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1.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360719

RESUMO

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.


Assuntos
Agonistas do Receptor A3 de Adenosina/uso terapêutico , Sinalização do Cálcio/efeitos dos fármacos , Gânglios Espinais , Dor , Receptor A3 de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Humanos , Microglia/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Dor/fisiopatologia
2.
Eur J Med Chem ; 223: 113607, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34171656

RESUMO

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A1, A2A, A2B and A3 adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A3 AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N6-(3-phenylpropyl)adenosine (18), N6-(3-phenylpropyl)-2-chloroadenosine (24) and N6-(3-phenylpropyl)adenosine (40) showed nanomolar A3 affinity (Ki 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Receptor A3 de Adenosina/metabolismo , Adenosina/metabolismo , Agonistas do Receptor A3 de Adenosina/síntese química , Agonistas do Receptor A3 de Adenosina/metabolismo , Animais , Compostos de Boro/síntese química , Compostos de Boro/metabolismo , Compostos de Boro/farmacologia , Células CHO , Cricetulus , Células HEK293 , Humanos , Ligantes , Estrutura Molecular , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Relação Estrutura-Atividade
3.
J Med Chem ; 64(12): 8161-8178, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34120444

RESUMO

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.


Assuntos
Antagonistas do Receptor A1 de Adenosina/farmacologia , Antagonistas do Receptor A3 de Adenosina/farmacologia , Sondas Moleculares/farmacologia , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Xantinas/farmacologia , Antagonistas do Receptor A1 de Adenosina/síntese química , Antagonistas do Receptor A3 de Adenosina/síntese química , Alcinos/química , Animais , Azidas/química , Células CHO , Química Click , Cricetulus , Desenho de Fármacos , Corantes Fluorescentes/química , Humanos , Ligantes , Sondas Moleculares/síntese química , Receptor A1 de Adenosina/química , Receptor A3 de Adenosina/química , Xantinas/síntese química
4.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073488

RESUMO

Kidney fibrosis is the final outcome of chronic kidney disease (CKD). Adenosine plays a significant role in protection against cellular damage by activating four subtypes of adenosine receptors (ARs), A1AR, A2AAR, A2BAR, and A3AR. A2AAR agonists protect against inflammation, and A3AR antagonists effectively inhibit the formation of fibrosis. Here, we showed for the first time that LJ-4459, a newly synthesized dual-acting ligand that is an A2AAR agonist and an A3AR antagonist, prevents the progression of tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed on 6-week-old male C57BL/6 mice. LJ-4459 (1 and 10 mg/kg) was orally administered for 7 days, started at 1 day before UUO surgery. Pretreatment with LJ-4459 improved kidney morphology and prevented the progression of tubular injury as shown by decreases in urinary kidney injury molecular-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion. Obstruction-induced tubulointerstitial fibrosis was attenuated by LJ-4459, as shown by a decrease in fibrotic protein expression in the kidney. LJ-4459 also inhibited inflammation and oxidative stress in the obstructed kidney, with reduced macrophage infiltration, reduced levels of pro-inflammatory cytokines, as well as reduced levels of reactive oxygen species (ROS). These data demonstrate that LJ-4459 has potential as a therapeutic agent against the progression of tubulointerstitial fibrosis.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Nefropatias/tratamento farmacológico , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Obstrução Ureteral/tratamento farmacológico , Agonistas do Receptor A3 de Adenosina/síntese química , Agonistas do Receptor A3 de Adenosina/química , Animais , Fibrose , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Ligantes , Masculino , Camundongos , Obstrução Ureteral/complicações , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
5.
FASEB J ; 35(4): e21211, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710641

