RESUMEN
Despite their acknowledged significance in the inflammatory signalling cascade across a range of disease states, P2X7R antagonists have not yet proven to be effective in clinical trials. In this study, we present findings on P2X7 receptor antagonists that are based on a core adamantyl-cyanoguanidine-quinoline lead. To investigate the specific features of the cyanoguanidine moiety that influence compound potency we carried out a structure-activity relationship (SAR) study. Compound potency was assessed using an inâ vitro dye-uptake assay measuring P2X7R pore formation. While none of the compounds displayed superior potency to the lead, we established key structural requirements for potent P2X7R antagonism. An additional SAR using different aryl groups was performed based on the promising activity displayed by the squaramide derivative.
Asunto(s)
Guanidinas , Antagonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X7 , Relación Estructura-Actividad , Antagonistas del Receptor Purinérgico P2X/química , Antagonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/síntesis química , Guanidinas/química , Guanidinas/farmacología , Guanidinas/síntesis química , Humanos , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/química , Estructura Molecular , Relación Dosis-Respuesta a Droga , Quinolinas/química , Quinolinas/farmacología , Quinolinas/síntesis química , Células HEK293RESUMEN
P2X7 is a member of the Ionotropic Purinergic Receptor (P2X) family. The P2X family of receptors is composed of seven (P2X1-7), ligand-gated, nonselective cation channels. Changes in P2X expression have been reported in multiple disease models. P2Xs have large complex extracellular domains that function as receptors for a variety of ligands, including endogenous and synthetic agonists and antagonists. ATP is the canonical agonist. ATP affinity ranges from nanomolar to micromolar for most P2XRs, but P2X7 has uniquely poor ATP affinity. In many physiological settings, it may be difficult to achieve the millimolar extracellular ATP concentrations needed for P2X7 channel activation; however, channel function is implicated in pain sensation, immune cell function, cardiovascular disease, cancer, and osteoporosis. Multiple high-resolution P2X7 structures have been solved in apo-, ATP-, and antagonist-bound states. P2X7 structural data reveal distinct allosteric and orthosteric antagonist-binding sites. Both allosteric and orthosteric P2X7 antagonists are well documented to inhibit ATP-evoked channel current. However, a growing body of evidence supports P2X7 activation by non-nucleotide agonists, including extracellular histone proteins and human cathelicidin-derived peptides (LL-37). Interestingly, P2X7 non-nucleotide agonism is not inhibited by allosteric antagonists, but is inhibited by orthosteric antagonists. Herein, we review P2X7 function with a focus on the efficacy of available pharmacology on P2X7 channel current activation by non-nucleotide agonists in effort to understand agonist/antagonist efficacy, and consider the impact of these data on the current understanding of P2X7 in physiology and disease given these limitations of P2X7-selective antagonists and incomplete knockout mouse models.
Asunto(s)
Agonistas del Receptor Purinérgico P2X , Receptores Purinérgicos P2X7 , Animales , Humanos , Adenosina Trifosfato/metabolismo , Agonistas del Receptor Purinérgico P2X/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismoRESUMEN
P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened ß2- and ß3-sheets and their linker (loop ß2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer ß2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer ß2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype.
Asunto(s)
Adenosina Trifosfato , Conformación Proteica en Lámina beta , Receptores Purinérgicos P2X7 , Adenosina Trifosfato/metabolismo , Humanos , Inflamación , Conformación Proteica en Lámina beta/genética , Estabilidad Proteica , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Activación TranscripcionalRESUMEN
The P2X7 receptor (P2X7R) is a calcium-permeable cation channel activated by high concentrations of extracellular ATP. It plays a role in vital physiological processes, particularly in innate immunity, and is dysregulated in pathological conditions such as inflammatory diseases, neurodegenerative diseases, mood disorders, and cancers. Structural modeling of the human P2X7R (hP2X7R) based on the recently available structures of the rat P2X7 receptor (rP2XR) in conjunction with molecular docking predicts the orientation of tyrosine at position 288 (Y288) in the extracellular domain to face ATP. In this short communication, we combined site-directed mutagenesis and whole-cell patch-clamp recording to investigate the role of this residue in the hP2X7R function. Mutation of this extracellular residue to amino acids with different properties massively impaired current responses to both ATP and BzATP, suggesting that Y288 is important for normal receptor function. Such a finding facilitates development of an in-depth understanding of the molecular basis of hP2X7R structure-function relationships.
