Comprehensive insights into potential roles of purinergic P2 receptors on diseases: Signaling pathways involved and potential therapeutics.

Yanshuo Guo : Conceptualization, Investigation, Visualization, Writing - original draft. Tianqi Mao : Conceptualization, Visualization, Writing - original draft. Yafei Fang : Investigation, Methodology, Writing - review & editing. Hui Wang : Investigation, Writing - review & editing. Jiayue Yu : Visualization. Yifan Zhu : Investigation. Shige Shen : Writing - review & editing. Mengze Zhou : Conceptualization, Supervision, Funding acquisition. Huanqiu Li : Conceptualization, Writing - review & editing, Supervision, Funding acquisition. Qinghua Hu : Conceptualization, Writing - review & editing, Supervision, Funding acquisition.

In this review, we discuss the critical physiological and pathological functions of purinergic P2 receptors in nervous system, digestive system, immune system, and cancer that include effects on various pathways such as cellular metabolism, nerve impulse conduction, protein synthesis, immune response, and mucosal barrier function. The roles of purinergic P2 receptor-mediated pathways in physiological and pathological processes are still being elucidated. Meanwhile, this review also analyzes the characteristics of purinergic P2 receptors and existing drug development in detail, as well as proposes novel drug development strategies for targeting purinergic P2 receptors, such as agonists, antagonists, pharmaceutical compositions and combination strategies, providing directions for safer and more effective targeted drug development. Altogether, purinergic P2 receptors have emerged as novel targets for therapeutic intervention in diseases. However, the wide distribution and pharmacological effects of purinergic P2 receptors pose challenges to develop targeted drugs that probably lead to the emergence of contradictory or conflicting pharmacological effects. The function of purinergic P2 receptors may be tissue-, cell-, or disease-specific, which could partly explain the conflicts that could be solved from the following perspectives. Firstly, to design rational drugs based on structure, combining molecular dynamics simulation, targeted mutagenesis and other techniques to discover structural sites associated with specific disease environments, such as cryptic allosteric modulation sites. Secondly, to optimize the drug delivery systems to specific tissues or cells in pathological states that concerns gene technology and material science approaches to optimize drug design and delivery methods, while minimizing side effects of drugs on non-targeted tissues or cells possibly. Thirdly, to adjust the factors that probably affect drug distribution, such as the mode of administration, the physicochemical properties of the drug ( e.g. molecular weight, lipid solubility), and membrane permeability. Meanwhile, in order to better address the risks of drugs targeting purinergic P2 receptors, more intensive studies are probably necessary to investigate their different functions in different tissues and biological development stages, as well as the dosage in non-target tissues. This review presents the latest research on the complex roles of purinergic P2 receptors in physiological and pathological states along with structure-based drug targeting, providing novel strategies for drug development based on safety, efficiency and rationalization. It should be noted that the available pharmacological tools are still limited, and the purified extraction and structural resolution of certain purinergic P2 receptors remain unclear, which posing challenges for pharmacological development. Unraveling the relationship between structure, localization and function of purinergic P2 receptors may provide new insights into their multiple roles in physiology and pathology by combining bioinformatics and artificial intelligence approaches. Furthermore, although some drugs targeting purinergic P2 receptors are in clinical or preclinical development, few studies have been conducted on their toxicity and target organs. In complex pathologies and mechanisms, candidate compounds may still have non-specific targeting and off-target effects that require further investigation to improve the benefit/risk ratio. In any case, more studies have shown that purinergic P2 receptors are involved in human diseases, future work should continue to elucidate their physiological and pathological roles with a focus on developing highly effective novel targeted drugs. Targeting purinergic P2 receptors for disease treatment is an attractive research direction.

Here, we compiled and analyzed the reported crystal structures of various purinergic P2 receptor subtypes. This section elucidated the intricate structural variations among purinergic P2 receptor subtypes, offering a perspective that enhances the development of targeted agents and deepens understanding of receptor functionality across physiological conditions. Over 15 purinergic P2 receptors, categorized into two signal transduction families, mediate diverse signal transduction. P2X receptor subtypes, such as P2X 1 R-P2X 7 R, form trimeric ion channel structures with both homomeric and heteromeric assemblies (P2X 1/2 , P2X 1/4 , P2X 1/5 , P2X 2/3 , P2X 2/5 , P2X 2/6 , and P2X 4/6 ). These receptors feature two transmembrane domains, intracellular termini, and a large extracellular ligand-binding loop specifically activated by ATP ( Fig. 3 A). The P2XRs, metaphorically described as a dolphin emerging from water, exhibit distinct domains: head, dorsal fin (DF), left flipper (LF), right flipper (RF), body, and fluke. These receptors are characterized by intricate inter- and intra-subunit interactions, delineated in a threefold structure. The ATP binding sites are constituted by the amalgamation of the head and LF domains from one subunit with the DF and upper body domains of an adjacent subunit, culminating in a trio of symmetrically aligned ATP binding sites. The receptor's core, formed by the intertwined body domains of the three subunits, is encircled by these ATP sites and bifurcated into upper and lower segments, analogous to the dolphin's body. The upper segment ensures receptor stability, while the lower segment conveys ATP-induced conformational shifts from the extracellular to the transmembrane (TM) domain ( Fig. 4 A). Distinct sites within the various essential residues of P2XRs lead to different enzyme inhibition conditions. These key sites offer a promising strategy for discovering updates on recent work that has identified the molecular basis of their properties. Fig. 3 The general of purinergic P2 receptors. (A) The general structures of P2X receptors consists of subunits composed of two transmembrane domains. In addition, the crystal structure is bound to ATP, with red ribbons representing α-helices. (B) The P2Y receptors consist of seven transmembrane segments. These segments are interconnected by three extracellular loops and three intracellular loops. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Fig. 4 The architecture and orthosteric ligand-binding sites of the major conformational states of P2X 3 R. (A) The P2X receptors subunit has a dolphin-like shape. The open state (B, J), desensitized state (C, K), and apo state (D) in the gating cycle are presented. The close-up view of the binding pocket reveals significant interactions involving ATP(E), TNP-ATP(F), and A-317491(G). (H) The crystal structure of h P2X 3 in complex with AF-219. (I) The interactions between AF-219 and h P2X 3 . (L) The overall structure of h P2X 3 MFCslow with ATP and Mg 2+ bound. (M) A closer examination reveals the binding site for Mg 2+ -ATP in h P2X 3 MFCslow (N) A closer examination reveals the binding site for Ca 2+ -ATP in h P2X 3 MFCslow. Each subunit's structural frame is represented as a cartoon in a distinct color. Oxygen atoms are highlighted in red, while nitrogen atoms are depicted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The general of purinergic P2 receptors. (A) The general structures of P2X receptors consists of subunits composed of two transmembrane domains. In addition, the crystal structure is bound to ATP, with red ribbons representing α-helices. (B) The P2Y receptors consist of seven transmembrane segments. These segments are interconnected by three extracellular loops and three intracellular loops. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The architecture and orthosteric ligand-binding sites of the major conformational states of P2X 3 R. (A) The P2X receptors subunit has a dolphin-like shape. The open state (B, J), desensitized state (C, K), and apo state (D) in the gating cycle are presented. The close-up view of the binding pocket reveals significant interactions involving ATP(E), TNP-ATP(F), and A-317491(G). (H) The crystal structure of h P2X 3 in complex with AF-219. (I) The interactions between AF-219 and h P2X 3 . (L) The overall structure of h P2X 3 MFCslow with ATP and Mg 2+ bound. (M) A closer examination reveals the binding site for Mg 2+ -ATP in h P2X 3 MFCslow (N) A closer examination reveals the binding site for Ca 2+ -ATP in h P2X 3 MFCslow. Each subunit's structural frame is represented as a cartoon in a distinct color. Oxygen atoms are highlighted in red, while nitrogen atoms are depicted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The P2YRs family includes eight members: P2Y 1, 2, 4, 6, 11-14 R. High-resolution studies, particularly on P2Y 1 R and P2Y 12 R, have shed light on their architecture. Structurally, each P2Y receptor consists of seven hydrophobic transmembrane regions linked by three extracellular loops (ELs) and three intracellular loops ( Fig. 3 B). The crystal structure of the P2X 3 R in its changes from apo to holo to desensitized states and the structural role of the intracellular residues in P2X receptor gating has been reported by Steven E. Mansoor et al . The P2X 3 R possesses ATP in its ligand-binding pocket and an open pore in the open state while the desensitized state has a closed pore. The cytoplasmic residues of the open-state structure of h P2X 3 were truncated into the form of a ‘cytoplasmic cap’ compared with zf P2X 4 ( Fig. 4 B). The apo structure of presence of an Mg 2+ ion within the head domain. These findings yield valuable insights into the modulation of P2X 3 R [176] . TNP-ATP and A-317491, two types of competitive antagonists, were used in the structural investigation of h P2X 3 . These antagonists and ATP share a ligand-binding pocket that is located between two protomers. ATP, on the other