RESUMO

Organization of G protein-coupled receptors at the plasma membrane has been the focus of much recent attention. Advanced microscopy techniques have shown that these receptors can be localized to discrete microdomains and reorganization upon ligand activation is crucial in orchestrating their signaling. Here, we have compared the membrane organization and downstream signaling of a mutant (R108A, R3.50A) of the adenosine A3 receptor (A3 AR) to that of the wild-type receptor. Fluorescence Correlation Spectroscopy (FCS) studies with a fluorescent agonist (ABEA-X-BY630) demonstrated that both wild-type and mutant receptors bind agonist with high affinity but in subsequent downstream signaling assays the R108A mutation abolished agonist-mediated inhibition of cAMP production and ERK phosphorylation. In further FCS studies, both A3 AR and A3 AR R108A underwent similar agonist-induced increases in receptor density and molecular brightness which were accompanied by a decrease in membrane diffusion after agonist treatment. Using bimolecular fluorescence complementation, experiments showed that the R108A mutant retained the ability to recruit ß-arrestin and these receptor/arrestin complexes displayed similar membrane diffusion and organization to that observed with wild-type receptors. These data demonstrate that effective G protein signaling is not a prerequisite for agonist-stimulated ß-arrestin recruitment and membrane reorganization of the A3 AR.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Compostos de Boro/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Receptor A3 de Adenosina/metabolismo , Adenosina/farmacologia , Animais , Arrestina/metabolismo , Células CHO , Cricetulus , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação , Ligação Proteica , Receptor A3 de Adenosina/genética
6.
Mol Immunol ; 132: 1-7, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33524770

RESUMO

Macrophages perform the fundamental function of sensing cellular damage, initiating and mediating immune response and tissue repair. Adenine nucleotides are in relatively high abundance in cells and are released from cells during tissue damage that are converted to adenosine in the extracellular environment. The A1, A2A, A2B and A3 adenosine receptors serve to regulate immune function. Despite characterization of the adenosine receptors, a comprehensive examination of adenosine receptor signaling in THP-1 macrophage cells has not been done. Moreover, previous studies employed chemical agonists and antagonists that have the potential for off-target affects. Here we systematically knockdown each of the four known adenosine receptors in THP-1 macrophages using validated siRNA and investigated their function under LPS stimulation. We demonstrate that the A1 receptor is required for adenosine-stimulated IL-10 and IL-1ß secretion indicating an important role of this receptor during resolution of inflammation and tissue repair in these cells. The A1 and A3 receptor were required for IL-6 and IL-1ß secretion showing a net pro-inflammatory role for these receptors. Finally, we present the novel finding that THP-1 macrophages lacking the A2B receptor undergo pyroptosis when exposed to LPS, demonstrating a novel role of the A2B receptor in regulation of programmed cell death during inflammation. This work underscores the fundamental importance of adenosine signaling and provides insight into the independent roles of the adenosine receptors in modulating cytokine signaling.


Assuntos
Citocinas/metabolismo , Macrófagos/metabolismo , Piroptose/imunologia , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Adenosina/farmacologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Piroptose/efeitos dos fármacos , Piroptose/genética , RNA Interferente Pequeno , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genética , Receptor A2B de Adenosina/genética , Receptor A3 de Adenosina/genética , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/imunologia
7.
PLoS One ; 16(2): e0247659, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33630907

