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1.
Microb Pathog ; 135: 103609, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31247255

RESUMO

This article reports the utilization of Malus domestica for the synthesis of silver nanoparticles (AgNPs) with cytotoxic activity against the Michigan Cancer Foundation-7 (MCF-7) cell line as well as their antibacterial and radical scavenging potential. The biosynthesized AgNPs were confirmed using various analytical characterization techniques. The cytotoxic effect of Malus domestica-AgNPs (M.d-AgNPs) was studied by MTT assay and scavenging efficacy was assessed by DPPH, nitric oxide radical and phosphomolybdate assays. Furthermore, green synthesized nanoparticles were evaluated for their antibacterial activity against multidrug resistant-clinical isolates. M.d-AgNPs were observed to be almost spherical in shape with an average diameter from 50 to 107.3 nm as assessed by TEM and DLS. M.d-AgNPs revealed the dose-dependent antioxidant activity and antimicrobial activity against multidrug-resistant bacterial strain viz. Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Also, in vitro studies revealed dose-dependent cytotoxic effects of M.d-AgNPs treated MCF-7 cell line. The data strongly suggest that M.d-AgNPs had a potential antioxidant, antimicrobial and cytotoxicity activity.


Assuntos
Antibacterianos/biossíntese , Antibacterianos/farmacologia , Química Verde/métodos , Células MCF-7/efeitos dos fármacos , Malus/metabolismo , Nanopartículas Metálicas/química , Prata/farmacologia , Antineoplásicos/farmacologia , Antioxidantes/análise , Biofilmes/efeitos dos fármacos , Análise Custo-Benefício , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Estabilidade de Medicamentos , Depuradores de Radicais Livres , Química Verde/economia , Células HEK293/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Compostos Fitoquímicos/farmacologia , Difração de Raios X
2.
Acta Trop ; 191: 243-247, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30659804

RESUMO

The present study aimed at isolation the phytocompounds from the aerial parts of Gastrocotyle hispida and to evaluate its antioxidant and anticancer potential using in vitro assay. Gastrocotyle hispida is belonging to the family Boraginaceae used as a refreshing drink like tea. The decoction of the leaves is diuretic and is used in the treatment of rheumatism. Phytochemical study of a methanol extract yielded five known compounds viz: ß-sitosterol (GH-1), ß-sitosterol 3-glucoside (GH-2), 1-O-ß-glucopyranosyl-1,4-dihydroxy-2-prenylbenzene (GH-3), 6-Hydroxy-2,2-dimethyl-3-chrom (GH-4) and rosmarinic acid (GH-5). Total phenolic and flavonoid contents were calculated for the extract and fractions, the methanolic extract contained the highest content of total flavonoids (178 mg/g, expressed as quercetin equivalents) and total polyphenol (98.4 mg/g, expressed as gallic acid equivalent). Compounds were isolated by using column chromatography. In vitro, antioxidant activity of the extract and isolated compounds was investigated by DPPH and ABTS radical scavenging assays. The four different cell lines HepG2 (Liver), HEK-293 (Kidney) MCF-7 (Breast) and MDA-MB 231 (Breast) were used against the compounds. The isolated compounds showed dose-dependent free radical scavenging property in all tested models with the IC50 values of 10.2 µg/mL rosmarinic acid (GH-5), 52.1 µg/mL ß-sitosterol (GH-1) and 85 µg/mL for ß-sitosterol 3-glucoside (GH-2). The ß-sitosterol (GH-1) showed significant activity against HepG2 and HEK 293 cell lines. Rosmarinic acid (GH-5) possesses potent anticancer activity against breast cancer cells (MCF7) with the IC50 value of 4.2 µg/mL. It can be concluded that Gastrocotyle hispida has potential antioxidant, anticancer activities and further used as an anticancer agent.


Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Boraginaceae/química , Células HEK293/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Flavonoides , Humanos , Fenóis , Compostos Fitoquímicos , Quercetina , Arábia Saudita , Sitosteroides
3.
Planta Med ; 85(2): 103-111, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30142660

RESUMO

Usnic acid, a lichen secondary metabolite produced by a whole number of lichens, has attracted the interest of researchers owing to its broad range of biological activity, including antiviral, antibiotic, anticancer properties, and it possessing a certain toxicity. The synthesis of new usnic acid derivatives and the investigation of their biological activity may lead to the discovery of compounds with better pharmacological and toxicity profiles. In this context, a series of new usnic acid derivatives comprising a terpenoid moiety were synthesized, and their ability to inhibit the catalytic activity of the human DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 was investigated. The most potent compounds (15A, 15B, 15G: , and 16A, 16B, 16G: ) had IC50 values in the range of 0.33 - 2.7 µM. The inhibitory properties were mainly dependent on the flexibility and length of the terpenoid moiety, but not strongly dependent on the configuration of the asymmetric centers. The synthesized derivatives showed low cytotoxicity against human cell lines in an MTT assay. They could be used as a basis for the development of more effective anticancer therapies when combined with topoisomerase 1 inhibitors.


Assuntos
Benzofuranos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/efeitos dos fármacos , Benzofuranos/síntese química , Benzofuranos/química , Linhagem Celular Tumoral/efeitos dos fármacos , Escherichia coli , Células HEK293/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , Microrganismos Geneticamente Modificados , Simulação de Acoplamento Molecular , Inibidores de Fosfodiesterase/química
4.
Clin Toxicol (Phila) ; 56(9): 828-840, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29451035

RESUMO

BACKGROUND: Although methylene blue (MB) had long been proposed to counteract the effects of cyanide (CN) intoxication, research on its mechanisms of action and efficacy has been abandoned for decades. Recent studies on the benefits of MB in post-anoxic injuries have prompted us to reexamine the relevance of this historical observation. METHODS: Our study was performed in adult male Sprague-Dawley rats and on HEK293T epithelial cells. First, the effects and toxicity of MB (0-80 mg/kg) on circulation and metabolism were established in four urethane-anesthetized rats. Then nine rats received a lethal infusion of a solution of KCN (0.75 mg/kg/min) and were treated by either saline or MB, at 20 mg/kg, a dose that we found to be innocuous in rat and to correspond to a dose of about 4 mg/kg in humans. MB was also administered 5 min after the end of a sub-lethal exposure to CN in a separate group of 10 rats. In addition, ATP/ADP ratio, ROS production, mitochondrial membrane potential (Δψm) and cellular O2 consumption rate (OCR) were determined in HEK293T cells exposed to toxic levels of CN (200 µM for 10 min) before and after applying a solution containing MB (1-100 µM for 10 min). RESULTS: Methylene blue was found to be innocuous up to 50 mg/kg. KCN infusion (0.75 mg/kg/min) killed all animals within 7-8 min. MB (20 mg/kg) administered at the same time restored blood pressure, cardiac contractility and limited O2 deficit, allowing all the animals to survive, without any significant methemoglobinemia. When administered 5 min after a non-lethal CN intoxication, MB sped up the recovery of lactate and O2 deficit. Finally, MB was able to decrease the production of ROS and restore the ATP/ADP ratio, Δψm as well as OCR of epithelial cells intoxicated by CN. CONCLUSIONS: The present observations should make us consider the potential interest of MB in the treatment of CN intoxication. The mechanisms of the antidotal properties of MB cannot be accounted for by the creation of a cyanomethemoglobinemia, rather its protective effects appears to be related to the unique properties of this redox dye, which, depending on the dose, could directly oppose some of the consequences of the metabolic depression produced by CN at the cellular level.


