P2X receptors (P2XR) are a family of seven cation channels gated by extracellular ATP (eATP). Activation of P2XRs results in diverse cellular responses, including cell signalling, proliferation, differentiation, and death-all critically important in multiple physiological and pathophysiological states. These receptors, therefore, represent therapeutic targets of considerable interest. However, P2XRs, while structurally related, exhibit highly divergent and context-dependent functions. Their spatiotemporal and functional complexity is evident by overlapping expression across multiple cell types that can shift dynamically during physiological processes or disease progression. Furthermore, P2XRs can assemble as homo- or hetero-trimers, with distinct functional properties. These factors complicate definitive identification of a given P2XR responsible for a specific pathophysiological effect. Receptor activity in vivo is transient because of receptor-specific mechanisms and follows eATP breakdown by ectonucleotidases. Any correlation of ATP release with receptor engagement, as assessed in vitro, often does not correspond with the in vivo dynamics. Translation from animal models to humans is complicated by the species-specific pharmacology of some P2XRs, confounded by many animal models in use not fully replicating human P2XR function and regulation in pathology. Furthermore, there are no clinical biomarkers to distinguish incomplete receptor blockade from lack of therapeutic effect. Thus, translation has been very limited. To identify and validate specific P2XR functionalities, future experimental designs should use approaches and assays that can reliably assess receptor involvement, while reducing methodologically flawed findings. We propose guidelines developed in consultation with the purinergic community for consistent and reliable research practices in P2XR studies.

The ATP-gated P2X7 receptor (P2X7R) activates inflammatory signaling pathways in the central nervous system. In particular, P2X7Rs drive chronic glia-mediated neuroinflammation, which is increasingly recognized as a key contributor to Alzheimer's disease, a neurodegenerative disorder that lacks effective disease-modifying therapies. Here we identify a potent and selective negative allosteric modulator of P2X7Rs with therapeutic potential. We synthesize a series of small molecules based on a polycyclic scaffold and confirm blood-brain barrier penetration by testing a radiolabeled analogue using positron emission tomography imaging. Through a structure-guided medicinal chemistry campaign centered on our scaffold, we identify four promising P2X7R antagonists. Of these, UB-ALT-P2 exhibits the most favorable safety profile, high oral bioavailability and robust brain penetration. High-resolution cryo-EM structures of UB-ALT-P2 bound to human, mouse, and rat P2X7Rs reveal a conserved antagonist binding mode with steric features that favor prolonged binding to human receptors. In the 5xFAD mouse model of AD, oral UB-ALT-P2 blunts weight loss, improves short- and long-term memory, reduces amyloid-β plaque burden, lowers hyperphosphorylated tau, and diminishes oxidative and inflammatory markers. These results establish UB-ALT-P2 as a potent and safe P2X7R antagonist that can mitigate core AD pathologies, providing a compelling foundation for further development.

P2X receptors are a family of seven trimeric non-selective cation channels that are activated by extracellular ATP to play roles in the cardiovascular, neuronal, and immune systems. Although it is known that the P2X1 receptor subtype has increased sensitivity to ATP and fast desensitization kinetics, an underlying molecular explanation for these subtype-selective features is lacking. Here we report high-resolution cryo-EM structures of the human P2X1 receptor in the apo closed, ATP-bound desensitized, and the high-affinity antagonist NF449-bound inhibited states. The apo closed and ATP-bound desensitized state structures of human P2X1 define subtype-specific properties such as distinct pore architecture and ATP-interacting residues. The NF449-bound inhibited state structure of human P2X1 reveals that NF449 has a unique dual-ligand supramolecular binding mode at the interface of neighboring protomers, inhibiting channel activation by overlapping with the canonical P2X receptor ATP-binding site. Altogether, these data define the molecular pharmacology of the human P2X1 receptor laying the foundation for structure-based drug design. The P2X1 receptor is an ATP-gated ion channel that is a target for anti-platelet therapy. Here, authors resolve cryo-EM structures of human P2X1 in multiple states, revealing subtype-specific features and supramolecular competitive antagonism.