RESUMO

Pulsed-electromagnetic-field (PEMF) treatment was found to enhance cellular differentiation of the mouse preosteoblast, MC3T3-E1, to a more osteoblastic phenotype. Differentiation genes such as Alp, BSPI, cFos, Ibsp, Osteocalcin, Pthr1 and Runx2 showed increased expression in response to PEMF stimulation. Detailed molecular mechanisms linking PEMF to the activation of these genes are limited. Two adenosine receptors known to be modulated in response to PEMF, Adora2A and Adora3, were functionally impaired by CRISPR-Cas9-mediated gene disruption, and the consequences of which were studied in the context of PEMF-mediated osteoblastic differentiation. Disruption of Adora2A resulted in a delay of Alp mRNA expression, but not alkaline phosphatase protein expression, which was similar to that found in wild type cells. However, Adora3 disruption resulted in significantly reduced responses at both the alkaline phosphatase mRNA and protein levels throughout the PEMF stimulation period. Defects observed in response to PEMF were mirrored using a chemically defined growth and differentiation-inducing media (DM). Moreover, in cells with Adora2A disruption, gene expression profiles showed a blunted response in cFos and Pthr1 to PEMF treatment; whereas cells with Adora3 disruption had mostly blunted responses in AlpI, BSPI, Ibsp, Osteocalcin and Sp7 gene activation. To demonstrate specificity for Adora3 function, the Adora3 open reading frame was inserted into the ROSA26 locus in Adora3 disrupted cells culminating in rescued PEMF responsiveness and thereby eliminating the possibility of off-target effects. These results lead us to propose that there are complementary and parallel positive roles for adenosine receptor A2A and A3 in PEMF-mediated osteoblast differentiation.


Assuntos
Radiação Eletromagnética , Osteoblastos/citologia , Osteogênese , Receptor A2A de Adenosina , Receptor A3 de Adenosina , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Regulação da Expressão Gênica , Camundongos , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo
8.
Int J Mol Med ; 47(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33537802

RESUMO

Paris saponin H (PSH) is a type of steroid saponin derived from Rhizoma Paridis (RP; the rhizome of Paris). In our previous studies, saponins from RP exerted antiglioma activity in vitro. However, the effects of PSH on glioma have not been elucidated. The aim of the present study was to evaluate the effects of PSH on U251 glioblastoma cells and elucidate the possible underlying mechanism. The cells were treated with PSH at various concentrations for 48 h, and the cell viability, invasion, apoptosis and cycle progression were assessed using specific assay kits. The activation of Akt, 44/42­mitogen­activated protein kinase (MAPK) and the expression levels of A1 adenosine receptor (ARA1) and ARA3 were assessed by western blotting. The results demonstrated that PSH inhibited cell viability, migration and invasion, and induced apoptosis. Treatment of U251 cells with PSH induced the upregulation of p21 and p27, and the downregulation cyclin D1 and S­phase kinase associated protein 2 protein expression levels, which induced cell cycle arrest at the G1 phase. The results also demonstrated that PSH inhibited the expression of ARA1, and the agonist of ARA1, 2­chloro­N6­cyclopentyladenosine, reversed the effects of PSH. Hypoxia induced increases in the ARA3, hypoxia­inducible factor­1α (HIF­1α) and vascular endothelial growth factor (VEGF) protein expression levels, which were associated with the activation of the Akt and P44/42 MAPK pathways. Compared with the hypoxia group, PSH inhibited the expression levels of ARA3, HIF­1α and VEGF, as well as the phosphorylation levels of Akt and 44/42 MAPK, and repressed HIF­1α transcriptional activity. Furthermore, the results demonstrated that PSH inhibited the expression of HIF­1α by inhibiting the phosphorylation of Akt and 44/42 MAPK mediated by ARA3. Taken together, these results suggested that PSH reduced U251 cell viability via the inhibition of ARA1 and ARA3 expression, and further inhibited Akt and 44/42 MAPK phosphorylation, induced apoptosis and cell cycle arrest.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Saponinas/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fase G1/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Mol Cell ; 81(4): 659-674.e7, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33472058

RESUMO

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Processamento Pós-Transcricional do RNA , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais , Adenosina/genética , Adenosina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Masculino , Coelhos , Receptor A3 de Adenosina/genética
10.
Exp Cell Res ; 399(2): 112482, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33434531