Assuntos
Antídotos/farmacologia , Antídotos/uso terapêutico , Cianetos/envenenamento , Células HEK293/efeitos dos fármacos , Azul de Metileno/farmacologia , Azul de Metileno/uso terapêutico , Envenenamento/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
5.
Microb Pathog ; 115: 12-18, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29223452

RESUMO

In the present study, antimicrobial activity of a common Himalayan lichen viz. Bulbothrix setschwanensis (Zahlbr.) Hale extract in three common solvents (acetone, chloroform and methanol) was evaluated against six bacterial and seven fungal clinical strains. The acetone extract showed promising antimicrobial activity against S. aureus (1.56 mg/mL) and C. neoformans (6.25 mg/mL). Further, GC-MS analysis revealed 2,3-bis(2-methylpentanoyloxy)propyl 2-methylpentanoate and Ethyl 2-[(2R,3R,4aR,8aS)-3-hydroxy-2,3,4,4a,6,7,8,8a-octahydropyrano [3,2-b]pyran-2-yl]acetate as the predominant compounds. The combination of acetone extract with antibacterial drugs [kanamycin (KAN), rifampicin (RIF)] and antifungal drugs [amphotericin B (Amp B) and fluconazole (FLC)] showed lysis of S. aureus and C. neoformans at non-inhibitory concentration (FICI values were 0.31 for KAN, 0.18 for RIF, 0.37 for Amp B and 0.30 for FLC, respectively). Notably, the acetone extract confirmed cell wall damage of both S. aureus and C. neoformans cells and was clearly visualized under scanning electron microscopy (SEM), flow cytometry and confocal microscopy. Besides this, the three extracts also have less significant cytotoxic activity at MIC concentrations against mammalian cells (HEK-293 and HeLa). This study for the first time suggests that the chemical compounds present in the acetone extract of B. setschwanensis could be used against S. aureus and C. neoformans infections.


Assuntos
Anti-Infecciosos/farmacologia , Parede Celular/efeitos dos fármacos , Cryptococcus neoformans/efeitos dos fármacos , Líquens/química , Saccharomycetales/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Antifúngicos/química , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Parede Celular/ultraestrutura , Cryptococcus neoformans/citologia , Fungos/efeitos dos fármacos , Células HEK293/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Cinética , Testes de Sensibilidade Microbiana , Staphylococcus aureus/citologia , Fatores de Tempo
6.
Mar Drugs ; 15(12)2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29194378

RESUMO

Pharmaceutical approaches based on nanotechnologies and the development of eye drops composed of the mucoadhesive polymers chitosan and hyaluronic acid are emerging strategies for the efficient treatment of ocular diseases. These innovative nanoparticulate systems aim to increase drugs' bioavailability at the ocular surface. For the successful development of these systems, the evaluation of mucoahesiveness (the interaction between the ocular delivery system and mucins present on the eye) is of utmost importance. In this context, the aim of the present work was to investigate the mucoadhesivity of a novel nanoparticle eye drop formulation containing an antibiotic (ceftazidime) intended to treat eye infections. Eye drop formulations comprised a polymer (hydroxypropyl) methyl cellulose (HPMC) 0.75% (w/v) in an isotonic solution incorporating chitosan/sodium tripolyphosphate (TPP)-hyaluronic acid-based nanoparticles containing ceftazidime. The viscosity of the nanoparticles, and the gels incorporating the nanoparticles were characterized in contact with mucin at different mass ratios, allowing the calculation of the rheological synergism parameter (∆η). Results showed that at different nanoparticle eye formulation:mucin weight ratios, a minimum in viscosity occurred which resulted in a negative rheological synergism. Additionally, the results highlighted the mucoadhesivity of the novel ocular formulation and its ability to interact with the ocular surface, thus increasing the drug residence time in the eye. Moreover, the in vitro release and permeation studies showed a prolonged drug release profile from the chitosan/TPP-hyaluronic acid nanoparticles gel formulation. Furthermore, the gel formulations were not cytotoxic on ARPE-19 and HEK293T cell lines, evaluated by the metabolic and membrane integrity tests. The formulation was stable and the drug active, as shown by microbiological studies. In conclusion, chitosan/TPP-hyaluronic acid nanoparticle eye drop formulations are a promising platform for ocular drug delivery with enhanced mucoadhesive properties.