Asunto(s)
Mutagénesis Sitio-Dirigida/métodos , Receptores Purinérgicos P2X7/química , Tirosina/química , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Humanos , Simulación del Acoplamiento Molecular , Mutación , Técnicas de Placa-Clamp , Unión Proteica , RatasRESUMEN
Age-related macular degeneration (AMD) is a common and severe blinding disease among people worldwide. Retinal inflammation and neovascularization are two fundamental pathological processes in AMD. Recent studies showed that P2X7 receptor was closely involved in the inflammatory response. Here, we aim to investigate whether A740003, a P2X7 receptor antagonist, could prevent retinal inflammation and neovascularization induced by oxidized low-density lipoprotein (ox-LDL) and explore the underlying mechanisms. ARPE-19 cells and C57BL/6 mice were treated with ox-LDL and A740003 successively for in vitro and in vivo studies. In this research, we found that A740003 suppressed reactive oxygen species (ROS) generation and inhibited the activation of Nod-like receptor pyrin-domain protein 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathway. A740003 also inhibited the generation of angiogenic factors in ARPE-19 cells and angiogenesis in mice. The inflammatory cytokines and phosphorylation of inhibitor of nuclear factor-κB alpha (IKBα) were repressed by A740003. Besides, ERG assessment showed that retinal functions were remarkably preserved in A740003-treated mice. In summary, our results revealed that the P2X7 receptor antagonist reduced retinal inflammation and neovascularization and protected retinal function. The protective effects were associated with regulation of NLRP3 inflammasome and the NF-κB pathway, as well as inhibition of angiogenic factors.
Asunto(s)
Inflamación/tratamiento farmacológico , Lipoproteínas LDL/toxicidad , Neovascularización Patológica/tratamiento farmacológico , Estrés Oxidativo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/química , Retinitis/tratamiento farmacológico , Animales , Citocinas/metabolismo , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Neovascularización Patológica/inducido químicamente , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Retinitis/inducido químicamente , Retinitis/metabolismo , Retinitis/patología , Transducción de SeñalRESUMEN
Oxysterols, or cholesterol oxidation products, are naturally occurring lipids which regulate the physiology of cells, including those of the immune system. In contrast to effects that are mediated through nuclear receptors or by epigenetic mechanism, which take tens of minutes to occur, changes in the activities of cell-surface receptors caused by oxysterols can be extremely rapid, often taking place within subsecond timescales. Such cell-surface receptor effects of oxysterols allow for the regulation of fast cellular processes, such as motility, secretion and endocytosis. These cellular processes play critical roles in both the innate and adaptive immune systems. This review will survey the two broad classes of cell-surface receptors for oxysterols (G-protein coupled receptors (GPCRs) and ion channels), the mechanisms by which cholesterol oxidation products act on them, and their presence and functions in the different cell types of the immune system. Overall, this review will highlight the potential of oxysterols, synthetic derivatives and their receptors for physiological and therapeutic modulation of the immune system.
Asunto(s)
Sistema Inmunológico/metabolismo , Oxiesteroles/metabolismo , Humanos , Canales Iónicos/metabolismo , Oxiesteroles/química , Unión Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/metabolismo , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
P2X7 receptors are trimeric ion channels activated by extracellular ATP. Upon activation, they trigger cytolysis and apoptosis but also control cell proliferation. To shed more light on channel gating and the underlying function of the individual subunits, receptors of concatenated subunits were built containing a defined number of functional binding sites. The currents evoked by ATP were obtained in the outside-out configuration of the patch-clamp technique, and steady-state activation, as well as time courses, were analyzed. Our results show that each occupied binding site contributes to channel activation. While the occupation of a single binding site can already activate the channels, three bound ligands maximally stabilize the open state. Hence, P2X7 receptors can be described by a stepwise activation process.
