RESUMO
Trypanosoma cruzi is the pathogen of Chagas disease, a neglected tropical disease that affects more than 6 million people worldwide. There are no vaccines to prevent infection, and the therapeutic arsenal is very minimal and toxic. The unique E-NTPDase of T. cruzi (TcNTPDase1) plays essential roles in adhesion and infection and is a virulence factor. Quercetin is a flavonoid with antimicrobial, antiviral, and antitumor activities. Its potential as a partial inhibitor of NTPDases has also been demonstrated. In this work, we synthesized the non-natural L-glycoside derivatives of quercetin and evaluated them as inhibitors of recombinant TcNTPDase1 (rTcNTPDase1). These compounds, and quercetin and miquelianin, a natural quercetin derivative, were also tested. Compound 16 showed the most significant inhibitory effect (94%). Quercetin, miquelianin, and compound 14 showed inhibition close to 50%. We thoroughly investigated the inhibitory effect of 16. Our data suggested a competitive inhibition with a Ki of 8.39 µM (± 0.90). To better understand the interaction of compound 16 and rTcNTPDase1, we performed molecular dynamics simulations of the enzyme and docking analyses with the compounds. Our predictions show that compound 16 binds to the enzyme's catalytic site and interacts with important residues for NTPDase activity. As an inhibitor of a critical T. cruzi enzyme, (16) could be helpful as a starting point in the developing of a future treatment for Chagas disease. Furthermore, the discovery of (16) as an inhibitor of TcNTPDase1 may open new avenues in the study and development of new inhibitors of E-NTPDases.
RESUMO
Ectonucleotidases are a plasma membrane-bound enzyme that hydrolyses extracellular adenosine triphosphate (eATP) and adenosine diphosphate (eADP) to adenosine monophosphate (AMP). It regulates normal function of lymphocytes, acts as an inflammatory marker and represents a molecular target for new therapeutics. Thus, this study sought to isolate lymphocytes from blood (BL), spleen (SL) and cervical lymph node (CLL), and characterize the eATP and eADP enzymatic hydrolysis in Wistar rats. The hydrolysis of the nucleotides occurred primarily at pH 8.0, 37°C in the presence of Ca2+ or Mg2+ . Chevillard-plot showed the hydrolysis of eATP and eADP at the same active site. The inhibitors of some classical ATDPases did not cause any significant change on enzymatic activity. Inhibitors of E-NTPDase (-1, -2, -3 isoforms) and E-NPP-1 decrease the enzyme activity in all resident lymphocytes. Furthermore, kinetic parameters (Vmax and Km) revealed that SL had significantly (P < .001) higher enzymatic activity when compared to BL and CLL. In conclusion, this study standardized kinetic values for eATP and eADP hydrolysis for resident lymphocytes isolated from BL, SL and CLL.
Assuntos
5'-Nucleotidase/metabolismo , Linfonodos/química , Linfócitos/química , Nucleotídeos/metabolismo , Baço/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Hidrólise , Cinética , Linfonodos/metabolismo , Linfócitos/citologia , Linfócitos/metabolismo , Nucleotídeos/sangue , Nucleotídeos/isolamento & purificação , Ratos , Ratos Wistar , Baço/metabolismoRESUMO
The nucleotides were discovered in the early 19th century and a few years later, the role of such molecules in energy metabolism and cell survival was postulated. In 1972, a pioneer work by Burnstock and colleagues suggested that ATP could also work as a neurotransmitter, which was known as the "purinergic hypothesis". The idea of ATP working as a signaling molecule faced initial resistance until the discovery of the receptors for ATP and other nucleotides, called purinergic receptors. Among the purinergic receptors, the P2Y family is of great importance because it comprises of G proteincoupled receptors (GPCRs). GPCRs are widespread among different organisms. These receptors work in the cells' ability to sense the external environment, which involves: to sense a dangerous situation or detect a pheromone through smell; the taste of food that should not be eaten; response to hormones that alter metabolism according to the body's need; or even transform light into an electrical stimulus to generate vision. Advances in understanding the mechanism of action of GPCRs shed light on increasingly promising treatments for diseases that have hitherto remained incurable, or the possibility of abolishing side effects from therapies widely used today.
Assuntos
Plasmodium falciparum/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
HIV replication promotes atherogenesis and participates in the immune response to the virus, thereby influencing the inflammatory profile. These changes may, in turn, contribute to the risk of cardiovascular diseases with involvement of platelets. However, adenine nucleotides and nucleosides involved in thromboregulation and modulation of immune response may therefore be affected by these alterations. OBJECTIVES: This study sought to evaluate the profile of pro and anti-inflammatory cytokines (IL-10, IL-6, IL-17, TNF, IL-4, IL-2 and IFN-gamma), cardiac markers (troponin, CK, CK MB, LDH, CRP) in HIV-positive patients and assess the in vitro effect of antiretroviral therapy on the activities of ectonucleotidases (E-NTPDase and E-5'-nucleotidase) in human platelets. DESIGN AND METHODS: Blood samples were obtained from ten HIV positive patients at the Infectious Disease Clinic of the University Hospital of Santa Maria, Brazil and ten HIV negative individuals (control group) for this study. RESULTS: The results revealed that there were significant (P < 0.05) increases in serum levels of IL-6 and IFN-gamma with no significant (P > 0.05) changes in the serum levels of the cardiac markers investigated (CK, CK-MB, troponin, LDH and CRP). In addition, the ectonucleotidases (E-NTPDase and E-5'-nucleotidase) activities were not altered (P > 0.05) in human platelets when incubated with different antiretroviral drugs in vitro. CONCLUSIONS: The results of this study suggest that, despite successful treatment, a proinflammatory state is not altered in HIV patients, and that antiretroviral therapy per se does not change the purinergic profile.