RESUMO

IL-6-triggered Th17 cell expansion is responsible for the pathogenesis of many immune diseases including rheumatoid arthritis (RA). Traditionally, IL-6 induces Th17 cell differentiation through JAK-STAT3 signaling. In the present work, PKA inhibition reduces in vitro induction of Th17 cells, while IL-6 stimulation of T cells facilitates the internalization of A3AR and increased cAMP production in a GRK2 dependent manner. Inhibition of GRK2 by paroxetine (PAR) or genetic depletion of GRK2 restored A3AR distribution and prevented Th17 cell differentiation. Furthermore, in vivo PAR treatment effectively reduced the splenic Th17 cell proportion in a rat model of collagen-induced arthritis (CIA) which was accompanied by a significant improvement in clinical manifestations. These results indicate that IL-6-induced Th17 cell differentiation not only occurs through JAK-STAT3-RORγt but is also mediated through GRK2-A3AR-cAMP-PKA-CREB/ICER-RORγt. This elucidates the significance of GRK2-controlled cAMP signaling in the differentiation of Th17 cells and its potential application in treating Th17-driven immune diseases such as RA.


Assuntos
Quinase 2 de Receptor Acoplado a Proteína G/genética , Interleucina-6/farmacologia , Receptor A3 de Adenosina/metabolismo , Células Th17/fisiologia , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Interleucina-6/fisiologia , Masculino , Ratos , Ratos Transgênicos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Th17/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética
11.
J Immunol Res ; 2020: 8632048, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33299899

RESUMO

Novel coronavirus disease 2019 (COVID-19) causes pulmonary and cardiovascular disorders and has become a worldwide emergency. Myocardial injury can be caused by direct or indirect damage, particularly mediated by a cytokine storm, a disordered immune response that can cause myocarditis, abnormal coagulation, arrhythmia, acute coronary syndrome, and myocardial infarction. The present review focuses on the mechanisms of this viral infection, cardiac biomarkers, consequences, and the possible therapeutic role of purinergic and adenosinergic signalling systems. In particular, we focus on the interaction of the extracellular nucleotide adenosine triphosphate (ATP) with its receptors P2X1, P2X4, P2X7, P2Y1, and P2Y2 and of adenosine (Ado) with A2A and A3 receptors, as well as their roles in host immune responses. We suggest that receptors of purinergic signalling could be ideal candidates for pharmacological targeting to protect against myocardial injury caused by a cytokine storm in COVID-19, in order to reduce systemic inflammatory damage to cells and tissues, preventing the progression of the disease by modulating the immune response and improving patient quality of life.


Assuntos
Trifosfato de Adenosina/metabolismo , COVID-19/imunologia , Doenças Cardiovasculares/virologia , Receptores Purinérgicos/metabolismo , SARS-CoV-2 , Agonistas do Receptor A2 de Adenosina/farmacologia , COVID-19/tratamento farmacológico , COVID-19/metabolismo , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/fisiopatologia , Citocinas/metabolismo , Humanos , Isquemia Miocárdica/imunologia , Isquemia Miocárdica/fisiopatologia , Isquemia Miocárdica/virologia , Pandemias , Antagonistas Purinérgicos/farmacologia , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia
12.
Sci Rep ; 10(1): 20781, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247159

RESUMO

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.


Assuntos
Antagonistas do Receptor A3 de Adenosina/química , Antagonistas do Receptor A3 de Adenosina/farmacologia , Antagonistas do Receptor A3 de Adenosina/farmacocinética , Animais , Sítios de Ligação/genética , Ligação Competitiva , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ensaio Radioligante , Ratos , Receptor A3 de Adenosina/química , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade
13.
Int J Mol Sci ; 21(22)2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33218074

RESUMO

Adenosine is a purine nucleoside, responsible for the regulation of multiple physiological and pathological cellular and tissue functions by activation of four G protein-coupled receptors (GPCR), namely A1, A2A, A2B, and A3 adenosine receptors (ARs). In recent years, extensive progress has been made to elucidate the role of adenosine in pain regulation. Most of the antinociceptive effects of adenosine are dependent upon A1AR activation located at peripheral, spinal, and supraspinal sites. The role of A2AAR and A2BAR is more controversial since their activation has both pro- and anti-nociceptive effects. A3AR agonists are emerging as promising candidates for neuropathic pain. Although their therapeutic potential has been demonstrated in diverse preclinical studies, no AR ligands have so far reached the market. To date, novel pharmacological approaches such as adenosine regulating agents and allosteric modulators have been proposed to improve efficacy and limit side effects enhancing the effect of endogenous adenosine. This review aims to provide an overview of the therapeutic potential of ligands interacting with ARs and the adenosinergic system for the treatment of acute and chronic pain.