Asunto(s)
Adenosina Trifosfato/farmacología , Activación del Canal Iónico/efectos de los fármacos , Mutación Missense , Oocitos/fisiología , Receptores Purinérgicos P2X7/genética , Adenosina Trifosfato/metabolismo , Algoritmos , Animales , Sitios de Unión/genética , Femenino , Activación del Canal Iónico/genética , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Oocitos/metabolismo , Técnicas de Placa-Clamp/métodos , Ratas , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.
Asunto(s)
Anoctaminas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Algoritmos , Animales , Anoctaminas/química , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular , Colesterol/metabolismo , Células HEK293 , Humanos , Inmunohistoquímica , Modelos Biológicos , Oocitos , Receptores Purinérgicos P2X7/químicaRESUMEN
The P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure-function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.
Asunto(s)
Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Líquido Extracelular/metabolismo , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/fisiología , Humanos , Estructura Secundaria de Proteína , Receptores Purinérgicos P2X7/genéticaRESUMEN
Postherpetic neuralgia (PHN) is the most common complication of acute herpes zoster. The treatment of PHN remains a challenge for clinical pain management. The present study investigated the P2X7 receptor antagonist brilliant blue G (BBG) whether inhibits endoplasmic reticulum stress and pyroptosis (a necrotic form of cell death) and alleviates PHN. Varicella zoster virus (VZV)-infected CV-1 cells were used to induce PHN model. Mechanical paw withdrawal thresholds were measured using an ascending series of von Frey filaments. Immunohistochemistry was used to detect the expression of P2X7R in nerve tissues. Western blot was used to determine the expression of endoplasmic reticulum (ER) stress and pyroptosis-related molecules. The expression of IL-1ß and IL-18 in tissue homogenate was detected by ELISA. The PHN rat has the lower paw withdrawal threshold, but higher expression of P2X7 in nerve tissues. And, endoplasmic reticulum stress was activated and pyroptosis was increased in PHN rats. BBG can decrease pain thresholds and reduce ER stress and pyroptosis in PHN rats. In addition, ER stress activator tunicamycin (TM) can reverse the effect of BBG on the paw withdrawal thresholds, endoplasmic reticulum stress, and pyroptosis. Therefore, P2X7 receptor antagonist BBG alleviates PHN by activating ER stress and reducing pyroptosis.
Asunto(s)
Estrés del Retículo Endoplásmico , Herpes Zóster/complicaciones , Neuralgia Posherpética/prevención & control , Antagonistas del Receptor Purinérgico P2X/farmacología , Piroptosis , Receptores Purinérgicos P2X7/química , Colorantes de Rosanilina/farmacología , Animales , Herpes Zóster/virología , Herpesvirus Humano 3/patogenicidad , Indicadores y Reactivos/farmacología , Neuralgia Posherpética/metabolismo , Neuralgia Posherpética/patología , Neuralgia Posherpética/virología , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Sistema Nervioso Central/metabolismo , Antagonistas del Receptor Purinérgico P2X/síntesis química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Línea Celular , Sistema Nervioso Central/efectos de los fármacos , Perros , Femenino , Semivida , Humanos , Interleucina-1beta/metabolismo , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/citología , Microglía/efectos de los fármacos , Microglía/metabolismo , Microsomas Hepáticos/metabolismo , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Antagonistas del Receptor Purinérgico P2X/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7/químicaRESUMEN
Extracellular nucleotides are important mediators of activation, triggering various responses through plasma membrane P2 and P1 receptors. P2 receptors are further subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors. P2X4 is an ATP-gated cation channel broadly expressed in most tissues of the body. Within the P2X family, P2X4 has a unique subcellular distribution, being preferentially localized in lysosomes. In these organelles, high ATP concentrations do not trigger P2X4 because of the low pH. However, when the pH increases to 7.4, P2X4 can be stimulated by intra-lysosomal ATP, which is in its active, tetra-anionic form. Elucidation of P2X4, P2X3 and P2X7 structures has shed some light on the functional differences between these purinergic receptors. The potential interaction between P2X4 and P2X7 has been extensively studied. Despite intensive effort, it has not been possible yet to determine whether P2X4 and P2X7 interact as heterotrimers or homotrimers at the plasma membrane. However, several publications have shown that functional interactions between P2X4 and P2X7 do occur. Importantly, these studies indicate that P2X4 potentiates P2X7-dependent activation of inflammasomes, leading to increased release of IL-1ß and IL-18. The role of P2X4 in various diseases could be beneficial or deleterious even though the pathophysiological mechanisms involved are still poorly defined. However, in diseases whose physiopathology involves activation of the NLRP3 inflammasome, P2X4 was found to exacerbate severity of disease. The recent production of monoclonal antibodies specific for the human and mouse P2X4, some of which are endowed with agonist or antagonist properties, raises the possibility that they could be used therapeutically. Analysis of single nucleotide polymorphisms of the human P2RX4 gene has uncovered the association of P2RX4 gene variants with susceptibility to several human diseases.
