RESUMO
Some studies aimed at revealing the relationship between protein structure and their functional properties. However, the majority of these reports have been carried out using protein isolates. There are limited reports on the possible relationship between the functional properties and the structure of a purified protein. In this work the amaranth 11S globulin acidic subunit (AAC) and five mutations of the same protein that were modified in their variable regions with antihypertensive peptides (VYVYVYVY and RIPP), were analyzed at two ionic strength (2.9 and 17.6 g/L NaCl) and pH (3.0-7.0). Results revealed better solubility for the proteins mutated at the terminal ends (AACM.1 and AACM.4) and lower solubility for the protein inserted with RIPP peptide. Spectroscopy studies revealed an increase of ß-sheet structure at high salt concentration for all proteins. It was also observed that salt concentration acted as a modulator, which allowed a better foam features for all modified proteins limiting movement of side chains and reducing red-shifted displacement of λmax. All proteins showed foam capacity ranging from 76 to 93% although foam stability was twofold better for modified proteins than for AAC at high salt concentration. This study allowed better understanding about the structural changes that influence the foaming properties of engineered proteins.
Assuntos
Amaranthus , Globulinas , Amaranthus/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Globulinas/química , Concentração de Íons de Hidrogênio , Concentração Osmolar , Peptídeos/metabolismo , Proteínas de Plantas/metabolismoRESUMO
Organic nitrates are widely used to restore endogenous nitric oxide (NO) levels reduced by endothelial nitric oxide synthase dysfunction. However, these drugs are associated with undesirable side effects, including tolerance. This study aims to investigate the cardiovascular effects of the new organic nitrate 1,3-diisobutoxypropan-2-yl nitrate (NDIBP). Specifically, we assessed its effects on blood pressure, vascular reactivity, acute toxicity, and the ability to induce tolerance. In vitro and ex vivo techniques showed that NDIBP released NO both in a cell-free system and in isolated mesenteric arteries preparations through a process catalyzed by xanthine oxidoreductase. NDIBP also evoked endothelium-independent vasorelaxation, which was significantly attenuated by 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO, 300 µM), a nitric oxide scavenger; 1-H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), a soluble guanylyl cyclase inhibitor; tetraethylammonium (TEA, 3 mM), a potassium channel blocker; febuxostat (500 nM), a xanthine oxidase inhibitor; and proadifen (10 µM), an inhibitor of cytochrome P450 enzyme. Furthermore, this organic nitrate did not induce tolerance in isolated vessels and presented low toxicity following acute oral administration. In vivo changes on cardiovascular parameters were assessed using normotensive and renovascular hypertensive rats. NDIBP evoked a reduction of blood pressure that was significantly higher in hypertensive animals. Our results suggest that NDIBP acts as a NO donor, inducing blood pressure reduction without having the undesirable effects of tolerance. Those effects seem to be mediated by activation of NO-sGC-cGMP pathway and positive modulation of K+ channels in vascular smooth muscle.
Assuntos
Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Artérias Mesentéricas/efeitos dos fármacos , Nitratos/uso terapêutico , Doadores de Óxido Nítrico/uso terapêutico , Vasodilatadores/uso terapêutico , Animais , Anti-Hipertensivos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Hipertensão/metabolismo , Masculino , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/metabolismo , Canais de Potássio/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel/metabolismo , Vasodilatadores/metabolismo , Xantina Desidrogenase/metabolismoRESUMO
BACKGROUND: The 11S globulin from amaranth is the most abundant storage protein in mature seeds and is well recognized for its nutritional value. We used this globulin to engineer a new protein by adding a four valinetyrosine antihypertensive peptide at its C-terminal end to improve its functionality. The new protein was named AMR5 and expressed in the Escherichia coli BL21-CodonPlus(DE3)-RIL strain using a custom medium (F8PW) designed for this work. RESULTS: The alternative medium allowed for the production of 652 mg/L expressed protein at the flask level, mostly in an insoluble form, and this protein was subjected to in vitro refolding. The spectrometric analysis suggests that the protein adopts a ß/α structure with a small increment of α-helix conformation relative to the native amaranth 11S globulin. Thermal and urea denaturation experiments determined apparent Tm and C1/2 values of 50.4°C and 3.04 M, respectively, thus indicating that the antihypertensive peptide insertion destabilized the modified protein relative to the native one. AMR5 hydrolyzed by trypsin and chymotrypsin showed 14- and 1.3-fold stronger inhibitory activity against angiotensin I-converting enzyme (IC50 of 0.034 mg/mL) than the unmodified protein and the previously reported amaranth acidic subunit modified with antihypertensive peptides, respectively. CONCLUSION: The inserted peptide decreases the structural stability of amaranth 11S globulin and improves its antihypertensive activity.