hand, has a U-shaped molecular structure, whereas TNP-ATP and A-317491 have a Y-shaped molecular structure with a “trunk”. Interestingly, antagonists impede binding cleft closure and the necessary conformational modifications for channel opening by penetrating further into the cleft ( Fig. 4 E-G). Jin Wang et al. found a negative allosteric site on the P2X 3 R using the subtype-specific allosteric inhibitor AF-219. With a buried 4-isopropyl-2-methoxybenzenesulfonamide ring and an exposed 2,4-diaminopyrimidine ring outside the binding pocket of the receptor, AF-219 has a unique structure. This allosteric region is composed of hydrophobic amino acids, including Val61, Leu191, Lys176, and Val238, which plays a vital role in AF-219′s binding and modulation. Furthermore, AF-219 establishes critical interactions with the receptor, including hydrogen bonds with Asn190 and Lys176, along with hydrophobic contacts involving Val61, Val238, and Leu265. These results offer important information on the process by which AF-219 modifies P2X 3 R ( Fig. 4 H-I) [177] . Mufeng Li et al. found an important acidic chamber next to the nucleotide-binding pocket, which is a noteworthy finding regarding h P2X 3 receptor. This chamber exhibits a capacity to hold divalent ions in two different configurations. It hosts divalent ions with the side chain pointed toward Glu109 when there is no nucleotide present. But when ATP is present, the chamber changes to accommodate divalent ions, with the side chain oriented in the direction of the ATP phosphate group. These results shed light on how the acidic chamber is dynamic and adapts to the presence or lack of nucleotide ligands while holding divalent ions ( Fig. 4 L-N) [178] . Toshimitsu Kawate et al. revealed the crystal structures of the closed, resting zebrafish P2X 4 R in 2009. Notably, the extracellular domain appears corrugated when viewed perpendicular to the crystallographic three-fold axis of symmetry. On the other hand, when viewed parallel to this axis, it takes on the shape of an equilateral triangle ( Fig. 5 A-B) [179] . Fig. 5 The architecture and orthosteric ligand-binding sites of the major conformational states of P2X 4 R and P2X 7 R . (A) A stereogram of the homotrimer P2X 4 viewed parallel to the membrane. Each subunit is represented by a different color. (B) Another stereogram of the homotrimer P2X 4 is viewed parallel to the triplet axis of the molecule. (C) Analysis of conformational difference between the apo state (left) and the ATP-bounded state (right) of P2X 4 R. (D) The crystal structure of P2X 4 R in complex with ATP. (E) Close-up views of the ATP binding sites. (F) Close-up views of the CTP binding sites. (G) Cartoon representation of the pdP2X 7 structure in TNP-ATP binding state. (H) The side view displays the binding sites of A804598 in the upper body domains, including the ATP-binding pockets. (I) The top view of the apo pdP2X 7 structure shows the ATP-binding pockets (indicated by red dashed lines) and the drug-binding pockets (indicated by a green dashed line). (J) The structure shows a close-up view of the TNP-ATP binding sites. In the structure, the TNP-ATP and amino acid residues are represented by stick models. (K) The coordination of P2X 7 specific antagonists is depicted as follows: Stick representations of the indicated P2X 7 specific antagonists (green) and the binding residues in the drug-binding pocket are shown. (L) The structure shows a close-up view of the ATP binding sites. (M−N) The binding pocket for rP2X 7 in the open state (M) is depicted alongside h P2X 3 in the open state (N) using surface representation. The binding pocket of rP2X 7 features a narrow channel (<11 Å orifice) that shields the pocket from solvent, thereby limiting ligand access. Conversely, the binding pocket of h P2X3 is significantly more solvent exposed (17 Å orifice). Each subunit's structural frame is represented as a cartoon in a distinct color. Oxygen atoms are highlighted in red, while nitrogen atoms are depicted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The architecture and orthosteric ligand-binding sites of the major conformational states of P2X 4 R and P2X 7 R . (A) A stereogram of the homotrimer P2X 4 viewed parallel to the membrane. Each subunit is represented by a different color. (B) Another stereogram of the homotrimer P2X 4 is viewed parallel to the triplet axis of the molecule. (C) Analysis of conformational difference between the apo state (left) and the ATP-bounded state (right) of P2X 4 R. (D) The crystal structure of P2X 4 R in complex with ATP. (E) Close-up views of the ATP binding sites. (F) Close-up views of the CTP binding sites. (G) Cartoon representation of the pdP2X 7 structure in TNP-ATP binding state. (H) The side view displays the binding sites of A804598 in the upper body domains, including the ATP-binding pockets. (I) The top view of the apo pdP2X 7 structure shows the ATP-binding pockets (indicated by red dashed lines) and the drug-binding pockets (indicated by a green dashed line). (J) The structure shows a close-up view of the TNP-ATP binding sites. In the structure, the TNP-ATP and amino acid residues are represented by stick models. (K) The coordination of P2X 7 specific antagonists is depicted as follows: Stick representations of the indicated P2X 7 specific antagonists (green) and the binding residues in the drug-binding pocket are shown. (L) The structure shows a close-up view of the ATP binding sites. (M−N) The binding pocket for rP2X 7 in the open state (M) is depicted alongside h P2X 3 in the open state (N) using surface representation. The binding pocket of rP2X 7 features a narrow channel (<11 Å orifice) that shields the pocket from solvent, thereby limiting ligand access. Conversely, the binding pocket of h P2X3 is significantly more solvent exposed (17 Å orifice). Each subunit's structural frame is represented as a cartoon in a distinct color. Oxygen atoms are highlighted in red, while nitrogen atoms are depicted in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) In 2012, Motoyuki Hattori et al. conducted a significant investigation in which they elucidated the crystal structure of the zebrafish P2X 4 R complexed with ATP, alongside a novel apo structure. The apo state of this trimeric receptor closely resembles the ATP-bound state in the upper domain; however, it undergoes outward flexing upon ATP binding. Within the ATP-binding pocket, the adenine base forms crucial hydrogen bond interactions with specific residues, namely Thr189, Lys70, and Thr189, located in the lower domain. Furthermore, the establishment of a U-shaped conformation in the P2X 4 R complex with ATP is facilitated by salt bridge and hydrogen bonding interactions involving phosphate groups. These findings provide valuable insights into the intricate structural dynamics and interactions that underlie the functionality of the P2X 4 R and its response to ATP binding ( Fig. 5 E). The crystal structure of P2X 4 R bound with the low-affinity agonist CTP was established in a work of Go Kasuya et al. . Recognition of these phosphate groups involves certain amino acids: Asn296, Arg298, Lys316, Lys70, and Lys72. This pattern of interaction is similar to that found in the ATP-bound states ( Fig. 5 F). The discovery of a hydrogen bond between the cytosine base of CTP and Arg143, a trait not anticipated in earlier models based on the ATP-bound structure, however, represents a notable departure ( Fig. 5 E-F). This work offers new structural insights into the mechanisms controlling nucleotide base specificity in P2XRs and modifies the previously suggested model [180] . By providing crystal structures of this mammalian receptor complexed with five structurally dissimilar antagonists, Akira Karasawa et al. improved our understanding of P2X 7 R. Interestingly, all of these antagonists target the same binding pocket, which is located next to the ATP-binding site. Although having identical hydrophobic characteristics to the P2X 7 R, A804598, the smallest P2X 7 R antagonist, cannot fit into the analogous pocket of the P2X 4 R due to its narrowness. This suggests that the size of the hydrophobic pocket between subunits plays a crucial role in determining the specificity of inhibitors ( Fig. 5 I-K) [181] . The crystal structure of the chicken P2X 7 R associated with the antagonist TNP-ATP has been established by Go Kasuya et al. . The adenine ring adopts a similar orientation to that observed in previously published ATP-bound P2X structures, resulting in hydrogen interactions with the side chains of highly conserved ck Thr177 and the main chain carbonyl groups of ck Thr64 and ck Thr177. TNP-ATP also establishes an additional hydrogen bond with ck Thr64′s side chain. Because of the different orientation of the adenine ring, it interacts with the side chain of h Thr172 ( ck Thr177) and the main chain carbonyl group of h Lys63 ( ck Thr64) but not with the main chain carbonyl group of h Thr172 ( ck Thr177) ( Fig. 4 F and 5J). P2X 7 R's distinct functional characteristics were thoroughly investigated by Alanna E. McCarthy et al. , with special attention paid to the protein's cytoplasmic domain. The crystal structures of rat P2X 7 R in both its apo and ATP-bound forms were solved as a result of their research. A comparative study of these apo state structures revealed a notable difference in the r P2X 7 R binding pocket entrance. It has a tight entry that protects the pocket from the surrounding solvent, in contrast to h P2X 3 , where the pocket is more exposed to solvent. This structural change may result in a shorter period for ATP binding, which could lead to decreased affinity, along with possible protein flexibility controlling pocket access ( Fig. 5 M-N) [182] , [183] . By revealing the crystal structures of h P2Y 1 R complexed with two different antagonists—BPTU and MRS2500— Dandan Zhang et al. have contributed significant insights. In the instance of MRS2500, a network of hydrogen bonds and hydrophobic contacts allow its adenine ring to interact with Arg287, Leu44, and Asn283 in vital ways. Of particular interest is the close fit of MRS2500′s 2-iodo group into a pocket formed by P2Y 1 R's N terminus, which permits interactions with Cys42. Furthermore, the 6 N-methyl group of MRS2500 creates connections with Ala286 and Asn299, whereas the (N)-methanocarba ring contacts Tyr203 situated in EL2. When nucleotide-like antagonists create high-affinity contacts with P2Y 1 R, their phosphate groups play a crucial role. For example, the 3′-phosphate group interacts via salt-bridge interactions with Lys46 and