Asunto(s)
Receptores Purinérgicos P2X4/química , Receptores Purinérgicos P2X4/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Degranulación de la Célula , Encefalomielitis Autoinmune Experimental/etiología , Etanol/farmacología , Humanos , Inflamasomas/fisiología , Inflamación/etiología , Mastocitos/fisiología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/fisiologíaRESUMEN
Breast cancer is currently the most common cancer and the leading cause of cancer death among women worldwide. Advanced breast cancer is prone to metastasis, and there is currently no drug to cure metastatic breast cancer. The purinergic ligand-gated ion channel 7 receptor is an ATP-gated nonselective cation channel receptor and is involved in signal transduction, growth regulation, cytokine secretion, and tumor cell development. Recent studies have shown that upregulation of the P2X7 receptor in breast cancer can mediate AKT signaling pathways, Ca2 þ-activated SK3 potassium channels, and EMT and regulate the secretion of small extracellular vesicles to promote breast cancer invasion and migration, which are affected by factors such as hypoxia and ATP. In addition, studies have shown that microRNAs can bind to the 3' untranslated region of the P2X7 receptor, which affects the occurrence and development of breast cancer by upregulating and downregulating P2X7 receptor expression. Studies have shown that new P2X7 receptor inhibitors, such as emodin and Uncaria tomentosa, can inhibit P2X7 receptor-mediated breast cancer invasion and are expected to be used clinically. This article reviews the research progress on the relationship between the P2X7 receptor and breast cancer to provide new ideas and a basis for clinical diagnosis and treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Terapia Molecular Dirigida/métodos , Proteínas de Neoplasias/fisiología , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Receptores Purinérgicos P2X7/fisiología , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Uña de Gato , Cationes/metabolismo , Progresión de la Enfermedad , Emodina/uso terapéutico , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Transporte Iónico , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/efectos de los fármacos , Extractos Vegetales/uso terapéutico , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-Actividad , Regulación hacia ArribaRESUMEN
P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside-compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. SIGNIFICANCE STATEMENT: Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.
Asunto(s)
Ginsenósidos/farmacología , Glicósidos/farmacología , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Ginsenósidos/química , Glicósidos/química , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , Receptores Purinérgicos P2X7/genética , Relación Estructura-ActividadRESUMEN
Activation of P2X7 signaling, due to high glucose levels, leads to blood retinal barrier (BRB) breakdown, which is a hallmark of diabetic retinopathy (DR). Furthermore, several studies report that high glucose (HG) conditions and the related activation of the P2X7 receptor (P2X7R) lead to the over-expression of pro-inflammatory markers. In order to identify novel P2X7R antagonists, we carried out virtual screening on a focused compound dataset, including indole derivatives and natural compounds such as caffeic acid phenethyl ester derivatives, flavonoids, and diterpenoids. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) rescoring and structural fingerprint clustering of docking poses from virtual screening highlighted that the diterpenoid dihydrotanshinone (DHTS) clustered with the well-known P2X7R antagonist JNJ47965567. A human-based in vitro BRB model made of retinal pericytes, astrocytes, and endothelial cells was used to assess the potential protective effect of DHTS against HG and 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP), a P2X7R agonist, insult. We found that HG/BzATP exposure generated BRB breakdown by enhancing barrier permeability (trans-endothelial electrical resistance (TEER)) and reducing the levels of ZO-1 and VE-cadherin junction proteins as well as of the Cx-43 mRNA expression levels. Furthermore, HG levels and P2X7R agonist treatment led to increased expression of pro-inflammatory mediators (TLR-4, IL-1ß, IL-6, TNF-α, and IL-8) and other molecular markers (P2X7R, VEGF-A, and ICAM-1), along with enhanced production of reactive oxygen species. Treatment with DHTS preserved the BRB integrity from HG/BzATP damage. The protective effects of DHTS were also compared to the validated P2X7R antagonist, JNJ47965567. In conclusion, we provided new findings pointing out the therapeutic potential of DHTS, which is an inhibitor of P2X7R, in terms of preventing and/or counteracting the BRB dysfunctions elicited by HG conditions.
