Involvement of microglial P2X7 receptor in pain modulation.

BACKGROUND: Pain is a rapid response mechanism that compels organisms to retreat from the harmful stimuli and triggers a repair response. Nonetheless, when pain persists for extended periods, it can lead to adverse changes into in the individual's brain, negatively impacting their emotional state and overall quality of life. Microglia, the resident immune cells in the central nervous system (CNS), play a pivotal role in regulating a variety of pain-related disorders. Specifically, recent studies have shed light on the central role that microglial purinergic ligand-gated ion channel 7 receptor (P2X7R) plays in regulating pain. In this respect, the P2X7R on microglial membranes represents a potential therapeutic target. AIMS: To expound on the intricate link between microglial P2X7R and pain, offering insights into potential avenues for future research. METHODS: We reviewed 140 literature and summarized the important role of microglial P2X7R in regulating pain, including the structure and function of P2X7R, the relationship between P2X7R and microglial polarization, P2X7R-related signaling pathways, and the effects of P2X7R antagonists on pain regulation. RESULTS: P2X7R activation is related to M1 polarization of microglia, while suppressing P2X7R can transfer microglia from M1 into M2 phenotype. And targeting the P2X7R-mediated signaling pathways helps to explore new therapy for pain alleviation. P2X7R antagonists also hold potential for translational and clinical applications in pain management. CONCLUSIONS: Microglial P2X7R holds promise as a potential novel pharmacological target for clinical treatments due to its distinctive structure, function, and the development of antagonists.

Structural Basis of the Negative Allosteric Modulation of 5-BDBD at Human P2X4 Receptors.

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site. SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor.

Synthesis and Characterization of the Novel Rodent-Active and CNS-Penetrant P2X7 Receptor Antagonist Lu AF27139.

There remains an insufficient number of P2X7 receptor antagonists with adequate rodent potency, CNS permeability, and pharmacokinetic properties from which to evaluate CNS disease hypotheses preclinically. Herein, we describe the molecular pharmacology, safety, pharmacokinetics, and functional CNS target engagement of Lu AF27139, a novel rodent-active and CNS-penetrant P2X7 receptor antagonist. Lu AF27139 is highly selective and potent against rat, mouse, and human forms of the receptors. The rat pharmacokinetic profile is favorable with high oral bioavailability, modest clearance (0.79 L/(h kg)), and good CNS permeability. In vivo mouse CNS microdialysis studies of lipopolysaccharide (LPS)-primed and 2'(3')-O-(benzoylbenzoyl)adenosine-5'-triphosphate (BzATP)-induced IL-1ß release demonstrate functional CNS target engagement. Importantly, Lu AF27139 was without effect in standard in vitro and in vivo toxicity studies. Based on these properties, we believe Lu AF27139 will be a valuable tool for probing the role of the P2X7 receptor in rodent models of CNS diseases.

Synthesis and Pharmacological Evaluation of Novel Non-nucleotide Purine Derivatives as P2X7 Antagonists for the Treatment of Neuroinflammation.

The ATP-gated P2X7 purinergic receptor (P2X7) is involved in the pathogenesis of many neurodegenerative diseases (NDDs). Several P2X7 antagonists have been developed, though none of them reached clinical trials for this indication. In this work, we designed and synthesized novel blood-brain barrier (BBB)-permeable derivatives as potential P2X7 antagonists. They comprise purine or xanthine cores linked to an aryl group through different short spacers. Compounds were tested in YO-PRO-1 uptake assays and intracellular calcium dynamics in a human P2X7-expressing HEK293 cell line, two-electrode voltage-clamp recordings in Xenopus laevis oocytes, and in interleukin 1ß release assays in mouse peritoneal macrophages. BBB permeability was assessed by parallel artificial membrane permeability assays and P-glycoprotein ATPase activity. Dichloroarylpurinylethanones featured a certain P2X7 blockade, being compound 6 (2-(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one), named ITH15004, the most potent, selective, and BBB-permeable antagonist. Compound 6 can be considered as a first non-nucleotide purine hit for future drug optimizations.