Arg195 and generates hydrogen bonds with Tyr110 and Tyr303. Likewise, the 5′-phosphate group forms hydrogen connections with Thr205 and a crucial salt-bridge interaction with Arg310. These interactions work together to facilitate the antagonist's binding and P2Y 1 R inhibition ( Fig. 6 B). Fig. 6 The architecture and orthosteric ligand-binding sites of the major conformational states of P2Y 1 R and P2Y 12 R. (A-D) Structural depiction of P2Y 1 R complexes and ligand-binding pockets. The side view of the P2Y 1 R-MRS2500 complex and the P2Y 1 R-BPTU complex. The ligand-binding pockets of P2Y 1 R for MRS2500 and BPTU are depicted in the image. (E-H) Structural depiction of P2Y 12 R complexes and ligand-binding pockets. The structures of the P2Y 12 R-2MeSADP and P2Y 12 R-2MeSATP complexes are depicted in this illustration. Additionally, a comparison between the binding poses of 2MeSADP and AZD1283 in the overlaid P2Y 12 R complexes is presented. (I) The structure of the P2Y 12 R-Selatogrel is depicted in this illustration. The receptor is represented in a blue color using a cartoon format, while the ligands are visualized as spheres with green carbons. In these figures, the receptors are depicted in blue using a cartoon representation. The ligands MRS2500 and BPTU are represented as spheres with green carbons. Additionally, specific elements are color-coded: oxygen (red), nitrogen (dark blue), sulfur (yellow), phosphorus (orange), and iodine (purple). Salt bridges are illustrated as red dashed lines, while hydrogen bonds are indicated by blue dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The architecture and orthosteric ligand-binding sites of the major conformational states of P2Y 1 R and P2Y 12 R. (A-D) Structural depiction of P2Y 1 R complexes and ligand-binding pockets. The side view of the P2Y 1 R-MRS2500 complex and the P2Y 1 R-BPTU complex. The ligand-binding pockets of P2Y 1 R for MRS2500 and BPTU are depicted in the image. (E-H) Structural depiction of P2Y 12 R complexes and ligand-binding pockets. The structures of the P2Y 12 R-2MeSADP and P2Y 12 R-2MeSATP complexes are depicted in this illustration. Additionally, a comparison between the binding poses of 2MeSADP and AZD1283 in the overlaid P2Y 12 R complexes is presented. (I) The structure of the P2Y 12 R-Selatogrel is depicted in this illustration. The receptor is represented in a blue color using a cartoon format, while the ligands are visualized as spheres with green carbons. In these figures, the receptors are depicted in blue using a cartoon representation. The ligands MRS2500 and BPTU are represented as spheres with green carbons. Additionally, specific elements are color-coded: oxygen (red), nitrogen (dark blue), sulfur (yellow), phosphorus (orange), and iodine (purple). Salt bridges are illustrated as red dashed lines, while hydrogen bonds are indicated by blue dashed lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) The P2Y 1 R-BPTU crystal structure provides important information about the binding mechanism of BPTU within the lipidic interface of P2Y 1 R's transmembrane domain. Interestingly, the antagonist securely anchors Leu102 within the binding region of the receptor by forming two hydrogen bonds between the nitrogen atoms of its urea group and the main chain of Leu102. Furthermore, BPTU's pyridyl ring interacts hydrophobically with Ala106 and Phe119, increasing its affinity for P2Y 1 R. In addition, the phenoxy group of benzene ring is positioned in a cavity created by helices in a way that interacts with important residues such as Thr103, Met123, and Leu126, which in turn enhances the stability of the entire complex ( Fig. 6 D) [184] . Important information about the structures of h P2Y 12 R when attached to two compounds—the ATP derivative 2MeSATP and 2MeSADP, a close analogue of endogenous agonist ADP—has been supplied by Jin Zhang et al. When it comes to 2MeSADP, interactions with Tyr105 are formed by its adenine group occupying the same binding site as the nicotinate group in the antagonist AZD1283. Additionally, the 2-thioether of 2MeSADP snugly inserts into a hydrophobic pocket comprised of Asn159, Leu155, Ser156, and Phe106. This greater complementarity results in a higher affinity of 2MeSADP compared to ADP ( Fig. 6 F-H). Positively charged and hydrophilic residues interact with the diphosphate group of 2MeSADP in the receptor. Cys97 establishes a hydrogen bond with the ribose 3′hydroxyl group, while Cys175 interacts with the β-phosphate group, demonstrating the accuracy of these interactions ( Fig. 6 F-H) [185] . The crystal structure of the P2Y 12 R bound to selatogrel, an inverse agonist, was analyzed by V́eronique Pons et al. to further understand its binding mode. The receptor conformation in complex with selatogrel was found to be highly similar to that of AZD1283, indicating that the receptor was clearly in the antagonized conformation ( Fig. 6 I). Selatogrel binds to the same pocket as AZD1283. The positioning of two moieties of selatogrel showed highly similar binding compared to AZD1283. This section provided an in-depth summary of the compounds that have been reported to target different purinergic P2 receptor subtypes, outlining their structural properties and the results of both in vitro and in vivo research. Through this work, we aimed to contribute to a better understanding of purinergic P2 receptor pharmacology and encourage continued innovation in this field of drug research. P2X receptors are a kind of ATP-gated ionotropic receptor. As a more powerful P2X receptor agonist, BzATP ( 1 ) is usually utilized instead of ATP, while it is practically inert at the rat P2X 4 R. In addition, selective agonists targeting specific P2X subtypes have not yet been reported [186] . Notably, BzATP finds clinical application in the treatment of sepsis-induced intestinal barrier disruption. The synthesis of 15 sulfonamide tethered (hetero)aryl ethylidenes derivatives utilizing a simple three-step technique has been established by Jamshed Iqbal et al. [187] . 2 was discovered to be the most effective and selective inhibitor of P2X 2 R (IC 50  = 0.32 ± 0.01 μM) and P2X 7 R (IC 50  = 1.10 ± 0.21 μM) in calcium signaling tests [188] ( Table S1 ). Furthermore, by investigating the mechanism by which 2 inhibits P2X 2 R, 2 possessed negative allostery. Gefapixant ( 3 ), an orally administered powerful and selective allosteric P2X 3 R antagonist, is anticipated to be the first pharmaceutical to be licensed for the treatment of refractory chronic cough. It has successfully passed a phase III clinical trial [189] . A phase Ⅱa clinical trial using the powerful and specific P2X 3 R antagonist elipixant ( 4 ) was completed to treat refractory persistent cough [190] . Clinical trials are currently being conducted to address ailments like endometriosis, overactive bladder, and neuropathic pain. TNP-ATP ( 5 ) and A-317491 ( 6 ) are two early reported P2X 3 R antagonists. TNP-ATP, a fluorescent ATP analog, has been shown to be an effective nanomolar antagonist of rat P2X 1 R, P2X 3 R and heteromultimeric combination of P2X 2 R and P2X 3 R [191] ( Table S1 ). In rats, A-317491, an effective and specific non-nucleotide antagonist of P2X 3 R, decreases neuropathic pain and chronic inflammation. However it exhibits limited therapeutic value due to its low oral and central nervous system bioavailability, along with significant plasma protein binding [192] . 7 and 8 were identified as selective inhibitors for P2X 7 R, which were reported together with 2 [187] . The amino group of 2 was replaced by hydrogen or bromine atoms to obtain 7 (IC 50  = 4.21 μM) and 8 (IC 50  = 5.54 μM). Due to their selectivities toward other subtypes were better, the clinical applications of 7 and 8 extend to the treatment of neuropathic pain and neurodegenerative disorders. A series of adamantyl cyanoguanidine compounds constructed from hybrids of the adamantyl amide scaffold identified by AstraZeneca ( 9 ) and cyanoguanidine scaffold identified by Abbott Laboratories ( 10 ) were synthesized to investigate inhibitory activity against P2X 7 R by Michael Kassiou et al. [193] . It was shown that the adamantyl moiety of P2X 7 R antagonists functions as an efficient bioisostere for the hydrophobic aryl group of the cyanoguanidine scaffold. 11 displayed five-fold greater inhibitory potency than the lead 10 in interleukin-1β release assays (IC 50  = 2 nM). The aryl group utilizes the direct binding of the guanidino nitrogen to benzene derivatives, while a methylene linker is beneficial for heteroaromatic analogues. In forced-swim test, 12 with pyridine group revealed the capacity to act centrally and produce an anti-depressant phenotype. These highly potent adamantyl-cyanoguanidine compounds indicate significant for developing an effective, CNS-penetranting P2X 7 R antagonist, as evidenced by positive in vivo outcomes. Adamantane acylbenzamide 13 was obtained by AstraZeneca through high-throughput screening. Despite its compromised metabolic stability, it holds promise for the treatment of chronic inflammatory diseases in clinical applications [194] . A number of derivatives were synthesized by using 13 as the lead [195] ( Table S1 ). By installing bioisosteric fluorine on the adamantane bridgeheads, Michael Kassiou et al. worked to develop a range of bioisosteres with improved physical properties and metabolic stability. Trifluorinated benzamide ( 14 ), for example, showed robust P2X 7 R inhibitor activity, a significant decrease in lipophilicity, and enhanced PK characteristics, and was effective in various polymorphs of the P2X 7 R. Furthermore, different P2YRs are activated by different nucleotides. P2Y 1,12,13 receptors are activated only by ADP, while P2Y 11 R is only activated by ATP. P2Y 4 R and P2Y 6 R are pyrimidine nucleotide receptors, which activated by UTP and UDP, respectively. P2Y 2 R is activated by both ATP and UTP. P2Y 14 R is respond to UDP, UDP-glucose and other UDP-sugers [196] . In recent years, drugs targeting P2YRs have been rapidly developed. Hong Liu et al. identified the 2-(phenoxyaryl)-3-urea derivatives with good in vitro potency against P2Y 1 R in low micromolar range. The best antagonistic potency was shown by 15 (IC 50  = 0.21 μM). Additionally, 15 also shows potential for application in antiplatelet therapy. Molecular docking simulation indicated that 15 shown a similar binding mode as the positive control BPTU [197] . As a selective, high-affinity, reversible antagonist of P2Y 2 R, 16 (AR-C118925) was shown by Muller et al. to be an effective pharmacological tool [198] . Stocks et al. developed and assessed the initial fluorescent antagonists of the P2Y 2 R based on 16 [199] . Fluorophores and slightly altered 16 were coupled to create fluorescent ligands. 