Assuntos
Peptídeos/metabolismo , Proteínas/metabolismo , Globulinas/metabolismo , Anti-Hipertensivos/metabolismo , Sementes , Temperatura , Meios de Cultura , Amaranthus , Estabilidade Proteica , Compostos FitoquímicosRESUMO
Hydrogen sulfide (H2S) is a vasorelaxant gas with therapeutic potential in several diseases. However, effects of H2S donors in hypertensive pregnancy complicated by feto-placental growth restriction are unclear. Therefore, we aimed to examine and compare the effects of fast-releasing H2S donor (sodium hydrosulfide-NaHS) and slow-releasing H2S donor (GYY4137) in hypertension-in-pregnancy. Pregnant rats were distributed into four groups: normal pregnancy (Norm-Preg), hypertensive pregnancy (HTN-Preg), hypertensive pregnancy + NaHS (HTN-Preg + NaHS), and hypertensive pregnancy + GYY4137 (HTN-Preg + GYY). Systolic blood pressure, plasma H2S levels, fetal and placental weights, number of viable fetuses, litter size, and endothelium-dependent vasodilation were examined. Also, oxidative stress was assessed in placenta. We found that GYY4137 attenuated hypertension on gestational days 16 and 18, while NaHS presented antihypertensive effect only on gestational day 18. GYY4137, but not NaHS, increased plasma H2S levels. Greater fetal and placental weights were found with GYY4137 than NaHS treatment. Also, HTN-Preg + NaHS presented further reductions in placental weights when compared to HTN-Preg group. Number of viable fetuses and litter size presented no significant changes. GYY4137 reduced placental oxidative stress caused by hypertension, while greater increases in oxidative stress were found in HTN-Preg + NaHS than HTN-Preg group. Hypertensive pregnancy caused impaired endothelium-dependent vasodilation, while GYY4137 and NaHS treatments blunted endothelial dysfunction. Endothelium-dependent vasodilation was completely blocked by the nitric oxide synthase inhibitor. We conclude that slow-releasing H2S donor GYY4137 is advantageous compared with fast-releasing H2S-donor NaHS to attenuate hypertension-in-pregnancy and to protect against feto-placental growth restriction and oxidative stress.
Assuntos
Anti-Hipertensivos/uso terapêutico , Retardo do Crescimento Fetal/tratamento farmacológico , Sulfeto de Hidrogênio , Hipertensão/tratamento farmacológico , Morfolinas/uso terapêutico , Compostos Organotiofosforados/uso terapêutico , Sulfetos/uso terapêutico , Animais , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/fisiologia , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/fisiopatologia , Feto/efeitos dos fármacos , Sulfeto de Hidrogênio/sangue , Hipertensão/sangue , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Malondialdeído/metabolismo , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Placenta/efeitos dos fármacos , Gravidez , Ratos Wistar , Sulfetos/farmacologiaRESUMO
In this work photo-electro-Fenton (PEF) processes using a dimensionally stable anode-gas diffusion electrode (DSA-GDE) system under light emission diodes (LED)-type radiation were used in the degradation of the angiotensin-II-receptor antagonists (ARA II), valsartan (VAL), and losartan (LOS), which are used in the treatment of hypertension diseases, and are considered among the emerging contaminants (ECs). Organic acids as citric, tartaric, and oxalic acids were used as complexing agents of iron ions in order to maintain the performance of the Fenton reaction at near-neutral pH value. The results show that at 3.42 mA/cm2 after 90 min of electro-Fenton (EF) treatment, degradation of 70% of VAL and 100% of LOS were observed. Total degradation of VAL and LOS was reached with a PEF process at the same time with mineralization of 30%. When citric and tartaric acids were used instead of oxalic acid, similar results were obtained, i.e., total degradation of both compounds, LOS and VAL, after 90 min of treatment. The degradation performance can be attributed to the increase of the initial dissolved iron in the system, facilitating the Fe3+/Fe2+ turnover in the catalytic photo-Fenton reaction and consequently, hydroxyl radical (â¢OH) production. In addition, the increased photo-activity of the complexes can be associated with their high capability to complex Fe3+ and to promote ligand-to-metal charge transfer, which is of key importance to feed Fe2+ to the Fenton process. The results show that the system evaluated was more efficient to eliminate sartan family compounds using LED lighting in comparison with traditional UV-A lamps used in this kind of work. Moreover, three transformation products of VAL degradation and two transformation products of LOS degradation were identified by high-resolution mass spectrometry (HRMS) using hybrid quadrupole-time-of-flight (QTOF) MS and, at the end of the PEF system, the several organic compounds accumulated and no mineralized were effectively treated in a subsequent aerobic biological system.