P2X3-Containing Receptors as Targets for the Treatment of Chronic Pain.

Current therapies for the treatment of chronic pain provide inadequate relief for millions of suffering patients, demonstrating the need for better therapies that will treat pain effectively and improve the quality of patient's lives. Better understanding of the mechanisms that mediate chronic pain is critical for developing drugs with improved clinical outcomes. Adenosine triphosphate (ATP) is a key modulator in nociceptive pathways. Release of ATP from injured tissue or sympathetic efferents has sensitizing effects on sensory neurons in the periphery, and presynaptic vesicular release of ATP from the central terminals can increase glutamate release thereby potentiating downstream central sensitization mechanisms, a condition thought to underlie many chronic pain conditions. The purinergic receptors on sensory nerves primarily responsible for ATP signaling are P2X3 and P2X2/3. Selective knockdown experiments, or inhibition with small molecules, demonstrate P2X3-containing receptors are key targets to modulate nociceptive signals. Preclinical studies have identified that P2X3-containing receptors are critical for sensory transduction for bladder function, and clinical studies have shown promise in treatment for bladder pain and pain associated with osteoarthritis. Further clinical characterization of antagonists to P2X3-containing receptors may lead to improved therapies in the treatment of chronic pain.

P2X7 receptor antagonism preserves retinal ganglion cells in glaucomatous mice.

To investigate the role of P2X7 receptor to preserve retinal ganglion cells (RGCs) structure and function in a genetic mouse model (DBA/2J mouse) of age-related glaucomatous neurodegeneration. Chronic treatment with P2X7 receptor antagonist eye drops was carried out in order to assess RGCs function and density by pattern electroretinogram (PERG) and RBPMS immunostaining, respectively. Further, microglia activation was assessed in flat-mounted retina by using Iba-1 immunostaining. Untreated glaucomatous eyes displayed significant microglia activation, alteration of PERG signal, and RGCs loss. In the P2X7 receptor antagonist-treated eyes, the PERG signal was significantly (p < 0.05) improved compared to controls, along with a significant (p < 0.05) reduction in terms of retinal microglial activation, and remarkable preservation of RGCs density. Altogether, these findings demonstrated that topical treatment with a P2X7 receptor antagonist has a neuroprotective effect on RGCs in glaucomatous mice, suggesting an appealing pharmacological approach to prevent retinal degenerative damage in optic neuropathy.

The role of P2X4 receptor in neuropathic pain and its pharmacological properties.

Neuropathic pain (NPP) is a common symptom of most diseases in clinic, which seriously affects the mental health of patients and brings certain pain to patients. Due to its pathological mechanism is very complicated, and thus, its treatment has been one of the challenges in the field of medicine. Therefore, exploring the pathogenesis and treatment approach of NPP has aroused the interest of many researchers. ATP is an important energy information substance, which participates in the signal transmission in the body. The P2 × 4 receptor (P2 × 4R) is dependent on ATP ligand-gated cationic channel receptor, which can be activated by ATP and plays an important role in the transmission of information in the nervous system and the formation of pain. In this paper, we provide a comprehensive review of the structure and function of the P2 × 4R gene. We also discuss the pathogenesis of NPP and the intrinsic relationship between P2 × 4R and NPP. Moreover, we explore the pharmacological properties of P2 × 4R antagonists or inhibitors used as targeted therapies for NPP.

Discovery and Structure Relationships of Salicylanilide Derivatives as Potent, Non-acidic P2X1 Receptor Antagonists.