17  were shown to have functional antagonist activity in Ca 2+ -mobilization tests, and the bioluminescence-energy-transfer assay revealed adequate affinity for h P2Y 2 R (pK d  = 6.32). 17 ′s superior imaging capabilities make it an ideal instrument for examining the composition and arrangement of h P2Y 2 R. Diquafosol tetrasodium( 18 ) is a potent selective agonist of the P2Y 2 R discovered by Inspire Pharmaceuticals and commercialization in Asian countries for the treatment of Xerophthalmia [200] ( Table S1 ). To date, with the efforts of Pengfei Xu et al. , diquafosol tetrasodium has made a significant breakthrough in total yield and the improved conditions are more suitable for large scale production [201] . Reactive Blue 2 ( 19 ) is a well-known non-selective P2YR antagonist [202] ( Table S1 ). In order to create a library of RB-2-related anthraquinone compounds, Younis Baqi et al. successfully optimized them as P2Y 4 R-selective antagonists [203] . Among them, 20 (IC 50  = 233 nM) is the most effective noncompetitive P2Y 4 R antagonist that has been found thus far. Therefore, it is hoped that 20 will be effective research tools for analyzing P2Y 4 R. Our group has essentially provided an overview of recent developments in the identification of P2Y 6 R antagonists [204] . There are very few known P2Y 6 R antagonists with constrained chemical structure designs. Among these, diisothiocyanate derivative MRS2578 ( 21 ) reported by Jacobson et al. showed potent inhibiting activity against P2Y 6 R (IC 50  = 37 nM) [205] . The electrophilic isothiocyanate group of MRS2578 is critical to antagonistic activity. Isothiocyanate groups covalently modified P2Y 6 R at Cys220, which promotes the ubiquitylation of Lys137 [206] . The non-substitutability of the isothiocyanate group limits the expansion of derivatives with MRS2578 as a scaffold. In 2017, TIM-38 ( 22 , IC 50  = 4.3 μM), a novel and efficient P2Y 6 R inhibitor, was discovered using high-throughput screening on chemical libraries including 141 700 compounds [207] . Without altering the reaction brought on by four additional h P2Y or muscarinic receptors, TIM-38 blocked P2Y 6 R and dose-dependently suppressed the increase of [Ca 2+ ] caused on by UDP. Moreover, TIM-38 has also been identified for its potential in treating ulcerative colitis and acute lung conditions, showcasing its therapeutic versatility [208] , [209] . Our group identified and synthesized the novel P2Y 6 R-specific antagonist 23 (IC 50  = 5.914 nM) by combining chemical optimization method with SBVS to overcome the drawbacks of the previously mentioned compounds. The direct binding of 23 to P2Y 6 R and its promising binding affinity were confirmed by chemical pull-down studies and binding assays. Significantly, in models of acute lung injury caused by lipopolysaccharide and ulcerative colitis induced by DSS, respectively, 23 demonstrated outstanding in vivo therapeutic efficacy. These results imply that 23 merits more optimization research and may be used as a particular P2Y 6 R antagonist for the treatment of inflammatory illnesses [102] . UTP has been proven to be potent in the activation of P2Y 6 R, and showed considerable non-selectivity in the UDP and UTP analogues [210] . The way to improve P2YR selectivity is to introduce conformational constraints in the nucleotide ligands, such as the replacement of the ribose ring by a bicyclo[3.1.0]hexane (methanocarba) ring system [211] . According to the location of the cyclopropyl ring fusion, Jacobson et al. discovered two isomeric forms of this modification in this work, which contain 24 (EC 50  = 6.90 μM) and 25 (EC 50  = 0.705 μM), allowing the ribose ring to maintain a distinct conformation. As UDP is susceptible to hydrolysis by exonucleases, they found through further research that 26 (EC 50  = 0.57 nM) has the highest potency among the substituents of UDP/CDP analogues containing a stabilizing α, β-methylene bridge and has been identified are highly selective over the P2Y 14 R as P2Y 6 R [212] . In terms of thrombus formation, P2Y 12 R decreases intracellular adenylate cyclase activity and prolongs intracellular calcium signal transduction, which amplifies platelet activation and aggregation [213] . Since P2Y 12 R is mainly concentratedly expressed in platelets, P2Y 12 R has been proved to be an excellent therapeutic target for antithrombotic drugs, as a result of a variety of clinical drugs on the market [214] , [215] . The first P2Y 12 R antagonist to be marketed was ticlodipine ( 27 ), which was launched as an antithrombotic drug in 1978. Unfortunately, the benefit-to-risk ratio of ticlodipine for patients was low, so clopidogrel ( 28 ), which was introduced as a back-up regimen in 1998, had a much better safety profile. Clopidogrel was first tested as a racemate, but its antithrombotic activity was later discovered to be present in the S-enantiomer. Such inhibitors require conversion to the active metabolite ( 29 ) for irreversible binding to the receptor. Clopidogrel is superior to ticlopidine in not causing neutropenia, however, some patients may experience insufficient activity, individual variability, and a delayed onset of effect. The introduction of a third thienopyridine, rasugrel ( 30 ), was launched in 2009, which has better activity than clopidogrel, with a faster onset, longer duration, and higher bioavailability. The only drawback to that it increases bleeding risk [216] , [217] . To overcome the side effects of the above-mentioned compounds, ticagrelor ( 31 ) was authorized by the European Medical Agency in 2010 as a ATP analogue P2Y 12 R antagonist. It binds to the receptor and prevents ADP signaling as well as any changes in the receptor's structure. Furthermore, ticagrelor has a higher bioavailability and less individual variation than clopidogrel, making it a superior option for treating individuals with acute coronary syndromes. The most recent, cangrelor ( 32 ), was authorized in 2015 as a genuine P2Y 12 R antagonist of the ATP analogue. It is the only intravenously authorized medication for percutaneous coronary intervention because of its quick start of action [216] , [217] . Active ingredient molecule of Traditional Chinese Medicines is becoming a popular direction in drug discovery. Xuyang Liu et al . reported that salvianolic acid A and C ( 33,34 ) could antagonize both P2Y 1 R and P2Y 12 R activity, while salvianolic acid B ( 35 ) could antagonize the P2Y 12 R activity. This differential receptor activity profiles 33 and 34 for broader cardiovascular applications due to their dual receptor antagonism, while 35 is positioned for targeted antiplatelet therapy [218] . 36 was reported by Eva Caroff’s research group in 2015, which was discovered to be a reversible and highly selective P2Y 12 R antagonist in vitro , with potency in human plasma at nanomolar doses. 37 inhibited thrombosis in a dose-dependent manner in the rat ferric chloride model, with a wide therapeutic window. As a result, they created a bis((isopropoxycarbonyl)oxy)methyl ester prodrug ( 37 ) that displayed antithrombotic properties in a rat ferric chloride model following oral treatment. Both 36 and 37 were tested in healthy individuals in clinical trials [219] . Since ticagrelor can cause shortness of breath and various types of bleeding, in addition, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and should be used with caution when combined with powerful CYP3A4 inducers/inhibitors. Deyu Kong et al. reported 38 as a novel morpholine partial scaffold compound that is pharmacologically distinct from ticagrelor. It antagonizes P2Y 12 R in a reversible manner, showcasing its role as an antiplatelet agent. It was reported that new skeleton P2Y 12 R antagonists, which contained tetrahydrothieno [3,2-c] pyridine analogs with numerous alicyclic and aromatic amines, was designed by Sujit B. Bhalekara et al. in 2019. 39 and 40 demonstrated significant antiplatelet activity that was superior to currently available medicines including aspirin and prasugrel. This finding may lead to a fresh perspective for future pharmacophores based on a straightforward design technique that avoids the arduous synthesis of clopidogrel and prasugrel [220] . AZD1283 ( 41 ) was reported by AstraZeneca as an oral, reversible P2Y 12 R antagonist, which demonstrated potent antithrombotic and hemorrhage reducing effects in animal models, and has entered human clinical trials. Since its poor absorption and low metabolic stability, AZD1283′s development ended before the Phase II study. A series of bicyclic pyridine analogues were developed and synthesized by Deyu Kong et al. to improve the metabolic stability of AZD1283. The ethyl nicotinate part of AZD1283 was replaced by nicotinic acid lactone, lactam, and cyclic ketone, and 42 displayed the most efficient antagonistic activity against human platelet-rich plasma (IC 50  = 2.94 μM). Compared to clopidogrel, 42 exhibited potent, dose-dependent inhibition of platelet aggregation in vivo , and reduced bleeding time and blood mass [221] , [222] . WSJ-557 ( 43 ) was reported as a nonpurine xanthine oxidase (XO) inhibitor in previous study by Yanhua Mou et al . Following that, an antiplatelet aggregation experiment generated by ADP revealed that 43 has antiplatelet activity (IC 50  = 15.727 μM). Through the structure–activity relationship study of 2-phenyl 1H-imidazole compounds, they turned XO inhibitor WSJ557 into dual-target antagonists of P2Y 1 R and P2Y 12 R. In an in vitro ADP-induced rabbit platelet-rich plasma aggregation assay, 44 and 45 (IC 50  = 4.237 and 3.875 μM, respectively) were identified as the most promising antiplatelet agents ( Table S1 ). The experiments flawlessly transitioned from a nonpurine imidazole XO inhibitor to dual-target P2Y 1 R and P2Y 12 R antagonists, implying that other nonpurine XO inhibitors with a chemical structure similar to WSJ-557 could be used to develop novel effective antagonists [223] . Further modification found that 46 (PPTN) has a good affinity for P2Y 14 R (Ki = 1.9 nM), and in the presence of 2 % HSA, it has a low change (Ki = 35 nM). However, the oral bioavailability of 46 is greatly affected (F = 5 %), which may be caused by the characteristics of zwitterionic polarity. Therefore, the acid moiety was modified with ester prodrug to obtain 47 . The pharmacokinetic features were markedly improved (F = 67 %), as contrasted with 46 . The research revealed that 48 which substituted the piperidine group of PPTN by N-acetyl had equivalent P2Y 14 R affinities in its binding assay ( h P2Y 14 R IC 50  = 27.6 nM, m P2Y 14 R IC 50  = 29.7 nM). Furthermore, 46 ′s bicyclic substituted derivative 49 of the piperidine group likewise maintains affinity ( h P2Y 14 R IC 50  = 20 nM), while 50 ′s PEG conjugate water solubility is improved 20 times greater than that of 46 . These modifications not only enhance the pharmacokinetic profiles but also underscore their potential clinical application targeting the P2Y 14 R to modulate platelet aggregation [224] . According to Jacobson et al. latest research, a zwitterion was nonessential for receptor binding, which was probed through docking and molecular dynamics simulation. 