Asunto(s)
Barrera Hematorretinal/efectos de los fármacos , Furanos/farmacología , Fenantrenos/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/toxicidad , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Sitios de Unión , Barrera Hematorretinal/citología , Barrera Hematorretinal/metabolismo , Permeabilidad Capilar , Línea Celular , Conexina 43/metabolismo , Citocinas/metabolismo , Citoprotección , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Furanos/química , Humanos , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Fenantrenos/química , Unión Proteica , Agonistas del Receptor Purinérgico P2X/toxicidad , Antagonistas del Receptor Purinérgico P2X/química , Quinonas , Especies Reactivas de Oxígeno/metabolismo , Receptores Purinérgicos P2X7/químicaRESUMEN
The P2X7 receptor (P2X7R) possesses a unique structure associated to an as yet not fully understood mechanism of action that facilitates cell permeability to large ionic molecules through the receptor itself and/or nearby membrane proteins. High extracellular adenosine triphosphate (ATP) levels-inexistent in physiological conditions-are required for the receptor to be triggered and contribute to its role in cell damage signaling. The inconsistent data on its activation pathways and the few studies performed in natively expressed human P2X7R have led us to review the structure, activation pathways, and specific cellular location of P2X7R in order to analyze its biological relevance. The ATP-gated P2X7R is a homo-trimeric receptor channel that is occasionally hetero-trimeric and highly polymorphic, with at least nine human splice variants. It is localized predominantly in the cellular membrane and has a characteristic plasticity due to an extended C-termini, which confers it the capacity of interacting with membrane structural compounds and/or intracellular signaling messengers to mediate flexible transduction pathways. Diverse drugs and a few endogenous molecules have been highlighted as extracellular allosteric modulators of P2X7R. Therefore, studies in human cells that constitutively express P2X7R need to investigate the precise endogenous mediator located nearby the activation/modulation domains of the receptor. Such research could help us understand the possible physiological ATP-mediated P2X7R homeostasis signaling.
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Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Polimorfismo Genético , Estructura Cuaternaria de Proteína , Receptores Purinérgicos P2X7/genética , Transducción de Señal , Transcripción GenéticaRESUMEN
Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.
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Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia Conservada , Células HEK293 , Humanos , Activación del Canal Iónico , Cinética , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Ratas , Receptores Purinérgicos P2X7/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Electricidad EstáticaRESUMEN
Vascular endothelial dysfunction is associated with increased mortality in patients with diabetes. Astragaloside IV (As-IV) is a bioactive saponin with therapeutic potential as an anti-inflammatory and antiendothelial dysfunction. However, the underlying mechanism for how As-IV ameliorated endothelial dysfunction is still unclear. Therefore, in this study, we examined the protective effect of As-IV against endothelial dysfunction and explored potential molecular biology mechanism. In vivo, rats were intraperitoneally injected with streptozotocin (STZ) at a dose of 65 mg/kg body weight to establish a diabetic model. In vitro studies, rat aortic endothelial cells (RAOEC) were pretreated with As-IV, SB203580 (p38 MAPK inhibitor) for 2 h prior to the addition of high glucose (33 mM glucose). Our findings indicated that As-IV improved impaired endothelium-dependent relaxation and increased the levels of endothelial NO synthase (eNOS) and nitric oxide (NO) both in vivo and in vitro. Besides, As-IV treatment inhibited the elevated inflammation and oxidative stress in diabetic model both in vivo and in vitro. Moreover, As-IV administration reversed the upregulated expression of P2X7R and p-p38 MAPK in vivo and in vitro. Additionally, the effects of both P2X7R siRNA and SB203580 on endothelial cells were similar to As-IV. Collectively, our study demonstrated that As-IV rescued endothelial dysfunction induced by high glucose via inhibition of P2X7R dependent p38 MAPK signaling pathway. This provides a theoretical basis for the further study of the vascular endothelial protective effects of As-IV.