Antagonists for the ATP-gated ion channel receptor P2X1 have potential as antithrombotics and for treating hyperactive bladder and inflammation. In this study, salicylanilide derivatives were synthesized based on a screening hit. P2X1 antagonistic potency was assessed in 1321N1 astrocytoma cells stably transfected with the human P2X1 receptor by measuring inhibition of the ATP-induced calcium influx. Structure-activity relationships were analyzed, and selectivity versus other P2X receptor subtypes was assessed. The most potent compounds, N-[3,5-bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (1, IC50 0.0192 µM) and N-[3,5-bis(trifluoromethyl)phenyl]-4-chloro-2-hydroxybenzamide (14, IC50 0.0231 µM), displayed >500-fold selectivity versus P2X2 and P2X3, and 10-fold selectivity versus P2X4 and P2X7 receptors, and inhibited collagen-induced platelet aggregation. They behaved as negative allosteric modulators, and molecular modeling studies suggested an extracellular binding site. Besides selective P2X1 antagonists, compounds with ancillary P2X4 and/or P2X7 receptor inhibition were discovered. These compounds represent the first potent, non-acidic, allosteric P2X1 receptor antagonists reported to date.

Pyroglutamide-Based P2X7 Receptor Antagonists Targeting Inflammatory Bowel Disease.

This report deals with the design, the synthesis, and the pharmacological evaluation of pyroglutamide-based P2X7 antagonists. A dozen were shown to possess improved properties, among which inhibition of YO-PRO-1/TO-PRO-3 uptake and IL1ß release upon BzATP activation of the receptor and dampening signs of DSS-induced colitis on mice, in comparison with reference antagonist GSK1370319A. Docking study and biological evaluation of synthesized compounds has highlighted new SAR, and low toxicity profiles of pyroglutamides herein described are clues for the finding of a usable h-P2X7 antagonist drug. Such a drug would raise the hope for a cure to many P2X7-dependent pathologies, including inflammatory, neurological, and immune diseases.

Brilliant Blue G Inhibits Inflammasome Activation and Reduces Disruption of Blood-Spinal Cord Barrier Induced by Spinal Cord Injury in Rats.

BACKGROUND Brilliant blue G (BBG) is a P2X7 receptor inhibitor that has been reported to improve spinal cord injury (SCI) in previous studies, but the specific mechanism has been unclear. In this study, we investigated the effects of BBG on inflammasomes and blood-spinal cord barrier (BSCB) permeability after SCI. MATERIAL AND METHODS The experimental rats were randomly divided into 3 groups: sham, SCI, and SCI+BBG. The expression of P2X7 and inflammasome-related proteins was measured by Western blot and immunohistochemistry, while IL-1ß and IL-18 levels were measured by using an enzyme-linked immunosorbent assay (ELISA) kit. The permeability of the BSCB was evaluated by Evans Blue (EB) exosmosis, and histological alterations were observed by hematoxylin-eosin staining. Motor function recovery was assessed by the Basso, Beattie, Bresnahan (BBB) scale after SCI. RESULTS The expression levels of P2X7, NLRP3, ASC, cleaved XIAP, caspase-1, caspase-11, IL-1ß, and IL-18 were increased significantly after SCI, and BBG administration inhibited this increase at 72 h after SCI. BBG administration significantly reduced EB leakage at 24 h after SCI. Furthermore, treatment with BBG significantly attenuated histological alterations and improved motor function recovery after SCI. CONCLUSIONS BBG administration promoted motor function recovery and alleviated tissue injury, and these effects might be related to the suppression of inflammasomes and the maintenance of BSCB integrity.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

8-Hydroxy-2-(1H-1,2,3-triazol-1-yl)-1,4-naphtoquinone derivatives inhibited P2X7 Receptor-Induced dye uptake into murine Macrophages.