Assuntos
Quitosana/química , Soluções Oftálmicas/química , Administração Oftálmica , Animais , Antibacterianos/administração & dosagem , Organismos Aquáticos , Ceftazidima/administração & dosagem , Sistemas de Liberação de Medicamentos , Células HEK293/efeitos dos fármacos , Humanos , Nanopartículas , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/farmacologia
7.
Sci Rep ; 7(1): 14669, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29116164

RESUMO

Exogenous nutrient elements modulate the energetic metabolism responses that are prerequisites for cellular homeostasis and metabolic physiology. Although zinc is important in oxidative stress and cytoprotection processes, its role in the regulation of energetic metabolism remains largely unknown. In this study, we found that zinc stimulated aspect in cell motility and was essential in restoring the Ochratoxin A (OTA)-induced energetic metabolism damage in HEK293 cells. Moreover, using zinc supplementation and zinc deficiency models, we observed that zinc is conducive to mitochondrial pyruvate transport, oxidative phosphorylation, carbohydrate metabolism, lipid metabolism and ultimate energy metabolism in both normal and toxic-induced oxidative stress conditions in vitro, and it plays an important role in restoring impaired energetic metabolism. This zinc-mediated energetic metabolism regulation could also be helpful for DNA maintenance, cytoprotection and hereditary cancer traceability. Therefore, zinc can widely adjust energetic metabolism and is essential in restoring the impaired energetic metabolism of cellular physiology.


Assuntos
Movimento Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ocratoxinas/farmacologia , Zinco/farmacologia , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Superóxido Dismutase/metabolismo
8.
FEBS Lett ; 591(15): 2279-2289, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28670736

RESUMO

The lysosome-associated transporter proton-coupled amino acid transporter 1 (PAT1) promotes nutrient recycling through releasing luminal amino acids into the cytosol. Using HEK293 cells expressing an EGFP-tagged PAT1 (EGFP-PAT1) as a model, we identified a consensus tyrosine-based targeting signal in the cytosolic N-terminal region of PAT1, which facilitates its expression on the lysosome. Interestingly, this signal can be removed via protein cleavage in an amino acid-sensitive manner. The cleavage is suppressed upon amino acid starvation and is induced by amino acid replenishment. However, amino acid deficiency does not suppress the cleavage of amino acid-binding mutants of EGFP-PAT1. Our data support a mechanism, whereby amino acid binding induces PAT1 cleavage to remove a targeting signal, thus suppressing the expression of PAT1 on the lysosome.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisossomos/metabolismo , Simportadores/metabolismo , Alanina/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Sistemas de Transporte de Aminoácidos/genética , Aminoácidos/farmacologia , Retroalimentação Fisiológica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Mutação , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Simportadores/genética , Serina-Treonina Quinases TOR/metabolismo , Tirosina/genética
9.
Fundam Clin Pharmacol ; 31(6): 695-700, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28755515

RESUMO

In this study, we investigated the effect of bufalin on the human ether-à-go-go-related gene (hERG) K+ channels using the perforated patch recording technique. We measured a half-maximal inhibitory concentration (IC50 ) of 24.83 µM and maximal inhibitory effect of 39.45 ± 1.14% with bufalin. These findings suggest that bufalin is a potent hERG K+ channel blocker and may provide a new way for understanding Chan Su-induced arrhythmia.


Assuntos
Bufanolídeos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Medicina Tradicional Chinesa , Glândula Parótida/química , Esteroides/farmacologia , Animais , Anuros , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/tratamento farmacológico , Bufanolídeos/efeitos adversos , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos
10.
Microbiol Res ; 201: 1-11, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28602396