Assuntos
Eletrodos , Losartan/química , Valsartana/química , Poluentes Químicos da Água/química , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Biodegradação Ambiental , Ácido Cítrico/química , Difusão , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Radical Hidroxila/química , Ferro/química , Iluminação/instrumentação , Iluminação/métodos , Losartan/metabolismo , Oxirredução , Tartaratos/química , Valsartana/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
OBJECTIVE: To evaluate, for the first time, the use of SCC4 cell monolayers as an alternative sublingual barrier model and study the influence of nanoencapsulation on carvedilol transport across SCC4 cell monolayers. SIGNIFICANCE: The sublingual cavity is an interesting route for administration of drugs with limited oral bioavailability due to hepatic first pass metabolism. By this route, the drug is directly absorbed into blood circulation. In this sense, mucoadhesive carvedilol-loaded nanocapsules (CAR-NC) were previously proposed for the administration of this drug by sublingual route. Carvedilol is used for cardiovascular diseases and suffers metabolism in liver when orally administrated. Nanoencapsulation of carvedilol controlled its permeation across porcine sublingual mucosa. METHODS: Carvedilol-loaded cationic nanocapsules were prepared by interfacial deposition of a preformed polymer. Drug permeation studies were carried out in Transwell® inserts. The integrity of cell monolayers after the drug transport was assessed by transepithelial electric resistance. Compatibility of the CAR-NC with the SCC4 cells was evaluated by the Sulforhodamine B assay. RESULTS: The drug permeated the cell monolayer by a controlled way when nanoencapsulated and this profile had a linear relation with those observed in porcine sublingual mucosa. The integrity of the cell monolayer was maintained after drug permeation and CAR-NC was no cytotoxic to SCC4 cells. CONCLUSION: Nanoencapsulated carvedilol permeated by a controlled and safe way by SCC4 cell monolayer. SCC4 cells monolayers may be used as in vitro model for sublingual drug transport studies in the development of novel formulations.
Assuntos
Anti-Hipertensivos/síntese química , Anti-Hipertensivos/metabolismo , Carvedilol/síntese química , Carvedilol/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Nanocápsulas/química , Administração Sublingual , Anti-Hipertensivos/administração & dosagem , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Carvedilol/administração & dosagem , Humanos , Nanocápsulas/administração & dosagem , Células Tumorais CultivadasRESUMO
The insertion of peptides is a biotechnology tool widely used to improve the nutraceutical properties of proteins. Because the effect of these insertions in protein stability and function is difficult to predict, it should be determined experimentally. In this study, we created two variants of amarantin acidic subunit and analyzed them along with other four proteins reported previously. We measured their response against two destabilizing agents: temperature and urea. The six proteins presented the insertion of antihypertensive peptides (VYVYVYVY or RIPP) in the variable regions of the protein. We observed that their effect strongly depended on the site of the insertion. The insertion in the variable region I stabilized the protein both thermally and chemically, but it affected the inhibitory activity of the angiotensin-converting enzyme in vitro. In contrast, insertions in other three regions were severely destabilizing, producing molten globules. Our findings reveal that the insertion of bioactive peptides in variable regions of a protein can increase or decrease the protein's thermal and chemical stability and that these conformational changes may also alter its final activity.