The P2X2 receptor (P2X2R) is a slowly desensitizing adenosine triphosphate (ATP)-gated ion channel that is highly expressed in the cochlea. When mutated, the P2X2R exacerbates age- and noise-related hearing loss, but selective modulators of the receptor are lacking, and the molecular basis of activation and desensitization remains poorly understood. Here, we determine high-resolution cryoelectron microscopy structures of the full-length wild-type human P2X2R in an apo closed state and two distinct ATP-bound desensitized states. In the apo closed state structure, we observe features unique to the P2X2R and locate disease mutations within or near the transmembrane domain. In addition, our ATP-bound structures show how free anionic ATP forms subtype-specific interactions with the orthosteric binding site. We identify and characterize two different ATP-bound desensitized state structures, one similar to published models for other P2XR subtypes, and a second alternate conformation not previously observed. A loop adjacent to the orthosteric binding site between these two ATP-bound desensitized state structures undergoes significant conformational changes. These movements are supported by multireplicate, microsecond-scale molecular dynamics simulation studies and suggest a path by which ATP could enter or leave the orthosteric pocket. Together, our results provide structural insights into the P2X2R, facilitating structure-based drug development for this therapeutically important target.

Extracellular ATP serves as a crucial signaling molecule, present in varying concentrations across diverse cellular environments. The P2X receptor (P2XR) family, which recognizes extracellular ATP, consists of seven subtypes (P2X1R - P2X7R) that form functional homo- and hetero- trimeric ion channels. These receptors are activated by distinct concentrations of extracellular ATP, ranging from low nanomolar to high micromolar levels, and are expressed in numerous cell types. They play key roles in a variety of pathophysiological conditions affecting the central nervous, immune, and cardiovascular systems. Beyond differences in ATP sensitivity, the kinetics of ion channel gating in response to agonists vary significantly among P2XR subtypes. For example, P2X1Rs and P2X3Rs exhibit rapid desensitization (milliseconds), while P2X2Rs and P2X4Rs undergo slower desensitization (seconds), and P2X7Rs show little to no desensitization. Although membrane-proximal regions within the cytoplasmic termini have long been known to influence P2XR desensitization, the lack of structural data for these domains in any P2XR subtype hindered the development of a detailed molecular explanation for the diverse desensitization profiles. Furthermore, the lack of desensitization observed in P2X7R remained particularly puzzling. In 2016, the first crystal structures of P2X3R in three functional conformational states, including a desensitized state, revealed a novel desensitization mechanism, termed the “helical recoil” model. Later, in 2019, Cryo-EM structures of full-length P2X7R not only confirmed this model but also provided additional insight into the mechanisms that prevent desensitization in P2X7Rs. Recent Cryo-EM structures of the human P2X4R not only refine the helical recoil model but also identify a putative lipid-binding site in the cytoplasmic domain that further modulates desensitization kinetics. Over the past decade, structural biology has significantly advanced our understanding of P2XR structure and function, particularly in relation to the molecular mechanisms of receptor gating.

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. Interspecies variation among existing antagonists has proven challenging, in part due to the dearth of molecular information on different 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 new and existing 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, UB-MBX-46, interacts with the classical allosteric pocket of the human P2X7 receptor with picomolar potency and high selectivity, revealing its significant therapeutic potential. ### Competing Interest Statement The authors have declared no competing interest.

P2X receptors (P2XRs) are adenosine 5'-triphosphate (ATP)-gated ion channels comprising homomeric and heteromeric trimers of seven subtypes (P2X1-P2X7) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-electron microscopy structures of full-length wild-type human P2X4 receptor in apo closed, antagonist-bound inhibited, and ATP-bound desensitized states. Because the apo closed and antagonist-bound inhibited state structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the ATP-bound desensitized state structure does not, the cytoplasmic cap is formed before agonist binding. Furthermore, structural and functional data suggest the cytoplasmic cap is stabilized by lipids to modulate desensitization, and P2X4 is modified by glycosylation and palmitoylation. Last, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that facilitates development of small-molecule modulators.

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.

P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X7 is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X7 activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X7 bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na+ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.

P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes. Here, we report high-resolution cryo-electron microscopy structures of the homomeric rat P2X(7 )receptor bound to five previously known small-molecule allosteric antagonists and a sixth antagonist that we identify. Our structural, biophysical, and electrophysiological data define the molecular determinants of allosteric antagonism in this pharmacologically relevant receptor, revealing three distinct classes of antagonists that we call shallow, deep, and starfish. Starfish binders, exemplified by the previously unidentified antagonist methyl blue, represent a unique class of inhibitors with distinct functional properties that could be exploited to develop potent P2X(7) ligands with substantial clinical impact.