Extracellular adenosine 5'-triphosphate (ATP) triggers the P2X7 receptor (P2X7R) ionic channel to stimulate the release of the interleukin-IL-1ß cytokine into macrophages. The current study explored the reaction of six structurally diverse triazole derivatives on P2X7-mediated dye uptake into murine peritoneal macrophages. P2X7R activity determined by ATP-evoked fluorescent dye uptake. Triazole derivatives toxicity measured using dextran rhodamine exclusion based colorimetric assay. A740004 and BBG, both P2X7R antagonist, inhibited ATP-induced dye uptake. In contrast, the derivatives 5a, 5b, 5e, and 5f did not diminish P2X7R activity in concentrations until 100 µM. 5c and 5d analogs caused a potent inhibitory activity on P2X7-induced dye uptake. Dextran Rhodamine exclusion measurements after 24 h of continuous treatment with triazole derivatives indicated a moderated toxicity for all molecules. In conclusion, this study showed that a series of new hybrid 1,2,3-triazolic naphthoquinones reduces P2X7R-induced dye uptake into murine macrophages. In silico analysis indicates a good pharmacokinetic profile and molecular docking results of these analogs indicate the potential to bind into an allosteric site located into the P2X7R pore and juxtaposed with the ATP binding pocket. In this manner, the compounds 5c and 5d may be used as a scaffold for new P2X7R inhibitors with reduced toxicity, and good anti-inflammatory activity.

Emerging role of the P2X7-NLRP3-IL1ß pathway in mood disorders.

The science of neuroimmunopsychiatry has evolved rapidly in the last few years with the hope of tackling the unmet need in mood disorders. This article focuses on an inflammatory pathway, highly conserved in myeloid cells that may play a role in neuroinflammatory disorders including depression. Within the brain tissue, microglia are the myeloid cells that express the P2X7 ion channel that is connected through the NLRP3 inflammasome complex leading to release of IL-1ß and IL-18. We present, in the way of reviewing relevant literature, the preclinical data and scientific rationale supporting the role of the P2X7-NLRP3-IL-1ß pathway in mood disorders. We also highlight recent advances in drug discovery and development of P2X7 small molecule antagonists and P2X7 PET ligands which provide optimism that clinical tools are availableto address critical proof-of-concept experiments in mood disorders.

A P2X7 receptor antagonist reverses behavioural alterations, microglial activation and neuroendocrine dysregulation in an unpredictable chronic mild stress (UCMS) model of depression in mice.

A polymorphism in the P2RX7 gene that encodes for the P2X7 ionotropic ATP-gated receptor (P2X7R) protein has been shown to be associated with an increased risk for developing depressive illnesses. However, the role of P2X7R in depression is still unclear. To better understand the role of P2X7R and its subsequent impact on microglial activation, we compared the effect of the P2X7R antagonist Brilliant Blue G (BBG) with that of fluoxetine in an unpredictable chronic mild stress (UCMS) model of depression in mice. Our results indicate that BBG (50 mg/kg body weight in 0.9% NaCl, 10 ml/kg/day) successfully reversed the degradation of coat states and nest-building scores induced by exposure to UCMS, similar to the conventional antidepressant fluoxetine (15 mg/kg body weight in 0.9% NaCl, 10 ml/kg/day). BBG also reversed the UCMS-induced microglial activation in cortical and hippocampal regions and the basal nuclei of mouse brains and corrected the UCMS-induced hypothalamo-pituitary-adrenal (HPA) axis dysregulation. In contrast to fluoxetine, however, BBG treatment did not increase the density of doublecortin-positive cells in the dentate gyrus, indicating that BBG had no impact on hippocampal neurogenesis. These results suggest that P2X7R is involved in recovery from depressive-like states caused by exposure to UCMS in a mechanism that involves restoration of the HPA axis but not hippocampal neurogenesis. These results add to the evidence that P2X7R antagonist agents may have potential value in the pharmacological management of depression.

1,4-Naphthoquinones potently inhibiting P2X7 receptor activity.

P2X7 receptor (P2X7R) is an ATP-gated ion-channel with potential therapeutic applications. In this study, we prepared and searched a series of 1,4-naphthoquinones derivatives to evaluate their antagonistic effect on both human and murine P2X7 receptors. We explored the structure-activity relationship and binding mode of the most active compounds using a molecular modeling approach. Biological analysis of this series (eight analogues and two compounds) revealed significant in vitro inhibition against both human and murine P2X7R. Further characterization revealed that AN-03 and AN-04 had greater potency than BBG and A740003 in inhibiting dye uptake, IL-1ß release, and carrageenan-induced paw edema in vivo. Moreover, we used electrophysiology and molecular docking analysis for characterizing AN-03 and AN-04 action mechanism. These results suggest 1,4-napthoquinones, mainly AN-04, as potential leads to design new P2X7R blockers and anti-inflammatory drugs.