51 which replaced the piperidine ring of PPTN by 5-(hydroxymethyl)isoxazol-3-yl showed high affinity at both h P2Y 14 R (IC 50  = 15 nM) and m P2Y 14 R (IC 50  = 18.6 nM). 52 , the amidomethyl ester prodrug of 51 , showed a significant effect in the protease mouse model of allergic asthma, which is better than the parent drug. Therefore, searching for piperidine bioisosteres, converting zwitterionic antagonists into neutral molecules, and applying a prodrug strategy are further research direction based on scaffold of PPTN to seek novel and potent P2Y 14 R antagonists [225] , [226] . Not long ago our research group reported a new scaffold, which 2-naphthalic acid of PPTN was substituted by 3-amide benzoic acid. The most promising antagonist compound 53 (IC 50  = 1.77 ± 0.256 nM) down regulated the expression of NLRP3, Caspase1 and ACS protein significantly through flow cytometry. The water solubility of 53 was improved comparing to PPTN. However, 53 had a poor bioavailability (F = 6 %) owing to its zwitterionic character. According to the reported research, in order to maintain the activity, the carboxyl group is necessary. In addition, in the previous SAR studies, it is expected that no charged amino group is required, and the 5- and 6-membered ring that connect the nucleus on the circulation side outside the recipient cell is also effective. Therefore, phenyl or bicyclic substituted phenylpyridines have been introduced to improve pharmacokinetic properties. 54 (IC 50  = 2.18 ± 0.42 nM), aimed at treating acute gouty arthritis, which replaced the 4-piperidine phenyl by 2-furanyl, showed intermediate bioavailability (F = 48 %) in rats and exceptional microsomal stability in human microsomes [227] , [228] . Our research group has also done some work on P2Y 14 R antagonists that we adopted Glide docking-based virtual screening strategy for finding potent P2Y 14 R antagonists using two well-established P2Y 14 R homology models, and 19 compounds were hit. After the relevant pharmacological evaluation test, 55 (IC 50  = 2.46 nM) was identified as the best antagonist of this series. The skeleton of 55 is different from all the above-mentioned reported compounds, which provides a new skeleton for future research of P2Y 14 R antagonists [19] . By combining known P2Y 14 R antagonists with the advantageous fragments of the latest virtual screening compounds, we continued to report a series of P2Y 14 R antagonists with a novel scaffold of 2-phenyl-benzoxazole structure. Effective P2Y 14 R antagonistic activity and a greater inhibitory effect on MSU-induced inflammatory responses were demonstrated by the optimized 56 in vitro . It also demonstrated outstanding therapeutic effects in reducing inflammatory responses and demonstrated higher potential P2Y 14 R inhibitory activities in vivo [19] . Numerous clinical illnesses are influenced in their course by signaling mediated by purinergic P2 receptors. Developing effective and safe pharmacological ligands for this purpose offers significant therapeutic potential, yet it is a challenging endeavor. Several analogues with high affinity and selectivity have been produced by significant research in medicinal chemistry. Therefore, the scarcity of selective ligands is not the primary issue. The main hurdle lies in addressing the wide expression of purinergic P2 receptors and the redundancy of purinergic P2 signaling. Numerous cells have purinergic P2 receptors, suggesting that a specific kind of receptor is present on target cells implicated in disorders as well as on cells taking part in a variety of healthy processes. That problem becomes worse when indiscriminate agonists activate each receptor rather than just the cells with robust, persistent activation. Tolerance of ligand may occur through been exposed repeatedly, resulting in the desensitization of receptor activation and a reduced signaling response over time. Receptor internalization, reduced expression, or other mechanisms that decrease the overall impact of a particular ligand dosage can all contribute to this. Tolerance has been observed with P2X 3 R agonists [176] . Conversely, using receptor antagonists lowers the potential of tolerance because desensitization would need enough endogenous agonist receptor occupancy. Different consequences on various cell types become evident when considering metabolic disorders. In addition, the progression of metabolic abnormalities is influenced by low-grade inflammation and oxidative stress. Given that purinergic P2 receptors are expressed in distinct metabolic organs and immune cells, the administration of a molecule targeting a specific receptor can lead to multiple and potentially conflicting outcomes. Exciting opportunities for pharmaceutical research in the fields of ion channels, GPCRs, and enzymes are presented by allostery. Among these, extracellular ATP-regulated cation channels known as P2XRs have garnered significant interest as potential drug targets. Although compounds that target P2XRs have made their way into clinical trials for conditions like rheumatoid arthritis, little is known about how these negative allosteric regulation works. Future P2X receptor small-molecule probe drug design will have special options due to the discovery of the negative allosteric site. First off, by using virtual screening and patch clamp recordings in conjunction with this pocket identification, lead compounds with novel structural cores can be found. The approach significantly enhances the likelihood of discovering novel drug candidates. Secondly, the ligand-receptor interactions observed offer crucial insights for further structure-based rational drug design. Lastly, compounds' hydrophobic group is able to modified according to the pocket's layout, leading to the creation of novel small-molecules with high selectivity. These findings are particularly noteworthy as compressing the observed allosteric site constitutes an essential stage in channel gating. It can be interfered with by inserting molecules into this region to effectively inhibit its degeneration. The distinct architectural features of the various P2XR subtypes present an excellent chance to develop subtype-specific allosteric inhibitors [177] . In order to comprehend purinergic P2 receptors, additional molecular probes are required. Stocks et al. presented the discovery of a novel category of non-nucleotide P2Y 2 R antagonists, founded on the previously identified antagonist 16 (AR-C118925). As a result, several fluorescent ligands containing different linkers and fluorophores were found. In a bioluminescence-energy-transfer (BRET) test, ligand 17 among them demonstrated high affinity for h P2Y 2 R (pK d  = 6.32) ( Table S1 ). Furthermore, astrocytoma cells expressing untagged h P2Y 2 R were shown to have displaceable membrane labeling via confocal imaging employing this ligand. Given these special characteristics, ligand 17 is among the foremost options for researching the distribution and organization of h P2Y 2 R. Additionally, those superior imaging characteristics make 17 the appropriate instruments for examining h P2Y 2 R's structure [199] . Alternatively, a prodrug, an inactive form, can be administered to promote more targeted tissue targeting of a purinergic P2 receptor ligand. An enzyme that is selectively expressed then activates this prodrug at the targeted tissue. The most potent compounds in the study were produced by replacing the carboxylic acid functionality with phosphonic acid, which represented a significant development in this strategy. Additionally, for the first time, poor clearance in vivo was achieved in animal models. However, due to the low bioavailability of compound 36 in rats and dogs, they devised a bis((isopropoxycarbonyl)oxy)methyl ester compound 37 ( Table S1 ). Compound 36 showed effectiveness in the rat ferric chloride thrombosis model when given intravenously as the parent chemical or orally via the prodrug 37 . Furthermore, compared to clopidogrel, 36 exhibited a broader therapeutic window in the rat surgical blood loss model [219] . The development of purinergic P2 receptor agonists is marked by the critical importance of specificity, pharmacokinetic optimization, and the management of receptor desensitization. Achieving high specificity is essential to minimize off-target effects and enhance therapeutic efficacy, as illustrated by research into nucleotide analog 18 for P2Y 2 R activation. This work highlights the need for a meticulous approach to selectively target receptor subtypes [201] . Receptor desensitization, where prolonged agonist exposure reduces receptor responsiveness, presents another hurdle. Studies on nucleotide-based P2Y 6 R agonists 24 – 26 emphasize the critical need for agents that maintain their efficacy over time without causing receptor downregulation [212] . Despite these obstacles, advancements are being made through sophisticated methodologies like high-throughput screening, structure-based drug design, and allosteric modulation exploration. These approaches are key in identifying new agonist compounds with enhanced specificity, better pharmacokinetics, and minimal desensitization risks. The development and assessment of pyrimidine nucleotides with methanocarba modifications for P2Y 6 R activation demonstrate the potential benefits of structural modifications [211] . In conclusion, the field is progressively overcoming challenges through a deep understanding of purinergic signaling, receptor biology, and cutting-edge drug discovery techniques. Recent studies not only shed light on the current advancements in agonist development but also highlight the strategic efforts to tackle the major obstacles within this domain, edging closer to realizing the therapeutic potential of purinergic P2 receptor agonists for various diseases.