RESUMO

Mycoplasma fermentans is a pathogenic bacterium that infects humans and has potential pathogenic roles in respiratory, genital and rheumatoid diseases. NAD+-dependent deacetylase is involved in a wide range of pathophysiological processes and our studies have demonstrated that expression of mycoplasmal deacetylase in mammalian cells inhibits proliferation but promotes anti-starvation stress tolerance. Furthermore, mycoplasmal deacetylase is involved in cellular anti-oxidation, which correlates with changes in the proapoptotic proteins BIK, p21 and BIM. Mycoplasmal deacetylase binds to and deacetylates the FOXO3 protein, similar with mammalian SIRT2, and affects expression of the FOXO3 target gene BIM, resulting in inhibition of cell proliferation. Mycoplasmal deacetylase also alters the performance of cells under drug stress. This study expands our understanding of the potential molecular and cellular mechanisms of interaction between mycoplasmas and mammalian cells.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Mycoplasma fermentans/enzimologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Anticorpos Antibacterianos , Antioxidantes/análise , Proteínas Reguladoras de Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , DNA Bacteriano , Regulação para Baixo , Tolerância a Medicamentos , Proteína Forkhead Box O3/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Células HCT116 , Células HEK293/efeitos dos fármacos , Humanos , Hidrolases/imunologia , Imunoprecipitação/métodos , Proteínas de Membrana/efeitos dos fármacos , Camundongos , Proteínas Mitocondriais , Infecções por Mycoplasma/microbiologia , Mycoplasma fermentans/patogenicidade , Estresse Oxidativo/efeitos dos fármacos , Sirtuína 2/efeitos dos fármacos , Inanição
11.
Homeopathy ; 106(1): 32-36, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28325222

RESUMO

BACKGROUND: Several recent studies reported the capability of high diluted homeopathic medicines to modulate gene expression in cell cultures. In line with these studies, we examined whether ultra-high dilutions (30C and 200C) of sodium butyrate (SB) can affect the expression levels of genes involved in acquisition of a senescence-associated secretory phenotype (SASP) in human embryonic kidney (HEK) 293 cells. METHODS: Cell viability was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The expression levels of TNF-α, interleukin (IL)-2, IL-4, IL-6 and IL-10 genes were determined by real-time PCR assay. RESULTS: Exposure to both 30C and 200C during 48 h led to a significant decrease of the level of expression of TNF-α gene, while expression of IL-2 gene was increased when exposed to 30C, and expression of IL-10 gene was decreased when exposed to 200C. No changes in expression levels of all genes studied were observed in cells treated with both 30C and 200C remedies of SB during the 24 h. CONCLUSION: Observed changes in gene expression levels after exposure to 30C and 200C remedies of SB during 48 h suggest that extremely low concentrations of this agent can modulate the transcriptome of HEK 293 cells. These results are in line with findings from other studies confirming the ability of homeopathic remedies to modulate gene expression in cell cultures.


Assuntos
Antineoplásicos/farmacologia , Ácido Butírico/farmacologia , Homeopatia , Apoptose/efeitos dos fármacos , Regulação da Expressão Gênica , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Reação em Cadeia da Polimerase , Fator de Necrose Tumoral alfa/metabolismo
12.
PLoS One ; 12(1): e0169480, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28099443

RESUMO

Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 µM (OCT1 inhibition by allethrin) to 77.6 µM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 µM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can act as regulators of the activity of various ABC and SLC drug transporters, but only when used at high and non-relevant concentrations, making unlikely any contribution of these transporter activity alterations to pyrethroid toxicity in environmentally exposed humans.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Praguicidas/toxicidade , Piretrinas/toxicidade , Proteínas Carreadoras de Solutos/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular , Dopamina/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Transportador 1 de Cátions Orgânicos/antagonistas & inibidores , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/metabolismo , Praguicidas/química , Piretrinas/química , Proteínas Carreadoras de Solutos/metabolismo , Relação Estrutura-Atividade , Testes de Toxicidade
13.
J Hypertens ; 35(2): 330-337, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28005703