Assuntos
Regulação Alostérica , Neuralgia/prevenção & controle , Agonistas do Receptor Purinérgico P1/farmacologia , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/metabolismo , Doença Aguda , Animais , Dor Crônica/metabolismo , Dor Crônica/prevenção & controle , Humanos , Ligantes , Neuralgia/metabolismo
14.
Molecules ; 25(18)2020 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-32961824

RESUMO

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin-chalcone hybrids were synthesized (compounds 1-8) and screened in radioligand binding (hA1, hA2A and hA3) and adenylyl cyclase (hA2B) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin-chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA1 or hA3 subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA1, while the methoxy counterparts were selective for the hA3. The most potent hA1 ligand was compound 7 (Ki = 17.7 µM), whereas compound 4 was the most potent ligand for hA3 (Ki = 2.49 µM). In addition, docking studies with hA1 and hA3 homology models were established to analyze the structure-function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity.


Assuntos
Chalcona/química , Chalconas/química , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Sítios de Ligação , Chalcona/metabolismo , Chalconas/metabolismo , Desenho de Fármacos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor A1 de Adenosina/química , Receptor A2A de Adenosina/química , Receptor A3 de Adenosina/química , Relação Estrutura-Atividade
15.
Int J Mol Sci ; 21(14)2020 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709103

RESUMO

The aim of this paper was to check the possible interaction of two of the four purinergic P1 receptors, the A2A and the A3. Discovery of the A2A-A3 receptor complex was achieved by means of immunocytochemistry and of bioluminescence resonance energy transfer. The functional properties and heteromer print identification were addressed by combining binding and signaling assays. The physiological role of the novel heteromer is to provide a differential signaling depending on the pre-coupling to signal transduction components and/or on the concentration of the endogenous agonist. The main feature was that the heteromeric context led to a marked decrease of the signaling originating at A3 receptors. Interestingly from a therapeutic point of view, A2A receptor antagonists overrode the blockade, thus allowing A3 receptor-mediated signaling. The A2A-A3 receptor heteromer print was detected in primary cortical neurons. These and previous results suggest that all four adenosine receptors may interact with each other. Therefore, each adenosine receptor could form heteromers with distinct properties, expanding the signaling outputs derived from the binding of adenosine to its cognate receptors.


Assuntos
Mapas de Interação de Proteínas , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Receptor A2A de Adenosina/análise , Receptor A3 de Adenosina/análise , Transdução de Sinais
16.
Int J Mol Sci ; 21(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604732

RESUMO

The adenosine A3 receptor (A3R) is the only adenosine receptor subtype to be overexpressed in inflammatory and cancer cells and therefore is considered a novel and promising therapeutic target for inflammatory diseases and cancer. Heterologous expression of A3R at levels to allow biophysical characterization is a major bottleneck in structure-guided drug discovery efforts. Here, we apply protein engineering using chimeric receptors to improve expression and activity in yeast. Previously we had reported improved expression and trafficking of the chimeric A1R variant using a similar approach. In this report, we constructed chimeric A3/A2AR comprising the N-terminus and transmembrane domains from A3R (residues 1-284) and the cytoplasmic C-terminus of the A2AR (residues 291-412). The chimeric receptor showed approximately 2-fold improved expression with a 2-fold decreased unfolded protein response when compared to wild type A3R. Moreover, by varying culture conditions such as initial cell density and induction temperature a further 1.7-fold increase in total receptor yields was obtained. We observed native-like coupling of the chimeric receptor to Gai-Gpa1 in engineered yeast strains, activating the downstream, modified MAPK pathway. This strategy of utilizing chimeric receptor variants in yeast thus provides an exciting opportunity to improve expression and activity of "difficult-to-express" receptors, expanding the opportunity for utilizing yeast in drug discovery.