Purines and their derivatives are key molecules that control intracellular energy metabolism and nucleotide synthesis [1] , [2] . Purinergic receptors activated by purines and their derivatives represent a critical pathway in response to extracellular purine signaling. Based on the differences in endogenous ligands, purinergic receptors are categorized into two groups: purinergic P1 receptors and purinergic P2 receptors [3] , [4] . Specifically, adenosine and adenosine monophosphate active purinergic P1 receptors, a family of G protein-coupled receptors (GPCR), while adenosine triphosphate (ATP) and other nucleotides mediate the function of purinergic P2 receptors including ionotropic P2X receptors (P2X 1-7 R) activated by ATP and metabotropic P2Y receptors (P2Y 1,2,4,6,11-14 R) activated by ATP, uridine triphosphate (UTP), adenosine diphosphate (ADP), uridine diphosphate (UDP) and UDP-glucose [5] , [6] , [7] , [8] . Purinergic P2 receptors are categorized into two signaling families. The P2X receptors are capable of forming trimeric ion channel structures through homomeric and heteromeric assembly, resulting in various subtypes. The P2Y receptors consist of seven hydrophobic transmembrane domains, a hallmark of the GPCR family. There is clear evidence for the widespread distribution of purinergic P2 receptors in human tissues [9] , [10] ( Fig. 1 ). Since being found in the 1970s, purinergic P2 receptors and related signal transduction have been gradually proved to be involved in physiological and pathological processes, playing beneficial or harmful roles in the pathogenesis of diseases [11] , [12] , [13] . In mammals, purinergic P2 receptors dysfunction contributes to the pathologic progression of central and peripheral nervous system disorders such as Alzheimer's disease, anxiety, schizophrenia, and epilepsy [14] , [15] . Further studies have also implicated roles of purinergic P2 receptors in the pathology of other diseases including atherosclerosis, gout, liver fibrosis, and cancer and so on [16] , [17] , [18] , [19] , [20] , [21] . Fig 1 Distribution of purinergic P2 receptors in cells of human tissues. Distribution of purinergic P2 receptors in cells of human tissues. Antagonists targeting P2Y 12 R such as Cangrelor, Clopidogrel, Ticagrelor, and Prasugrel have been approved for the treatment of thrombosis. Gefapixant, a selective P2X 3 R antagonist, has been approved in Japan to treat refractory or unexplained chronic cough [22] . Meanwhile, many drug candidates targeting purinergic P2 receptors are now in clinical study trials [23] , [24] . Purinergic P2 receptors have become attractive therapeutic targets, and a better understanding of their complex pathophysiological roles will help to develop ideal therapeutic agents for related diseases [25] , [26] , [27] . This review mainly summarizes the mechanisms of purinergic P2 receptors in recent years, mainly concerning nervous system, digestive system, immune system, and cancer, moreover, this review also proposes available drug design strategies on the basis of the protein structure of purinergic P2 receptors. The development of novel purinergic P2 receptor modulators, including agonists, antagonists, pharmaceutical compositions and combination strategies, will possibly improve the specificity of drug-receptor interactions and contribute to the development of innovative drugs.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ATP and other nucleotides are mediators of the signaling of the purine system, thereby contributing to brain development and neurological disorders [28] , [29] . In recent years, purinergic P2 receptors have been found to participate in neurons’ growth and survival in the physiological state, which in turn coordinates the communication among nerve cells [28] , [30] . Huang L et al. described in detail that purinergic P2 receptors are involved in several physiological processes including neuronal proliferation, differentiation, and migration [31] , [32] , [33] . Relying on the structural features and classical pathways, more studies have found that neurotransmitters, cytokines, or neurotoxic substances regulate neuronal behavior through purinergic P2 receptors and participate in diseases. Under the condition of P2X 3 R activated by tumor necrosis factor-α, sensory neuron excitability is significantly increased [34] . Regarding the P2X 7 R, it can mediate neuronal apoptosis and injury through the P2X 7 R- nucleotide binding oligomerization domain-like receptor protein 1 (NLRP1) - cysteinyl aspartate specific proteinase 1 (Caspase1) and mitogen-activated protein kinases (MAPKs) signaling pathways [35] , [36] , [37] . More importantly, P2X 7 R is involved in promoting the survival of dopaminergic neurons, which is mainly mediated by the activation of Calmodulin/Ras protein specific guanine nucleotide-releasing factor 1 (RasGRF1), providing a new option for targeted therapy of Parkinson's disease [38] . P2XRs are channel receptors that can mediate the entry and exit of calcium ions (Ca 2+ ), an ion associated with neuronal excitability, into and out of cells. ATP-activated P2X 7 R in sensory neurons promotes vagal physiology that has also been implicated in developing amyotrophic sclerosis [39] , [40] . In cholinergic neurons, P2X 2 R can mediate the entry of choline, which in turn promotes the synthesis of acetylcholine [41] , [42] . In summary, purinergic P2 receptors mediate a variety of neuronal behaviors and may promote the release of neurotransmitters, and through this pathway, are extensively involved in intercellular signaling and neurological disorders ( Fig. 2 A). Fig. 2 Physiological and pathological mechanisms of purinergic P2 receptors. (A) The molecular mechanisms of purinergic P2 receptors in nervous system. (B) The molecular mechanisms of purinergic P2 receptors in digestive system. (C) The molecular mechanisms of purinergic P2 receptors in immune system. (D) The molecular mechanisms of purinergic P2 receptors in cancer. IL-6, interleukin-6; BDNF, brain-derived neurotrophic factor; TrkB, tropomyosin receptor kinase B; SOD1, superoxide dismutase 1; NLRP1, nucleotide binding oligomerization domain-like receptor protein 1; Caspase1, cysteinyl aspartate specific proteinase 1; Ca 2+ , calcium ions; CamKII, almodulin-dependent protein kinase II; RasGRF1, Ras protein specific guanine nucleotide-releasing factor 1; ERK1/2, extracellular regulated protein kinases1/2; p38, mitogen-activated protein kinase 14; CAN, calcineurin; DRP1, dynamin-related protein 1; AMPK, adenosine 5‘-monophosphate (AMP)-activated protein kinase; CatS, cathepsin S; FKN, fractalkine; HIF1α, hypoxia-induced factor-1α; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; IL-1β, interleukin-1β； RhoA, Ras homolog gene family member A; ROCK, Rho-associated coiled-coil-containing protein kinase; JNK, c-Jun N-terminal kinase; IL-33, interleukin-33；PKA, protein kinase A; HMGB1, high mobility group box-1; CREB, cyclic adenosine monophosphate response element binding protein; nSMase, neutral sphingomyelinase; MAPK, mitogen-activated protein kinases; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; PKC, protein kinase C; GSK-3β, hlycogen synthase kinase 3β; STAT2, signal transducer and activator of transcription 2; IRF6, interferon regulatory factor 6; CXCL5, C-X-C motif chemokine ligand 5; NF-κB, nuclear factor kappa-B; STAT1, signal transducer and activator of transcription 1; NFATC2, nuclear factor of activated T-cells 2; NFAT, nuclear factor of activated T-cells; PBX3, Pre-B cell leukemia homeobox 3; BCAT1, branched-chain aminotransferase 1; mTOR, mammalian target of rapamycin; ATF4, activating transcription factor 4; MMP9, matrix metallopeptidase 9; p-ERK1/2, phosphorylation-extracellular regulated protein kinases1/2; TGF-β, transforming growth factor-β; SMAD, mothers against DPP homolog; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; FAK, focal adhesion kinase; MMP2, matrix metallopeptidase 2; GM-CSF, granulocyte–macrophage colony-stimulating factor; VEGF, vascular endothelial-derived growth factor; EGFR, epidermal growth factor receptor; Src, protein kinase Src; RTK, receptor tyrosine kinase; ASC, apoptosis-associated speck-like protein containing CARD; YAP, Yes-associated protein; SERPINE1, serine protease inhibitor clade E member 1; MMP1, matrix metallopeptidase 1；CD40L, CD40 ligand. Physiological and pathological mechanisms of purinergic P2 receptors. (A) The molecular mechanisms of purinergic P2 receptors in nervous system. (B) The molecular mechanisms of purinergic P2 receptors in digestive system. (C) The molecular mechanisms of purinergic P2 receptors in immune system. (D) The molecular mechanisms of purinergic P2 receptors in cancer. IL-6, interleukin-6; BDNF, brain-derived neurotrophic factor; TrkB, tropomyosin receptor kinase B; SOD1, superoxide dismutase 1; NLRP1, nucleotide binding oligomerization domain-like receptor protein 1; Caspase1, cysteinyl aspartate specific proteinase 1; Ca 2+ , calcium ions; CamKII, almodulin-dependent protein kinase II; RasGRF1, Ras protein specific guanine nucleotide-releasing factor 1; ERK1/2, extracellular regulated protein kinases1/2; p38, mitogen-activated protein kinase 14; CAN, calcineurin; DRP1, dynamin-related protein 1; AMPK, adenosine 5‘-monophosphate (AMP)-activated protein kinase; CatS, cathepsin S; FKN, fractalkine; HIF1α, hypoxia-induced factor-1α; NLRP3, NACHT, LRR, and PYD domains-containing protein 3; IL-1β, interleukin-1β； RhoA, Ras homolog gene family member A; ROCK, Rho-associated coiled-coil-containing protein kinase; JNK, c-Jun N-terminal kinase; IL-33, interleukin-33；PKA, protein kinase A; HMGB1, high mobility group box-1; CREB, cyclic adenosine monophosphate response element binding protein; nSMase, neutral sphingomyelinase; MAPK, mitogen-activated protein kinases; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; PKC, protein kinase C; GSK-3β, hlycogen synthase kinase 3β; STAT2, signal transducer and activator of transcription 2; IRF6, interferon regulatory factor 6; CXCL5, C-X-C motif chemokine ligand 5; NF-κB, nuclear factor kappa-B; STAT1, signal transducer and activator of transcription 1; NFATC2, nuclear factor of activated T-cells 2; NFAT, nuclear factor of activated T-cells; PBX3, Pre-B cell leukemia homeobox 3; BCAT1, branched-chain aminotransferase 1; mTOR, mammalian target of rapamycin; ATF4, activating transcription factor 4; MMP9, matrix metallopeptidase 9; p-ERK1/2, phosphorylation-extracellular regulated protein kinases1/2; TGF-β, transforming growth factor-β; SMAD, mothers against DPP homolog; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; FAK, focal adhesion kinase; MMP2, matrix metallopeptidase 2; GM-CSF, granulocyte–macrophage