P2X receptors (P2XRs) are a family of ATP-gated ion channels comprising homomeric and heteromeric trimers of seven subunits (P2X 1 - P2X 7 ) that confer different rates of desensitization. The helical recoil model of P2XR desensitization proposes the stability of the cytoplasmic cap sets the rate of desensitization, but timing of its formation is unclear for slow-desensitizing P2XRs. We report cryo-EM structures of full-length, wild-type human P2X 4 receptor in apo, antagonist-bound, and desensitized states. Because the apo and antagonist-bound structures of this slow-desensitizing P2XR include an intact cytoplasmic cap while the desensitized state structure does not, the cytoplasmic cap forms before agonist binding. Furthermore, structural and functional data suggests the cytoplasmic cap is stabilized by lipids to slow desensitization and that P2X 4 is further modified by glycosylation and palmitoylation. Finally, our antagonist-bound inhibited state structure reveals features specific to the allosteric ligand-binding pocket in human receptors that empower the development of small-molecule modulators.

P2X receptors are trimeric ligand-gated ion channels, activated by wide-ranging concentrations of extracellular ATP, that play roles in numerous physiological and pathophysiological processes from asthma to hearing loss to neuropathic pain. Acting as a novel node of inflammation, P2X7 is a prominent pharmaceutical target for atherosclerosis, neurodegeneration, and cancer. However, the molecular pharmacology of P2X7 activation and antagonism is not fully defined. We report high-resolution cryo-EM structures of full-length wild-type rat P2X7 in apo, agonist-, and antagonist-bound states.

Extracellular ATP is a critical signaling molecule that is found in a wide range of concentrations across cellular environments. The family of nonselective cation channels that sense extracellular ATP, termed P2X receptors (P2XRs), is composed of seven subtypes (P2X1-P2X7) that assemble as functional homotrimeric and heterotrimeric ion channels. Each P2XR is activated by a distinct concentration of extracellular ATP, spanning from high nanomolar to low millimolar. P2XRs are implicated in a variety of physiological and pathophysiological processes in the cardiovascular, immune, and central nervous systems, corresponding to the spatiotemporal expression, regulation, and activation of each subtype. The therapeutic potential of P2XRs is an emerging area of research in which structural biology has seemingly exceeded medicinal chemistry, as there are several published P2XR structures but currently no FDA-approved drugs targeting these ion channels. Cryogenic electron microscopy is ideally suited to facilitate structure-based drug design for P2XRs by revealing and characterizing novel ligand-binding sites. This review covers structural elements in P2XRs including the extracellular orthosteric ATP-binding site, extracellular allosteric modulator sites, channel pore, and cytoplasmic substructures, with an emphasis on potential therapeutic ligand development.

P2X receptors are ATP-gated ion channels expressed in a wide variety of eukaryotic cells. They play key roles in diverse processes such as platelet activation, smooth muscle contraction, synaptic transmission, nociception, cell proliferation, and inflammation making this receptor family an important pharmacological target. Structures of P2X receptors solved by X-ray crystallography have been instrumental in helping to define mechanisms of molecular P2X receptor function. In 2009, the first X-ray structure of the P2X4 receptor subtype confirmed a trimeric stoichiometry and revealed the overall architecture of the functional ion channel. Subsequent X-ray structures have provided the molecular details to define the orthosteric ATP binding pocket, the orthosteric antagonist binding pocket, an allosteric antagonist binding pocket, and the pore architecture in each of the major conformational states of the receptor gating cycle. Moreover, the unique gating mechanism by which P2X receptor subtypes desensitize at differing rates, referred to as the helical recoil model of receptor desensitization, was discovered directly from X-ray structures of the P2X3 receptor. However, structures of P2X receptors solved by X-ray crystallography have only been able to provide limited information on the cytoplasmic domain of this receptor family, as this domain was always truncated to varying degrees in order to facilitate crystallization. Because the P2X7 receptor subtype has a significantly larger cytoplasmic domain that has been shown to be necessary for its ability to initiate apoptosis, an absence of structural information on the P2X7 receptor cytoplasmic domain has limited our understanding of its complex signaling pathways as well as its unusual ability to remain open without undergoing desensitization. This absence of cytoplasmic structural information for P2X7 receptors was recently overcome when the first full-length P2X7 receptor structures were solved by single-particle cryogenic electron microscopy. These structures finally provide insight into the large and unique P2X7 receptor cytoplasmic domain and revealed two novel structural elements and several surprising findings: first, a cytoplasmic structural element called the cytoplasmic ballast was identified that contains a dinuclear zinc ion complex and a high affinity guanosine nucleotide binding site and second, a palmitoylated membrane proximal structural element called the C-cys anchor was identified which prevents P2X7 receptor desensitization. This chapter will highlight the major structural and functional aspects of P2X receptors discovered through structural biology, with a key emphasis on the most recent cryogenic electron microscopy structures of the full-length, wild-type P2X7 receptor.

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.