Assuntos
Adenosina/metabolismo , Membrana Celular/metabolismo , Mutação , Receptor A2A de Adenosina/metabolismo , Receptor A3 de Adenosina/química , Receptor A3 de Adenosina/metabolismo , Saccharomyces cerevisiae/metabolismo , Quimera , Humanos , Dobramento de Proteína , Receptor A2A de Adenosina/genética , Receptor A3 de Adenosina/genética , Saccharomyces cerevisiae/genética
17.
Arch Pharm Res ; 43(5): 540-552, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32430718

RESUMO

Chronic obstructive pulmonary disease (COPD) is the leading cause of human death worldwide. Currently available therapies for COPD mainly relieve symptoms and preserve lung function, suggesting the need to develop novel therapeutic or preventive regimens. Because chronic inflammation is a mechanism of emphysematous lesion formation and because adenosine A3 receptor signaling and peroxisome proliferator-activated receptor gamma (PPARγ) regulate inflammation, we investigated the effect of LJ-529, a selective adenosine A3 receptor agonist and partial PPARγ agonist, on inflammation in vitro and elastase-induced pulmonary emphysema in vivo. LJ-529 markedly ameliorated elastase-induced emphysematous lesion formation in the lungs in vivo, as indicated by the restoration of pulmonary function, suppression of airspace enlargement, and downregulation of elastase-induced matrix metalloproteinase activity and apoptotic cell death in the lungs. LJ-529 induced the expression of PPARγ target genes, the activity of PPARγ and several cytokines involved in inhibiting inflammation and inducing anti-inflammatory M2-like phenotypes. Moreover, LJ-529 did not exhibit significant cytotoxicity in normal cell lines derived from various organs in vitro and induced minimal changes in body weight in vivo, suggesting no overt toxicity of LJ-529 in vitro or in vivo. These results indicate the potential of LJ-529 as a novel therapeutic/preventive agent for emphysema with limited toxicity.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Anti-Inflamatórios/farmacologia , PPAR gama/agonistas , Enfisema Pulmonar/tratamento farmacológico , Receptor A3 de Adenosina/metabolismo , Tionucleosídeos/farmacologia , Adenosina/administração & dosagem , Adenosina/farmacologia , Agonistas do Receptor A3 de Adenosina/administração & dosagem , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos , PPAR gama/genética , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Enfisema Pulmonar/metabolismo , Tionucleosídeos/administração & dosagem
18.
J Pharmacol Exp Ther ; 374(2): 331-341, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434943

RESUMO

Treating chronic pain by using opioids, such as morphine, is hampered by the development of opioid-induced hyperalgesia (OIH; increased pain sensitivity), antinociceptive tolerance, and withdrawal, which can contribute to dependence and abuse. In the central nervous system, the purine nucleoside adenosine has been implicated in beneficial and detrimental actions of morphine, but the extent of their interaction remains poorly understood. Here, we demonstrate that morphine-induced OIH and antinociceptive tolerance in rats is associated with a twofold increase in adenosine kinase (ADK) expression in the dorsal horn of the spinal cord. Blocking ADK activity in the spinal cord provided greater than 90% attenuation of OIH and antinociceptive tolerance through A3 adenosine receptor (A3AR) signaling. Supplementing adenosine signaling with selective A3AR agonists blocked OIH and antinociceptive tolerance in rodents of both sexes. Engagement of A3AR in the spinal cord with an ADK inhibitor or A3AR agonist was associated with reduced dorsal horn of the spinal cord expression of the NOD-like receptor pyrin domain-containing 3 (60%-75%), cleaved caspase 1 (40%-60%), interleukin (IL)-1ß (76%-80%), and tumor necrosis factor (50%-60%). In contrast, the neuroinhibitory and anti-inflammatory cytokine IL-10 increased twofold. In mice, A3AR agonists prevented the development of tolerance in a model of neuropathic pain and reduced naloxone-dependent withdrawal behaviors by greater than 50%. These findings suggest A3AR-dependent adenosine signaling is compromised during sustained morphine to allow the development of morphine-induced adverse effects. These findings raise the intriguing possibility that A3AR agonists may be useful adjunct to opioids to manage their unwanted effects. SIGNIFICANCE STATEMENT: The development of hyperalgesia and antinociceptive tolerance during prolonged opioid use are noteworthy opioid-induced adverse effects that reduce opioid efficacy for treating chronic pain and increase the risk of dependence and abuse. We report that in rodents, these adverse effects are due to reduced adenosine signaling at the A3AR, resulting in NOD-like receptor pyrin domain-containing 3-interleukin-1ß neuroinflammation in spinal cord. These effects are attenuated by A3AR agonists, suggesting that A3AR may be a target for therapeutic intervention with selective A3AR agonist as opioid adjuncts.