colony-stimulating factor; VEGF, vascular endothelial-derived growth factor; EGFR, epidermal growth factor receptor; Src, protein kinase Src; RTK, receptor tyrosine kinase; ASC, apoptosis-associated speck-like protein containing CARD; YAP, Yes-associated protein; SERPINE1, serine protease inhibitor clade E member 1; MMP1, matrix metallopeptidase 1；CD40L, CD40 ligand. Glial cells are widely distributed in the nervous system, where they connect and support various neural components and mediate nervous system signalling [43] , [44] , [45] . P2X 7 R underlies microglia activation and proliferation [46] . Indeed, P2X 7 R often crosstalks with NACHT, LRR, and PYD structural domain-containing protein 3 (NLRP3) inflammasome during microglia activation, engaging in the release of inflammatory factors and contributing to the development of neuro-inflammation [47] , [48] . P2X 7 R can exacerbate deleterious oxidative stress by upregulating the activity of NADPH oxidase 2 through the phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2), leading to secondary injury after intracerebral haemorrhage [49] . Additionally, P2X 7 R regulates microglia polarization, migration, phagocytosis, and many other behaviors, suggesting a dominant role in neuro-inflammation and neurological disorders [17] , [50] , [51] . Therefore, inhibiting P2X 7 R can reduce the neuro-inflammatory response, microglia activation, and chemokine release, which can improve cognitive deficits [52] , [53] , [54] , [55] ( Fig. 2 A). Some tracers of P2X 7 R have been developed as neurological markers, and they have strong applications in determining pathogenic mechanisms and disease prognosis [56] , [57] . Meanwhile, the complexity of the disease-related environment is also of interest due to the wide distribution of purinergic P2 receptors. For instance, P2X 7 R is significantly upregulated in response to Alzheimer's disease-specific pathologies such as amyloid β accumulation [58] . The expression of P2X 7 R on microglia is critical for the pathogenesis that involves the phosphorylation of Tau proteins, the release of inflammatory factors and chemokines [52] , [53] , [54] , [55] , [59] , [60] . In the absence of ATP, P2X 7 R in microglia functions primarily as a scavenger receptor. However, upon binding to ATP, the non-myosin heavy chain of P2X 7 R dissociates thus losing its phagocytic capacity [53] , [61] . Under pathological conditions, specifically in the presence of high concentrations of extracellular ATP (eATP), the function of P2X 7 R in microglia shifts to promote the release of inflammatory mediators [62] , [63] , [64] . It has been found that activated P2X 7 R leads to increased production of toxic β-amyloid through glycogen synthase kinase 3(GSK-3) [65] . However, short-term stimulation of P2X 7 R activates α-secretase, which in turn produces harmless soluble amyloid [66] . The conflicting result may be attributed to the wide expression of P2X 7 R and its various roles in the brain, and may be related to factors such as the concentration of local ATP and the activation state of P2X 7 R [67] . The function of P2X 7 R expressed on astrocytes, neurons, and leukocytes and their association in Alzheimer's disease remains unclear. Although alterations in purinergic P2 receptor expression and activity may contribute to the onset and progression of disease, it is unclear whether alterations in purinergic P2 receptors are induced by disease or are the result of disease flare-ups, because the etiology of most neurologic disorders is unknown and the complex coordinated multicellular interactions are involved. Future studies may need to focus on single-cell function and the development of cell-specific targeting compounds or blocking antibodies to further explore the direct or indirect role of purinergic P2 receptors. The digestive system comprises the digestive tract and glands, mainly including the esophagus, stomach, liver, pancreas, and colon, that is mainly responsible for the effective acquisition, digestion, and ingestion of nutrients [68] , [69] , [70] , [71] . Purinergic P2 receptors are distributed in both hepatic parenchymal and non-parenchymal cells of liver [72] , [73] ( Fig. 2 B). P2X 4 R and P2Y 1,2,4,6,13 R, control various essential hepatocyte functions, such as glucose metabolism, bile secretion, and cholesterol transport, as well as maintaining hepatocyte-bile duct cell communication [74] , [75] , [76] , [77] . A recent study reported a novel role for P2Y 13 R in liver glucose metabolism. ADP activates the P2Y 13 R-3′, 5′-cyclic adenosine monophosphate (cAMP)-cAMP-dependent protein kinase (PKA)-cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in hepatocytes to induce hepatic gluconeogenesis [78] . Hepatic fatty acid oxidation, cholesterol and fatty acid biosynthesis genes are aberrantly expressed in P2Y 13 R knockout mice. In the pathological state, ligand-activated purinergic P2 receptors lead to signaling disorders. UTP, a specific agonist of P2Y 2 R, increases the phosphorylation activity of ERK, which is related to alcoholic liver inflammation [79] , [80] . Additionally, ATP released from dying cells activates P2X 4 R, which regulates calcium inward flow, leading to myofibroblast activation and maintenance of the fibrotic phenotype [81] . Ingmar Mederacke et al. discovered that dying hepatocytes release UDP-glucose and UDP-galactose, which activate P2Y 14 R on adjacent hepatic stellate cells and link hepatocyte death to liver fibrosis via activation of the ERK pathway [82] . Most purinergic P2 receptors in the liver promote inflammation, which can both strengthen the body's resistance to infection and disrupt the balance of health. P2X 2 R and P2X 5 R are protective mediator receptors that benefit the organism in the inflammatory state [83] , [84] . However, there is a significant upregulation of P2X 4 R expression in cells infected with hepatitis C virus (HCV), and mediates upstream cysteinyl aspartate specific proteinase 3 (Caspase-3) - pannexin 1 (PANX1) signaling, leading to the release of pro-hepatic disease development cellular exosomes in HCV-infected Huh7.5.1 cells [85] , [86] . Several studies have reported the pro-inflammatory effects of P2X 4 R, P2X 7 R, and P2Y 6 R in alcoholic hepatitis and liver fibrosis, which involve the coordination of monocytes, mast cells, and stellate cells [20] , [87] , [88] , [89] , [90] , [91] , [92] , [93] . Purinergic P2 receptors are found in the enteric spinal plexus, the submucosal plexus of the intestine, the colonic longitudinal muscle and the circular muscle that are activated by nucleotides released from immune cells and microorganisms to maintain intestinal homeostasis or mediate diseases [73] . P2X 4 R in the colonic lumen promotes neutrophil glycolysis by increasing Ca 2+ inward flow in response to excessive activation of ATP, which promotes reactive oxygen species production and aggravates dextrose sodium sulfate (DSS) -induced colitis [94] . P2Y 6 R promotes the secretion of mucus in goblet cells, thereby forming a protective mucus layer from secretory agents in colitis [95] . P2X 7 R or P2Y 6 R knockout mice exhibit exacerbated of colonic ulcers, increased accumulation of colonic immune cells and elevated secretion of pro-inflammatory cytokines [96] , [97] , [98] . However, in intestinal epithelial cells, P2Y 6 R exerts the opposite effect. As an inhibitory receptor downstream of ectonucleoside triphosphate diphosphohydrolase 8 (Entpd8), pharmacological inhibition of P2Y 6 R attenuates the expression of pro-inflammatory factors and chemokines, thereby alleviating intestinal inflammation [99] . The reason for this difference may be the distribution of nucleotidases at the apical surface of colonic epithelium as well as the different roles on different cells, since P2Y 6 R in deleted intestinal epithelial cells completely alleviates DSS-induced colonic inflammation [99] . In general, inhibiting purinergic P2 receptors is the primary strategy for treating colonic disease [100] . In recent years, fewer studies have been conducted on purinergic P2 receptors in the esophagus, pancreas, and stomach, likely due to limitations in disease types and available tools. Activation of purinergic P2 receptors play an important role in orchestrating physiopathological functions, but its specific role may vary from disease to disease or cell to cell. For example, P2X 4 R deficiency protects mice from hepatic fibrosis induced by bile duct ligation or feeding a methionine- and choline-deficient diet, but has no significant effect on carbon tetrachloride-induced hepatic fibrosis, which indicating that purinergic signaling affects biliary and non-biliary fibrosis differently, yet the underlying mechanisms are not understood [20] , [81] , [101] . Similarly, the different functions and opposite results of P2Y 6 R in goblet and intestinal epithelial cells have been described in detail [95] , [99] . Furthermore, Mabrouka Salem found P2Y 6 R deficiency leads to intestinal inflammation through increased Th17/Th1 recruitment [98] . Although P2Y 6 R antagonists are currently available for the relief of ulcerative colitis, there is a difference between pharmacological inhibition and systemic knockdown because of the heterogeneous tissue distribution and drug affinity [102] . In practice, it is important to clarify the specific cells in which purinergic P2 receptors play pathological roles, and to consider the pathogenic stage of the disease and the cellular environment rather than making generalizations that could be achieved by real-time monitoring of the pathological environment. As an organismal system composed of immune organs, immune cells and immunologically active substances, the immune system has immune surveillance, defense and regulatory functions [103] . It is widely recognized that extracellular ATP released by activated or damaged cells is a signal that promotes inflammation [104] , [105] . Since 1978, studies have identified that ATP promotes T lymphocyte growth and effector functions [18] , [106] . Purinergic P2 receptors (P2X 1,4,7 R and P2Y 1,11,12,14 R) mediate nucleotide release and downstream signaling channels, with critical roles in activation, migration, or antigen recognition of T cells [106] , [107] , [108] ( Fig. 2 C). In the physiological state, activated P2X 1 R mediates inward Ca 2+ flow to maintain the basal metabolic demand of T lymphocytes, whereas when stimulated by chemokines or antigens in T cells, P2X 4 R actively upregulates ATP levels and cooperates with P2Y 1 R and P2Y 11 R to promote T cell activation, differentiation and migration [106] , [107] . The ATP-P2X 7 R axis broadly regulates T cell activation, differentiation, migration, and