P2X7 receptor antagonism: Implications in diabetic retinopathy.

Diabetic retinopathy (DR) is the most frequent complication of diabetes and one of leading causes of blindness worldwide. Early phases of DR are characterized by retinal pericyte loss mainly related to concurrent inflammatory process. Recently, an important link between P2X7 receptor (P2X7R) and inflammation has been demonstrated indicating this receptor as potential pharmacological target in DR. Here we first carried out an in silico molecular modeling study in order to characterize the allosteric pocket in P2X7R, and identify a suitable P2X7R antagonist through molecular docking. JNJ47965567 was identified as the hit compound in docking calculations, as well as for its absorption, distribution, metabolism and excretion (ADME) profile. As an in vitro model of early diabetic retinopathy, human retinal pericytes were exposed to high glucose (25mM, 48h) that caused a significant (p<0.05) release of IL-1ß and LDH. The block of P2X7R by JNJ47965567 significantly (p<0.05) reverted the damage elicited by high glucose, detected as IL-1ß and LDH release. Overall, our findings suggest that the P2X7R represents an attractive pharmacological target to manage the early phase of diabetic retinopathy, and the compound JNJ47965567 is a good template to discover other P2X7R selective antagonists.

Escape from adamantane: Scaffold optimization of novel P2X7 antagonists featuring complex polycycles.

The adamantane scaffold, despite being widely used in medicinal chemistry, is not devoid of problems. In recent years we have developed new polycyclic scaffolds as surrogates of the adamantane group with encouraging results in multiple targets. As an adamantane scaffold is a common structural feature in several P2X7 receptor antagonists, herein we report the synthesis and pharmacological evaluation of multiple replacement options of adamantane that maintain a good activity profile. Molecular modeling studies support the binding of the compounds to a site close to the central pore, rather than to the ATP-binding site and shed light on the structural requirements for novel P2X7 antagonists.

Role of the purinergic signaling in epilepsy.

Adenine nucleotides and adenosine are signaling molecules that activate purinergic receptors P1 and P2. Activation of A1 adenosine receptors has an anticonvulsant action, whereas activation of A2A receptors might initiate seizures. Therefore, a significant limitation to the use of A1 receptor agonists as drugs in the CNS might be their peripheral side effects. The anti-epileptic activity of adenosine is related to its increased concentration outside the cell. This increase might result from the inhibition of the equilibrative nucleoside transporters (ENTs). Moreover, the implantation of implants or stem cells into the brain might cause slow and persistent increases in adenosine concentrations in the extracellular spaces of the brain. The role of adenosine in seizure inhibition has been confirmed by results demonstrating that in patients with epilepsy, the adenosine kinase (ADK) present in astrocytes is the only purine-metabolizing enzyme that exhibits increased expression. Increased ADK activity causes intensified phosphorylation of adenosine to 5'-AMP, which therefore lowers the adenosine level in the extracellular spaces. These changes might initiate astrogliosis and epileptogenesis, which are the manifestations of epilepsy. Seizures might induce inflammatory processes and vice versa. Activation of P2X7 receptors causes intensified release of pro-inflammatory cytokines (IL-1ß and TNF-α) and activates metabolic pathways that induce inflammatory processes in the CNS. Therefore, antagonists of P2X7 and the interleukin 1ß receptor might be efficient drugs for recurring seizures and prolonged status epilepticus. Inhibitors of ADK would simultaneously inhibit the seizures, prevent the astrogliosis and epileptogenesis processes and prevent the formation of new epileptogenic foci. Therefore, these drugs might become beneficial seizure-suppressing drugs.

Structural basis for subtype-specific inhibition of the P2X7 receptor.

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.