RESUMO

BACKGROUND: The function of prorenin, the inactive precursor of renin, remains unclear after many decades of research. The discovery of a (pro)renin receptor suggested that prorenin, by binding to this receptor, would become active, that is, obtain an 'open' conformation. However, the receptor only interacted with prorenin at levels that were many orders of magnitude above its normal levels, making such interaction in-vivo unlikely. Prorenin occurs in two conformations, an open, active form, and a closed, inactive form. Under physiological conditions (pH 7.4, 37 °C), virtually all prorenin occurs in the closed conformation. This study investigated to what degree prorenin-synthesizing cells release prorenin in an open conformation. METHODS AND RESULTS: Renin plus prorenin-synthesizing human mast cells, and prorenin-synthesizing HEK293 cells (transfected with the mammalian expression vector pRhR1100, containing human prorenin) and human decidua cells were incubated with the renin inhibitor VTP-27999. This inhibitor will trap open prorenin, as after VTP-27999 binding, prorenin can no longer return to its closed conformation, thus allowing its detection in a renin immunoradiometric assay. No evidence for the release of open prorenin was found. Moreover, incubating decidua cells with angiotensinogen yielded low angiotensin levels, corresponding with the activity of ≈1% of prorenin in the medium, that is, the amount of open prorenin expected based upon the equilibrium between open and closed prorenin under physiological conditions. CONCLUSION: Our study does not reveal evidence for the release of open, active prorenin by prorenin-synthesizing cells, at least under cell culture conditions. This argues against prorenin activity at the site of its release.


Assuntos
Renina/biossíntese , Renina/química , Adulto , Angiotensinogênio/farmacologia , Angiotensinas/metabolismo , Anti-Hipertensivos , Carbamatos/farmacologia , Decídua/citologia , Decídua/efeitos dos fármacos , Decídua/metabolismo , Feminino , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Pessoa de Meia-Idade , Piperidinas/farmacologia , Renina/antagonistas & inibidores
14.
Chem Biol Drug Des ; 89(3): 353-364, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27543496

RESUMO

In this study, a series of novel flavanone derivatives were designed and synthesized, and the antipsychotic activities of the target compounds were evaluated in vitro and in vivo. The results showed that synthesized compounds 7a-7g decreased the activity of dopamine D2 receptors in HEK293 cells co-transfected with D2 receptor/G protein α16a, with IC50 values of 0.051-0.35 µm. Compounds 7a-7g inhibited the over-production of nitric oxide stimulated by lipopolysaccharide/interferon-γ in BV-2 microglial cells. In mice, intragastric administration of 7d, 7e, and 7g reversed the increase in locomotor activity induced by MK-801 (an antagonist of NMDA receptors) and decreased the hyperactivity of climbing behavior induced by apomorphine (a dopamine receptor agonist). These results suggest that some of the novel flavanone derivatives have potential antipsychotic effects and may be useful in the treatment of schizophrenia.


Assuntos
Antipsicóticos/química , Antipsicóticos/farmacologia , Flavanonas/química , Animais , Antipsicóticos/síntese química , Apomorfina/efeitos adversos , Apomorfina/farmacologia , Técnicas de Química Sintética , Modelos Animais de Doenças , Maleato de Dizocilpina/efeitos adversos , Maleato de Dizocilpina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Receptores de Dopamina D2/metabolismo , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico
15.
Bioconjug Chem ; 27(12): 2850-2853, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27801580

RESUMO

A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Anfotericina B/química , Anfotericina B/farmacologia , Antibacterianos/efeitos adversos , Antifúngicos/química , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Ácido Cólico/química , Células HEK293/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
16.
Hypertension ; 68(6): 1365-1374, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27698068

RESUMO

Hyperactivity of the renin-angiotensin-aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin-angiotensin-aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1-7) in AT1-R-expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective ß-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1-7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin-angiotensin-aldosterone system peptides act as agonists on the AT1-R/ß-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/ß-arrestin2 intracellular routing. Importantly, we reveal Ang-(1-7) a known Mas receptor-specific ligand, as an AT1-R-biased agonist, selectively promoting ß-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1-7) at AT1-R, similar to that of synthetic AT1-R-biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1-7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1-7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin-angiotensin-aldosterone system.