death [109] . P2X 7 R knockout mice exhibit impaired activity of effector T cells [110] . Low doses of ATP inhibit chemokine receptors 7 (CCR7)-mediated lymphocyte migration in the physiological state via P2X 7 R [109] . Notably, the sensitivity of P2X 7 R to ATP differs among different subpopulations of T lymphocytes, which is associated with the degree of T cell activation and differentiation [111] , [112] , [113] . P2X 7 R is also highly sensitive to ATP on Tγδ and MAIT cells [114] . Macrophages are cells with strong phagocytic ability and antigen-presenting function. Purinergic P2 receptors can be activated by signaling molecules, which commonly regulate various behaviors of macrophages [115] . The expression of purinergic P2 receptors on macrophages of different phenotypes, related to pro- and anti-inflammation, reflects the body’s adaptive response to changes in the external environment [116] . P2X 4 R, P2X 7 R, and P2Y 11 R are the major purine receptors that induce intracellular Ca 2+ response under ATP stimulation [117] . P2X 4 R responds to Ca 2+ in macrophages and mediates the propagation of C-X-C motif chemokine ligand (CXCL) 5 pro-inflammatory signals [118] , [119] . Transfer of P2X 4 R-deficient macrophages to wild-type mice by Balázs Csóka exacerbates bacterial load, inflammation, and organ damage in mice during sepsis [120] . P2X 4 R and P2X 7 R also promote macrophages phagocytosis [121] . A recent study has identified the essential role of reactive oxygen species as chaperones of P2X 7 R to assist in the proper folding of P2X 7 R to promote phosphatidylserine efflux and accompany Ca 2+ entry into cells [122] . Another study found that activated P2X 7 R, in concert with PANX1, mediates calcium inward flow, which in turn triggered Lysosomal Leakage via TRP family channel (TRPM2) and two-pore channel, leading to an inflammatory response [123] . P2X 7 R functions as an ATP receptor to sense tissue damage, mediates the inward flow of cation channels such as sodium and calcium, and works with potassium efflux channel (TWIK2) to activate NLRP3 inflammasome [124] . In disease states, the expression level of P2X 7 R in macrophages is actively upregulated [125] , [126] . The P2X 7 R-NLRP3 signaling pathway mediates classical inflammatory response in macrophages, specific inhibition of which has emerged as a viable approach to control excessive inflammation [127] , [128] , [129] . There are complex interactions between immune cell populations. Macrophage-induced inflammatory responses promote neutrophil recruitment and increased inflammation, while the movement of macrophages to neutrophils is a key component of timely clearance of apoptotic neutrophils [130] . The production of CXCL2 by the macrophage ADP-P2Y 1 R pathway induces neutrophil infiltration at joint sites in arthritic mice, relying on the Ca 2+ - nuclear factor kappa-B (NF-κB) pathway [131] . P2XRs are generally considered to be pro-inflammatory, while P2YRs are anti-inflammatory. P2Y 6,11,12,14 R all convert extracellular danger signals into anti-inflammatory responses [132] , [133] . However, there is no absolute monolithic anti-inflammatory or pro-inflammatory effects of purinergic P2 receptors. For example, P2X 7 R promotes active reduction of CD14-dependent pro-inflammatory signaling in macrophages via the extracellular vesicle pathway during sepsis [134] . Additionally, the UDPG/P2Y 14 R signaling pathway is thought to be involved in lipopolysaccharide, monosodium urate-induced acute inflammatory responses [135] , [136] . Purinergic P2 receptors are expressed on nearly all the immune cells. The challenge probably limits development is achieving targeted and controllable regulation, since the ultimate goal of therapy is to enhance the body's immunity and attenuate unwanted immune responses in autoimmune diseases. The final effect of purinergic P2 receptors depends on the amount and timing of the released nucleotides, as well as the coordinated action of other immune environmental factors [113] , [137] , [138] . In T cells, P2X 1 R, P2X 4 R, and P2X 7 R coordinately amplify TCR-mediated T lymphocyte activation signals [139] , [140] . However, activated P2X 7 R can also induce T-cell death, which is mainly related to specific subpopulations of T-cells, differentiation status, and concentration of ATP [113] , [114] , [141] , [142] . A study conducted by Hanaa Safya et al . found that activated mouse CD45RB low T cells were significantly more sensitive to ATP-induced cell death than naive CD45RB high T cells [143] . In the intestine, P2X 7 R activation effectively promotes pathogenic intestinal effector T cell-dependent apoptosis [144] . Thus, purinergic P2 receptors as extremely sensitive environmental sensors that maintain immune cell homeostasis, can help to activate the body's defense system and stop unwanted immune-inflammatory responses in time, which have potential to be key targets for disease treatment [145] , [146] . Cancer is considered a process of abnormal gene expression triggered by a combination of factors over time that ultimately manifest as abnormal cell proliferation [147] , [148] , [149] , [150] . In the tumor microenvironment, released ATP and other nucleotides act as autocrine or paracrine signals to promote or inhibit tumor cell survival and growth, depending on the concentration of nucleotides, the expression of purinergic P2 receptors, and the infiltration of immune cells [151] , [152] . ATP released from dead cells can activate P2X 4 R through mammalian target of rapamycin (mTOR), facilitating the survival of neighboring tumor cells [152] . There is substantial evidence indicating that purinergic P2 receptors can promote tumor cell proliferation, mainly by regulating the second messenger signaling or downstream proteases [153] , [154] , [155] ( Fig. 2 D). In head and neck squamous cell carcinoma, activated P2Y 2 R-induced downstream signaling molecules can promote epidermal growth factor receptor (EGFR) phosphorylation, resulting in cell proliferation [155] . Although most current studies have focused only on a single purinergic P2 receptor, tumor cells may express multiple purinergic P2 receptors, in which complexity, modulation of only a single receptor may not alter disease progression. In gastric cancer cells, inhibition of key purinergic P2 receptors, such as P2Y 2 R, coupled with activation of other purinergic P2 receptors, such as P2X 4 R, is the best strategy for treatment, as the two play opposite roles [151] . P2Y 2 R shows a high correlation with hypoxia genes that is frequently reported in recent years as cancer-related gene, mainly involved in pancreatic cancer, gastric cancer, leukemia, breast cancer, liver cancer [154] , [156] , [157] , [158] , [159] , [160] . Tumors are often accompanied by an altered cellular metabolic profile, which often leads to an overproduction of ATP. For example, the accumulation of ATP induced by ubiquinol-cytochrome c reductase core protein I in pancreatic ductal adenocarcinoma is released extracellularly, which activates P2Y 2 R located on the cell membrane through autocrine or paracrine pathways, and possibly promote cell proliferation via receptor tyrosine kinase (RTK)-protein kinase B (AKT) signaling pathway [160] . Activated P2Y 2 R under the above conditions also promotes vascular remodelling [161] . In addition, P2Y 2 R activates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through P2Y 2 R- tyrosine-protein kinase Src (Src)-EGFR axis to promote oral squamous cell carcinoma invasion and migration [162] . The effects of P2Y 2 R-PI3K-AKT signaling pathway in promoting the proliferation of leukemia cells and enhancing glycolysis of tumor cells have also been reported [158] , [159] . Some downstream signals, P2Y 2 R-AKT-GSK-3β-vascular endothelial-derived growth factor (VEGF), can promote the proliferation and migration of gastric cancer cells [157] . P2Y 2 R shows enormous role in tumors, inhibiting which has become an effective mean of inhibiting tumor cell proliferation, migration and angiogenesis. Several databases and clinical sample-based tools of analysis have identified the therapeutic potential of purinergic P2 receptors in tumors, i.e. modulation of tumor immune response. P2X 2,7 R and P2Y 12-14 R have been reported to regulate the tumor microenvironment and promote immune cell infiltration [163] , [164] , [165] , [166] , [167] . P2X 7 R is a key determinant of neighborhood tumor microenvironment composition [168] . eATP, A signaling molecule present in tumor tissues but not in normal tissues, activates P2X 7 R [169] . eATP-activated P2X 7 R was found to probably enhance the effector function of intra-tumor T cells through the P2X 7 R-inflammasome-interleukin-18 (IL-18) axis [170] , [171] . For effective tumor elimination, CD8 + T cells require P2X 7 R to sense eATP and to regulate mitochondrial function [172] . Short-term activation of P2X 7 R upregulates the immune response and promotes anti-tumor immune response, while excessive activation of P2X 7 R leads to cell death, which may result in premature cellular senescence [169] . Meanwhile, eATP-activated P2X 7 R mediated the translocation of 2′3′-cyclic GMP-AMP to macrophages and the subsequent activation of stimulator of interferon genes (STING), which in turn enhanced the macrophage clearance [173] . The effects of eATP-P2X 7 R axis on tumor progression is dependent on cell class and timing of receptor activation. Furthermore, P2X 7 R knockdown in mice leads to a decrease in ATP concentration, which is different from P2X 7 R inhibitors, which return ATP to a state of homeostasis that combine immune effects and as well as direct effects of P2X 7 R on tumor cells, ultimately producing a powerful oncogenic effect [168] . Combined with the recent years' work on the roles of purinergic P2 receptors in the nervous system, digestive system, immune system and cancer, we propose the following points. First, the roles of purinergic P2 receptors in physiopathology may not be absolutely singular, depending on the cell type, cellular environment and receptor activation time [95] , [99] , [113] , [169] . Secondly, the roles of purinergic P2 receptors in physiopathology are not be completely independent, and the end result may be the summation of multiple purinergic P2 receptors-mediated signals induced by similar ligands [174] , [175] . There is an urgent need to investigate the structural features of purinergic P2 receptors and the complex environment in which they reside in physiological and pathological states to further elucidate the diverse functions of purinergic P2 receptors, as well as the complex inter-receptor coordination linkages in different stages of diseases.