A polycyclic scaffold identified by structure-based drug design effectively inhibits the human P2X7 receptor

Abstract

The P2X7 receptor is an ATP-gated ion channel that activates inflammatory pathways involved in diseases such as cancer, atherosclerosis, and neurodegeneration. However, despite the potential benefits of blocking overactive signaling, no P2X7 receptor antagonists have been approved for clinical use. Understanding species-specific pharmacological effects of existing antagonists has been challenging, in part due to the dearth of molecular information on receptor orthologs. Here, to identify distinct molecular features in the human receptor, we determine high-resolution cryo-EM structures of the full-length wild-type human P2X7 receptor in apo closed and ATP-bound open state conformations and draw comparisons with structures of other orthologs. We also report a cryo-EM structure of the human receptor in complex with an adamantane-based inhibitor, which we leverage, in conjunction with functional data and molecular dynamics simulations, to design a potent and selective antagonist with a unique polycyclic scaffold. Functional and structural analysis reveal how this optimized ligand, termed UB-MBX-46, interacts with the classical allosteric pocket of the human P2X7 receptor with subnanomolar potency and high selectivity, revealing its significant therapeutic potential.

We thank O. Davulcu, C. Yoshioka, and C. López at PNCC for access and microscopy assistance. Electron microscopy grid screening was performed at the Multiscale Microscopy Core within Oregon Health & Science University (OHSU). The authors would like to acknowledge the contributions of the OHSU Biophysics Shared Resource Core (Research Resource ID: RRID: SCR_022744) in facilitating this work. We thank L. Anson for comments on the manuscript. A portion of this research was supported by NIH grant U24GM129547 and performed at the PNCC at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. This research was supported by the National Heart, Lung, and Blood Institute (R00HL138129, S.E.M.), the National Institute of General Medical Sciences (DP2GM149551, S.E.M.), and the American Heart Association (24PRE1195450, A.C.O.). Part of this work was funded by the Spanish Ministerio de Ciencia, Innovación y Universidades, MICIU/AEI/10.13039/501100011033 and by ERDF/EU: grant PID2023-147004OB-I00 (to S.V.). The Kolocouris Lab thanks Chiesi Hellas for funding (SERG No. 10354). C.E.M. is grateful to the German Federal Ministery of Education and Research (BMBF) for support (Biopharma Neuroallianz, 0315606B), the Deutsche Forschungsgemeinschaft (SFB 1328) for supporting this work, and the COST Action CA21130 "P2X receptors as a therapeutic opportunity (PRESTO)". We thank Andhika B. Mahardhika for support in some of the biological experiments. A.N. is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID 335447717 - SFB 1328, project A15. The Mansoor Lab would like to thank Steve Janik and Sheryl Manning, Barbara Allen and Jim Batzer, and Randy and Barbara Lovre for their generous support.