Assuntos
Angiotensina II/farmacologia , Angiotensina I/metabolismo , Cardiotônicos/metabolismo , Células HEK293/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Animais , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Células HEK293/efeitos dos fármacos , Humanos , Músculos , Fenilefrina/farmacologia , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Sensibilidade e Especificidade , Transdução de Sinais , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , beta-Arrestinas/metabolismo
17.
J Microbiol Methods ; 130: 100-105, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27609715

RESUMO

The aim of this study was to produce a fusion protein consisting of the catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, and evaluation of its cytotoxic effects for targeted eradication of cancer cells. For this purpose, The DT386-BR2 structure was predicted using Modeller 9.14 and the best predicted model was selected based on the minimum DOPE score. A synthetic gene encoding DT386-BR2 was cloned in pET28a expression vector, expressed and purified by affinity chromatography. SDS-PAGE and Western blotting confirmed the expression of the DT386-BR2 fusion protein by revealing a band of about 47kDa after the induction of the expression. Finally, the purified protein was subjected to MTT assay for evaluation of its cyto-lethal effects on cancer and normal cell lines. Statistical analysis showed significant reduction in survival percent of HeLa and MCF-7 cancer cells in comparison to negative control (PBS), while the cytotoxic effect was not significant on the normal cells, i.e. HUVEC and HEK 293. The IC50 of DT386-BR2 for HeLa and MCF-7 was about 0.55 and 2.08µg/ml, respectively. In conclusion, the production and purification of DT386-BR2 fusion protein was successfully achieved and its cytotoxic effects on the studied cancer cell lines was established. The promising cytotoxic effects of this newly constructed fusion protein made it a suitable candidate for targeted therapy of cancer, and further in vitro and in vivo studies on this fusion protein is underway.


Assuntos
Antineoplásicos/farmacologia , Toxina Diftérica/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos/administração & dosagem , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Células HEK293/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Imunotoxinas/genética , Imunotoxinas/metabolismo , Imunotoxinas/farmacologia , Concentração Inibidora 50 , Células MCF-7/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico
18.
Antimicrob Agents Chemother ; 60(12): 7105-7114, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27645247

RESUMO

It is largely unknown if simultaneous administration of tuberculosis (TB) drugs and metformin leads to drug-drug interactions (DDIs). Disposition of metformin is determined by organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). Thus, any DDIs would primarily be mediated via these transporters. This study aimed to assess the in vitro inhibitory effects of TB drugs (rifampin, isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin, and linezolid) on metformin transport and whether TB drugs are also substrates themselves of OCTs and MATEs. HEK293 cells overexpressing OCT1, OCT2, OCT3, MATE1, and MATE2K were used to study TB drug-mediated inhibition of [14C]metformin uptake and to test if TB drugs are transporter substrates. Metformin uptake was determined by quantifying [14C]metformin radioactivity, and TB drug uptake was analyzed using liquid chromatography-tandem mass spectrometry. DDI indices were calculated (plasma maximum concentrations [Cmax]/50% inhibitory concentrations [IC50]), and based on the literature, a cutoff of >0.1 was assumed to warrant further in vivo investigation. Moxifloxacin was the only TB drug identified as a potent inhibitor (DDI index of >0.1) of MATE1- and MATE2K-mediated metformin transport, with IC50s of 12 µM (95% confidence intervals [CI], 5.1 to 29 µM) and 7.6 µM (95% CI, 0.2 to 242 µM), respectively. Of all TB drugs, only ethambutol appeared to be a substrate of OCT1, OCT2, OCT3, MATE1, and MATE2K. MATE1-mediated ethambutol uptake was inhibited strongly (DDI index of >0.1) by moxifloxacin (IC50, 12 µM [95% CI, 3.4 to 43 µM]). Our findings provide a mechanistic basis for DDI predictions concerning ethambutol. According to international guidelines, an in vivo interaction study is warranted for the observed in vitro interaction between ethambutol and moxifloxacin.