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Sustancias Protectoras/farmacología , Receptores Purinérgicos P2X7/metabolismo , Saponinas/farmacología , Transducción de Señal/efectos de los fármacos , Triterpenos/farmacología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/patología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Glucosa/farmacología , Interleucina-18/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Sustancias Protectoras/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno , Receptores Purinérgicos P2X7/química , Receptores Purinérgicos P2X7/genética , Saponinas/uso terapéutico , Triterpenos/uso terapéutico , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: Sepsis induces pulmonary P2X7 receptor (P2X7 R) expression and P2X7 R-knockout reduced lung inflammation in mice. The present study investigated the expression of circular RNA (circRNA) and mRNA in sepsis-induced acute lung injury (ALI) treated with a P2X7 R antagonist. METHODS: Sepsis was induced by tracheal administration of lipopolysaccharide (LPS), and the mice were then divided into two groups: without [sepsis + dimethyl sulfoxide (DMSO)] or with P2X7 R antagonist treatment (sepsis + P2X7 A). Sham mice were administrated sterile normal saline. Serum levels of interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, pathological changes, cell apoptosis and P2X7 R expression in lung were assessed, followed by RNA sequencing (RNA-seq) and bioinformatics analyses. A quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to validate circRNAs and mRNAs. RESULTS: Compared to the sham group, LPS-induced sepsis produced obvious pathological changes in lung tissue, as well as increased apoptotic lung cells, serum TNF-α and IL-1ß levels, and P2X7 R expression; P2X7 R antagonism significantly ameliorated these changes. RNA-seq identified many dysregulated circRNAs and mRNAs during sepsis, whereas this changed with P2X7 R antagonism. RT-qPCR confirmed that Mus musculus (mmu)_circ_0001679, mmu_circ_0001212, phospholamban (Pln), cadherin-2 (Cdh2) and nitrogen permease regulator 3-like (Nprl3) expression were significantly increased in the sepsis + DMSO group compared to that in the sham group but were decreased in the sepsis + P2X7 A group compared to that in the sepsis + DMSO group. The circRNA-microRNA-mRNA coexpression network indicated that mmu_circ_0001679 may regulate Nprl3 and that mmu_circ_0001212 may similarly regulate Pln, Cdh2 and Nprl3 as a competing endogenous RNA. CONCLUSIONS: P2X7 R antagonism attenuates sepsis-induced ALI by inhibiting dysregulated expression of circRNA (circ_0001679, circ_0001212) and mRNA (Pln, Cdh2 and Nprl3).
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Lesión Pulmonar Aguda/tratamiento farmacológico , Biomarcadores/metabolismo , Regulación de la Expresión Génica , Piridinas/farmacología , ARN Circular/genética , Receptores Purinérgicos P2X7/química , Sepsis/complicaciones , Tetrazoles/farmacología , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sustancias Protectoras/farmacología , Receptores Purinérgicos P2X7/metabolismo , Análisis de Secuencia de ARNRESUMEN
The coronavirus SARS-CoV-2 at the origin of COVID-19 shares more than 70% genetic similarity with SARS-CoV-1 that was at the origin of 2003 SARS. Infection-associated symptoms are very similar between SARS and COVID-19 diseases and are the same as community-acquired pneumonia symptoms. Antibiotics were empirically given to SARS patients in the early stages of the pathology whereas a different strategy has been decided in the management of COVID-19 pandemic with a worldwide shutdown. The cytokine storm, both identified in SARS and COVID-19 severe cases, is generated through inflammasome activation, which opens therapeutic perspectives to counteract the pathogenic inflammation. As corticoids have numerous side effects that limit their use, focusing on anti-inflammasome agents could represent a safer alternative for patients with severe COVID-19.