Assuntos
Analgésicos/efeitos adversos , Tolerância a Medicamentos , Hiperalgesia/induzido quimicamente , Morfina/efeitos adversos , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/etiologia , Adenosina/metabolismo , Animais , Feminino , Hiperalgesia/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/biossíntese , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
19.
Cell Death Dis ; 11(5): 401, 2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32461578

RESUMO

Glaucoma is a progressive chronic retinal degenerative disease and a leading cause of global irreversible blindness. This disease is characterized by optic nerve damage and retinal ganglion cell (RGC) death. The current treatments available target the lowering of intraocular pressure (IOP), the main risk factor for disease onset and development. However, in some patients, vision loss progresses despite successful IOP control, indicating that new and effective treatments are needed, such as those targeting the neuroprotection of RGCs. Adenosine A3 receptor (A3R) activation confers protection to RGCs following an excitotoxic stimulus. In this work, we investigated whether the activation of A3R could also afford protection to RGCs in the laser-induced ocular hypertension (OHT) model, a well-characterized animal model of glaucoma. The intravitreal injection of 2-Cl-IB-MECA, a selective A3R agonist, abolished the alterations induced by OHT in the negative and positive components of scotopic threshold response (STR) without changing a- and b-wave amplitudes both in scotopic and photopic conditions. Moreover, the treatment of OHT eyes with the A3R agonist promoted the survival of RGCs, attenuated the impairment in retrograde axonal transport, and improved the structure of the optic nerve. Taking into consideration the beneficial effects afforded by 2-Cl-IB-MECA, we can envisage that A3R activation can be considered a good therapeutic strategy to protect RGCs from glaucomatous damage.


Assuntos
Neuroproteção , Hipertensão Ocular/complicações , Receptor A3 de Adenosina/metabolismo , Degeneração Retiniana/etiologia , Células Ganglionares da Retina/patologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Agonistas do Receptor A3 de Adenosina/farmacologia , Animais , Transporte Axonal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Neuroproteção/efeitos dos fármacos , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Ratos Sprague-Dawley , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/ultraestrutura , Regulação para Cima/efeitos dos fármacos
20.
Biochem Pharmacol ; 177: 113934, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32224136

RESUMO

The A3 adenosine receptor (A3AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable ß-arrestin 2 (ßarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGαi protein to activated A3AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N6-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the ßarr2 or miniGαi pathway.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Receptor A3 de Adenosina/metabolismo , beta-Arrestina 2/metabolismo , Adenosina/análogos & derivados , Agonistas do Receptor A3 de Adenosina/síntese química , Citometria de Fluxo/métodos , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Ligantes , Medições Luminescentes/métodos , Receptor A3 de Adenosina/genética , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Transdução Genética/métodos , Transfecção/métodos , beta-Arrestina 2/genética
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