Assuntos
Interações de Medicamentos , Etambutol/farmacocinética , Fluoroquinolonas/farmacocinética , Metformina/farmacocinética , Antituberculosos/farmacocinética , Células HEK293/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacocinética , Moxifloxacina , Fator 1 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico
19.
Brain ; 139(11): 2891-2908, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27645800

RESUMO

No disease-modifying treatment exists for the fatal neurodegenerative polyglutamine disease known both as Machado-Joseph disease and spinocerebellar ataxia type 3. As a potential route to therapy, we identified small molecules that reduce levels of the mutant disease protein, ATXN3. Screens of a small molecule collection, including 1250 Food and Drug Administration-approved drugs, in a novel cell-based assay, followed by secondary screens in brain slice cultures from transgenic mice expressing the human disease gene, identified the atypical antipsychotic aripiprazole as one of the hits. Aripiprazole increased longevity in a Drosophila model of Machado-Joseph disease and effectively reduced aggregated ATXN3 species in flies and in brains of transgenic mice treated for 10 days. The aripiprazole-mediated decrease in ATXN3 abundance may reflect a complex response culminating in the modulation of specific components of cellular protein homeostasis. Aripiprazole represents a potentially promising therapeutic drug for Machado-Joseph disease and possibly other neurological proteinopathies.


Assuntos
Antipsicóticos/uso terapêutico , Aripiprazol/uso terapêutico , Ataxina-3/metabolismo , Doença de Machado-Joseph/tratamento farmacológico , Doença de Machado-Joseph/metabolismo , Proteínas Mutantes/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Ataxina-3/genética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Drosophila , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Células HEK293/ultraestrutura , Humanos , Doença de Machado-Joseph/genética , Camundongos , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Cultura de Órgãos , Peptídeos/genética , Piperidinas/farmacologia , Piranos/farmacologia , Pirazóis/farmacologia
20.
Exp Neurol ; 283(Pt A): 151-6, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27317297

RESUMO

INTRODUCTION: TRPM2 channels have been suggested to play a role in ischemic neuronal injury, specifically in males. A major hindrance to TRPM2 research has been the lack of specific TRPM2 inhibitors. The current study characterized the specificity and neuroprotective efficacy of a novel TRPM2 inhibitor. METHODS: Fluorescent calcium imaging (Fluo5F) was used to determine inhibitor efficacy of the TRPM2 peptide inhibitor (tat-M2NX) in HEK293 cells stably expressing hTRPM2. Adult (2-3months) and aged (18-20months) mice were subjected to 60min middle cerebral artery occlusion (MCAO) and injected with tat-M2NX, control scrambled peptide (tat-SCR) or clotrimazole (CTZ) either 20min prior or 3h after reperfusion. Infarct size was assessed using TTC staining. RESULTS: TRPM2 inhibition by tat-M2NX was observed by decreased Ca(2+) influx following H2O2 exposure human TRPM2 expressing cells. Male mice pre-treated with tat-M2NX had smaller infarct volume compared to tat-SCR. No effect of tat-M2NX on infarct size was observed in female mice. Importantly, male TRPM2(-/-) mice were not further protected by tat-M2NX, demonstrating selectivity of tat-M2NX. Administration of tat-M2NX 3h after reperfusion provided significant protection to males when analyzed at 24h or 4days after MCAO. Finally, we observed that tat-M2NX reduced ischemic injury in aged male mice. CONCLUSIONS: These data demonstrate the development of a new peptide inhibitor of TRPM2 channels that provides protection from ischemic stroke in young adult and aged male animals with a clinically relevant therapeutic window.


Assuntos
Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Peptídeos/uso terapêutico , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Fatores Etários , Animais , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/etiologia , Isquemia Encefálica/complicações , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Fatores Sexuais , Canais de Cátion TRPM/genética , Fatores de Tempo , Transfecção
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