling pathways for apoptosis and inflammation, and is predicted to play a key role in the link between inflammatory disease and atherosclerosis.In a mouse model of coronary artery disease, gene knock-out P2X7 receptor deficiency abolished atherosclerosis, suggesting that development of P2X7-specific antagonists to inhibit P2X7-signaling could result in a novel therapy for the prevention of coronary artery disease. However, despite being an actively pursued pharmacologic target, there are no FDA-approved drugs targeting the P2X7 receptor. Utilizing P2X receptors as a target for therapy has been hampered by a lack of information defining the receptor’s structure and molecular mechanisms of function. Recently, my group published the first atomic-resolution structures of full-length P2X7 receptor using single particle cryogenic electron microscopy, changing this outlook. My structures demonstrated why the P2X7 receptor subtype does not undergo desensitization and revealed that the cytoplasmic domain of P2X7 receptor has a novel fold without structural homology to any fold in the Protein Data Bank, containing a high-affinity (nanomolar) guanosine nucleotide binding site. This surprising finding in the cytoplasmic domain may begin to explain how activation of the ionotropic P2X7 receptor recruits metabotropic secondary messenger systems, with the location of the guanosine nucleotide binding site revealing where the receptor interfaces with intracellular signaling partners. Understanding these processes and molecular interactions wouldexpand pharmaceutical strategies beyond antagonism of P2X7 receptor at the extracellular domain to include modulating signaling at the cytoplasmic domain. The major aim of this grant is to use my structures of P2X7 receptor as a platform to investigate its unique signal transduction pathways and, using structure-based drug design, to develop novel ligands to modulate P2X7 receptor function for therapeutic purposes. A transformative aspect of this proposal is expanding the search of ligands targeting P2X7 receptor beyond small-molecules to include nucleic acid aptamers, a Identification of ligands (either small molecule or aptamer) that successfully inhibit P2X7 receptor activation or modulate P2X7 receptor intracellular signaling will provide invaluable research tools for studying P2X receptors with the potential to be developed into therapies for vascular inflammation and to prevent atherosclerosis. promising paradigm heretofore unexplored in P2X receptors.

PROJECT NARRATIVE The P2X7 receptor is a P2X purinergic receptor predicted to play a key role in the link between inflammation and atherosclerosis, making it an exciting pharmacological target for the prevention of chronic inflammation and coronary artery disease. However, despite its importance, no drugs targeting P2X7 receptors are currently FDA-approved, as utilizing P2X receptors as a target for therapy has been hampered by a dearth in selective small molecules that modulate P2X receptor function. Structural, functional, and selective inhibition studies on the P2X7 receptor are proposed herein to investigate the apparent dual ionotropic and metabotropic properties of this receptor’s signaling pathways, and rationally develop novel small-molecule and nucleic acid aptamer ligands to selectively modulate P2X7 receptor function for therapeutic benefit.