Assuntos
Biomarcadores/sangue , Infecções por HIV/sangue , Infecções por HIV/imunologia , Adulto , Idoso , Fármacos Anti-HIV/administração & dosagem , Terapia Antirretroviral de Alta Atividade , Citocinas/sangue , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Inflamação/sangue , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Sepsis is a potentially lethal condition, and it is associated with platelet alterations. The present study sought to investigate the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA) in the platelets of rats that were induced with sepsis. Male Wistar rats were divided into three groups of ten animals each: a negative control group (normal; NC); a group that underwent surgical procedures (sham); and a group that underwent cecal ligation and perforation (CLP). The induction of sepsis was confirmed by bacteremia, and the causative pathogen identified was Escherichia coli. Hematological parameters showed leukocytosis and thrombocytopenia in animals in the septic group. The results also revealed that there were significant (p < 0.05) increases in adenosine triphosphate (ATP) and adenosine monophosphate (AMP) hydrolyses, and in the deamination of adenosine in the CLP group compared to the sham and control groups. Conversely, ADP hydrolysis was significantly decreased (p < 0.05) in the CLP group compared to the sham and control groups. Purine levels were analyzed by high-performance liquid chromatography (HPLC) in serum samples from control, sham, and CLP groups. Increased concentrations of ATP, adenosine, and inosine were found in the CLP group compared to the sham and control groups. Conversely, the concentrations of ADP and AMP in the CPL group were not significantly altered. We suggest that alterations in hematological parameters, nucleotide hydrolysis in platelets, and nucleotide concentrations in serum samples of rats with induced sepsis may be related to thromboembolic events.
Assuntos
5'-Nucleotidase/metabolismo , Plaquetas/enzimologia , Ceco/cirurgia , Ligadura/efeitos adversos , Complicações Pós-Operatórias/enzimologia , Sepse/enzimologia , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/metabolismo , Humanos , Masculino , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/microbiologia , Ratos , Ratos Wistar , Sepse/etiologia , Sepse/metabolismo , Sepse/microbiologiaRESUMO
This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E-NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+ . A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E-NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells.
Assuntos
Adenosina Trifosfatases/metabolismo , Linfócitos/enzimologia , Macrófagos/enzimologia , Neutrófilos/enzimologia , Cavidade Peritoneal/citologia , Animais , Cálcio/química , Cátions/química , Sobrevivência Celular , Células Cultivadas , Feminino , Concentração de Íons de Hidrogênio , Cinética , Linfócitos/citologia , Macrófagos/citologia , Magnésio/química , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/citologia , Especificidade por Substrato , TemperaturaRESUMO
Plasmodium falciparum is the causative agent of the most dangerous form of malaria in humans. It has been reported that the P. falciparum genome encodes for a single ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), an enzyme that hydrolyzes extracellular tri- and di-phosphate nucleotides. The E-NTPDases are known for participating in invasion and as a virulence factor in many pathogenic protozoa. Despite its presence in the parasite genome, currently, no information exists about the activity of this predicted protein. Here, we show for the first time that P. falciparum E-NTPDase is relevant for parasite lifecycle as inhibition of this enzyme impairs the development of P. falciparum within red blood cells (RBCs). ATPase activity could be detected in rings, trophozoites, and schizonts, as well as qRT-PCR, confirming that E-NTPDase is expressed throughout the intraerythrocytic cycle. In addition, transfection of a construct which expresses approximately the first 500 bp of an E-NTPDase-GFP chimera shows that E-NTPDase co-localizes with the endoplasmic reticulum (ER) in the early stages and with the digestive vacuole (DV) in the late stages of P. falciparum intraerythrocytic cycle.