Assuntos
Amaranthus/genética , Anti-Hipertensivos/metabolismo , Peptídeos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Engenharia de Proteínas/métodos , Estabilidade Proteica , Temperatura , UreiaRESUMO
Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Hipertensão/tratamento farmacológico , Precursores de Proteínas/química , Superóxido Dismutase/química , Teprotida/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anticorpos/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Bothrops , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cobaias , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Natriurético Tipo C/química , Peptídeo Natriurético Tipo C/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Teprotida/metabolismo , Teprotida/farmacologiaRESUMO
AIM: To investigate the influence of gestational diabetes mellitus (GDM) on the kinetic disposition and transplacental and amniotic fluid distribution of metoprolol and its metabolites O-desmethylmetoproloic acid and α-hydroxymetoprolol stereoisomers in hypertensive parturients receiving a single dose of the racemic drug. METHODS: The study was conducted on hypertensive parturients with well-controlled GDM (n = 11) and non-diabetic hypertensive parturients (n = 24), all receiving a single 100 mg oral dose of racemic metoprolol tartrate before delivery. Serial maternal blood samples (0-24 h) and umbilical blood and amniotic fluid samples were collected for the quantitation of metoprolol and its metabolite stereoisomers using LC-MS/MS or fluorescence detection. RESULTS: The kinetic disposition of metoprolol and its metabolites was stereoselective in the diabetic and control groups. Well-controlled GDM prolonged tmax for both enantiomers of metoprolol (1.5 vs. 2.5 h R-(+)-MET; 1.5 vs. 2.75 h S-(-)-MET) and O-desmethylmetoproloic acid (2.0 vs. 3.5 h R-(+)-AOMD; 2.0 vs. 3.0 h S-(-)-OAMD), and for the four stereoisomers of α-hydroxymetoprolol (2.0 vs. 3.0 h for 1'S,2R-, 1'R,2R- and 1'R,2S-OHM; 2.0 vs. 3.5 h for 1'S,2S-OHM) and reduced the transplacental distribution of 1'S,2S-, 1'R,2R-, and 1'R,2S-OHM by approximately 20%. CONCLUSIONS: The kinetic disposition of metoprolol was enantioselective, with plasma accumulation of the S-(-)-MET eutomer. Well-controlled GDM prolonged the tmax of metoprolol and O-desmethylmetoproloic acid enantiomers and the α-hydroxymetoprolol stereoisomers and reduced by about 20% the transplacental distribution of 1'S,2S-, 1'R,2R-, and 1'R,2S-OHM. Thus, well-controlled GDM did not change the activity of CYP2D6 and CYP3A involved in metoprolol metabolism.
Assuntos
Anti-Hipertensivos/sangue , Diabetes Gestacional/metabolismo , Hipertensão/tratamento farmacológico , Metoprolol/sangue , Placenta/metabolismo , Adulto , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/uso terapêutico , Diabetes Gestacional/sangue , Diabetes Gestacional/tratamento farmacológico , Feminino , Sangue Fetal/química , Humanos , Hipertensão/sangue , Hipertensão/complicações , Hipertensão/metabolismo , Recém-Nascido , Metoprolol/química , Metoprolol/metabolismo , Metoprolol/uso terapêutico , Parto , Gravidez , Estereoisomerismo , Distribuição TecidualRESUMO
AT1 receptor is an interesting biological target involved in several important diseases, such as blood hypertension and cardiovascular pathologies. In this study we investigated the main electrostatic and steric features of a series of AT1 antagonists related to hypertensive activity using structure and ligand-based strategies (docking and CoMFA). The generated 3D model had good internal and external consistency and was used to predict the potency of an external test set. The predicted values of pIC50 are in good agreement with the experimental results of biological activity, indicating that the 3D model can be used to predict the biological property of untested compounds. The electrostatic and steric CoMFA maps showed molecular recognition patterns, which were analyzed with structure-based molecular modeling studies (docking). The most and the least potent compounds docked into the AT1 binding site were subjected to molecular dynamics simulations with the aim to verify the stability and the flexibility of the ligand-receptor interactions. These results provided valuable insights on the electronic/structural requirements to design novel AT1 antagonists.