Assuntos
Apirase/metabolismo , Eritrócitos/parasitologia , Malária/parasitologia , Plasmodium falciparum/parasitologia , Animais , Células Cultivadas , Eritrócitos/metabolismo , Hidrólise , ParasitosRESUMO
The activity of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5) was characterized in hepatic lymphocytes (HL) of rats. For this purpose, a specific method for the isolation of lymphocytes from hepatic tissue was developed. Subsequently, E-NTPDase activity of rat HL was compared with that of rat peripheral lymphocytes. The HL showed high cell count and viability. Also, the characterization test revealed that the optimal E-NTPDase activities were attained at 37°C and pH 8.0 in the presence of Ca2+ . In addition, in the presence of specific E-NTPDase inhibitors (20mM sodium azide and 0.3mM suramin), there were significant inhibitions in nucleotide hydrolysis. However, there was no significant change in adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolysis in the presence of inhibitors of other E-ATPase (0.1mM Ouabain, 0.5mM orthovanadate, and 1mM, 5mM, and 10mM sodium azide). Furthermore, the kinetic behavior of the enzyme in HL showed apparent Km of 134.90 ± 0.03µM and 214.40 ± 0.06µM as well as Vmax of 345.0 ± 28.32 and 242.0 ± 27.55 Æmol Pi/min/mg of protein for ATP and ADP, respectively. The Chevillard plot revealed that ATP and ADP were hydrolyzed at the same active site of the enzyme. Our results suggest that the degradation of extracellular nucleotides in HL may have been primarily accomplished by E-NTPDase. The higher E-NTPDase activity observed in HL may be attributed to the important physiological functions of ATP and ADP in HL. SIGNIFICANCE OF THE STUDY: Extracellular purine nucleotides are able to interact with specific receptors and trigger a number of important physiological functions in cells. This interaction is controlled by ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), enzyme that present their catalytic site at the extracellular space and degrades nucleotides. This purinergic signaling has important functions in peripheral lymphocytes and may represent an important new therapeutic target for the treatment of immunological diseases. However, there is dearth of information on the involvement of E-NTPDase in liver lymphocytes. The liver is an important organ, which performs both metabolic and toxicological roles in living organism, and hepatic lymphocytes may play crucial action in the regulation of immune responses in the liver tissue. Furthermore, various chronic diseases such as cirrhosis may be treated with novel pharmacotherapy by targeting the modulation of hepatic lymphocytes. Thus, the significance of this study is to evaluate the activity of E-NTPDase in liver lymphocyte and compare its activity with the peripheral lymphocytes.
Assuntos
Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Células Sanguíneas/enzimologia , Fígado/enzimologia , Linfócitos/enzimologia , Animais , Antígenos CD/genética , Apirase/antagonistas & inibidores , Apirase/genética , Células Sanguíneas/citologia , Células Sanguíneas/efeitos dos fármacos , Cálcio/metabolismo , Cátions Bivalentes , Separação Celular/métodos , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Fígado/citologia , Fígado/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Especificidade de Órgãos , Ouabaína/farmacologia , Ratos , Ratos Wistar , Azida Sódica/farmacologia , Especificidade por Substrato , Suramina/farmacologia , Vanadatos/farmacologiaRESUMO
UNLABELLED: The effect of vitamin D3 in oral solution (VD3 ) and vitamin D3 -loaded nanocapsules (NC-VD3 ) was analysed in animals with complete Freund's adjuvant (CFA) induced arthritis (AR). For this purpose, we evaluated scores for arthritis, thermal hyperalgesia and paw oedema, as well as histological analyses and measurements of the activity of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) enzymes in rat lymphocytes. Haematological and biochemical parameters were also determined. The doses administered were 120 UI/day of VD3 and 15.84 UI/day of NC-VD3 . Fifteen days after the induction of AR, the groups were treated for 15 days with vitamin D3 . The results demonstrated that VD3 was able to reduce arthritis scores, thermal hyperalgesia and paw oedema in rats with CFA-induced arthritis. However, treatment with NC-VD3 did not reduce arthritis scores. The histological analyses showed that both formulations were able to reduce the inflammatory changes induced by CFA. The activity of E-NTPDase in rat lymphocytes was higher in the AR compared with the control group, while the activity of E-ADA was lower. This effect was reversed after the 15-day treatment. Data from this study indicates that both forms of vitamin D3 seem to contribute to decreasing the inflammatory process induced by CFA, possibly altering the activities of ectoenzymes. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: The effects promoted by both formulations of vitamin D3 , either in oral solution or nanoencapsulated form, strongly suggests the softening of the inflammatory process induced by complete Freund's adjuvant (CFA), possibly altering the E-NTPDase and E-ADA activities. However, it is known that vitamin D has a beneficial effect on the modulation of the immune system components responsible for the inflammatory process. Moreover, the establishment of responses to treatment with vitamin D3 may provide an alternative for inhibiting the proinflammatory response, assisting in our understanding of the immunopathology of this disease and possibly improving the signs and symptoms that hinder the quality of life of patients with rheumatoid arthritis. HIGHLIGHTS: Evaluation of the effects on the E-NTPDase and E-ADA activities in an animal model of induced arthritis. Two formulations of vitamin D3 were used: form oral solution and nanoencapsulated. Vitamin D3 seems to contribute to the inflammatory process induced by CFA. Vitamin D3 possibly alters the E-NTPDase and E-ADA activities. Vitamin D3 may be an alternative supplementary treatment for chronic arthritis.
Assuntos
Adenosina Desaminase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Colecalciferol/uso terapêutico , Nanopartículas/química , Administração Oral , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artrite Reumatoide/sangue , Artrite Reumatoide/patologia , Colecalciferol/sangue , Colecalciferol/farmacologia , Modelos Animais de Doenças , Feminino , Adjuvante de Freund , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Nanocápsulas/química , Ratos Wistar , SoluçõesRESUMO
Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.