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1.
J Enzyme Inhib Med Chem ; 34(1): 1131-1139, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31169043

RESUMO

The antitumor agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (1) is a potent inhibitor of GSTP1-1, a glutathione S-transferase capable of inhibiting apoptosis by binding to JNK1 and TRAF2. We recently demonstrated that, unlike its parent compound, the benzoyl ester of 1 (compound 3) exhibits negligible reactivity towards GSH, and has a different mode of interaction with GSTP1-1. Unfortunately, 3 is susceptible to rapid metabolic hydrolysis. In an effort to improve the metabolic stability of 3, its ester group has been replaced by an amide, leading to N-(6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexyl)benzamide (4). Unlike 3, compound 4 was stable to human liver microsomal carboxylesterases, but retained the ability to disrupt the interaction between GSTP1-1 and TRAF2 regardless of GSH levels. Moreover, 4 exhibited both a higher stability in the presence of GSH and a greater cytotoxicity towards cultured A375 melanoma cells, in comparison with 1 and its analog 2. These findings suggest that 4 deserves further preclinical testing.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Glutationa S-Transferase pi/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
2.
ACS Chem Biol ; 13(10): 2939-2948, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30208272

RESUMO

It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Cloroquina/análogos & derivados , Cloroquina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Protozoários/metabolismo , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/farmacologia , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimaláricos/farmacologia , Cloroquina/síntese química , Cloroquina/farmacologia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Espectrometria de Massas , Microscopia Confocal , Plasmodium falciparum/química , Plasmodium falciparum/efeitos dos fármacos , Ligação Proteica , Proteômica/métodos
3.
Phytochemistry ; 150: 1-11, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29524794

RESUMO

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.


Assuntos
Citocininas/química , Receptores de Citocinas/antagonistas & inibidores , 4-Cloro-7-nitrobenzofurazano/farmacologia , Adenina/análogos & derivados , Adenina/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Carbocianinas/química , Corantes/química , Citocininas/farmacologia , Corantes Fluorescentes/química , Regulação da Expressão Gênica de Plantas , Isopenteniladenosina/síntese química , Isopenteniladenosina/química , Isopenteniladenosina/farmacologia , Microscopia Confocal , Estrutura Molecular , Desenvolvimento Vegetal , Reguladores de Crescimento de Planta/metabolismo , Purinas/química , Receptores de Citocinas/química , Rodaminas/química , Plântula/metabolismo , Terpenos/metabolismo , Zea mays/metabolismo
4.
Biochim Biophys Acta Biomembr ; 1860(5): 1135-1142, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29338975

RESUMO

The understanding of lipid bilayer structure and function has been advanced by the application of molecular fluorophores. However, the effects of these probe molecules on the physicochemical properties of membranes being studied are poorly understood. A quartz crystal microbalance with dissipation monitoring instrument was used in this work to investigate the impact of two commonly used fluorescent probes, 1­palmitoyl­2­{12­[(7­nitro­2­1,3­benzoxadiazol­4­yl)amino]dodecanoyl}­sn­glycero­3­phosphocholine (NBD-PC) and 1,2­dipalmitoyl­sn­glycero­3­phosphoethanolamine­n­(lissamine rhodamine­B­sulfonyl) (Lis-Rhod PE), on the formation and physicochemical properties of a 1­palmitoyl­2­oleoyl­sn­glycero­3­phosphocholine supported lipid bilayer (POPC-SLB). The interaction of the POPC-SLB and fluorophore-modified POPC-SLB with docosahexaenoic acid, DHA, was evaluated. The incorporation of DHA into the POPC-SLB was observed to significantly decrease in the presence of the Lis-Rhod PE probe compared with the POPC-SLB. In addition, it was observed that the small concentration of DHA incorporated into the POPC:NBD-PC SLB can produce rearrangement processes followed by the lost not only of DHA but also of POPC or NBD-PC molecules or both during the washing step. This work has significant implications for the interpretation of data employing fluorescent reporter molecules within SLBs.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Corantes Fluorescentes/farmacologia , Bicamadas Lipídicas/metabolismo , Fosfatidilcolinas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/farmacologia , Ácidos Docosa-Hexaenoicos/farmacocinética , Corantes Fluorescentes/química , Bicamadas Lipídicas/química , Conformação Molecular , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacocinética , Fosfatidilcolinas/farmacologia , Técnicas de Microbalança de Cristal de Quartzo , Rodaminas/química , Rodaminas/farmacologia
5.
J Cell Biochem ; 118(8): 2037-2043, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-27791278

RESUMO

The link between insulin resistance (IR) and type 2 diabetes has been recognized for a long time. Type 2 diabetes is often associated with basal hyperinsulinemia, reduced sensitivity to insulin, and disturbances in insulin release. There are evidences showing the reversal of IR by mesenchymal stem cells. However, the effect of conditioned media from adipose derived mesenchymal stem cells (ADSCs-CM) in reversal of IR has not been established. We established an insulin resistant model of 3T3L1 and C2C12 cells and treated with ADSCs-CM. 2-NBDG (2-[N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl]Amino]-2-Deoxyglucose) uptake was performed to assess improvement in glucose uptake. Genes involved in glucose transport and in inflammation were also analysed. Western blot for glucose transporter-4 and Akt was performed to evaluate translocation of Glut4 and insulin signaling respectively. We found that the ADSCs-CM treated cells restored insulin, stimulated glucose uptake as compared to the untreated control indicating the insulin sensitizing effect of the CM. The treated cells also showed inhibition adipogenesis in 3T3L1 cells and significant reduction of intramuscular triglyceride accumulation in C2C12 cells. Gene expressions studies revealed the drastic upregulation of GLUT4 gene and significant reduction in IL6 and PAI1 gene in both 3T3L1 and C2C12 cells, indicating possible mechanism of glucose uptake with concomitant decrease in inflammation. Enhancement of GLUT4 and phospho Akt protein expression seems to be responsible for the increment in glucose uptake and enhanced insulin signaling, respectively. Our study revealed for the first time that ADSCs-CM acts as an alternative insulin sensitizer providing stem cell solution to IR. J. Cell. Biochem. 118: 2037-2043,2017. © 2016 Wiley Periodicals, Inc.


Assuntos
Adipócitos/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Resistência à Insulina , Insulina/farmacologia , Células-Tronco Mesenquimais/metabolismo , Mioblastos/efeitos dos fármacos , Células 3T3-L1 , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Células Cultivadas , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Regulação da Expressão Gênica , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo
6.
Cell Physiol Biochem ; 39(3): 1209-28, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27595398

RESUMO

BACKGROUND: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. METHODS: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. RESULTS: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. CONCLUSIONS: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.


Assuntos
Glucose/imunologia , Janus Quinase 3/genética , Oócitos/metabolismo , Transportador 1 de Glucose-Sódio/genética , Linfócitos T Citotóxicos/imunologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Transporte Biológico , Brefeldina A/farmacologia , Células CACO-2 , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Regulação da Expressão Gênica , Glucose/farmacologia , Humanos , Janus Quinase 3/imunologia , Células Jurkat , Ativação Linfocitária , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Florizina/farmacologia , Cultura Primária de Células , Quinazolinas/farmacologia , Transdução de Sinais , Transportador 1 de Glucose-Sódio/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Transgenes , Xenopus laevis
7.
Glia ; 64(11): 1962-71, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27462832

RESUMO

Previous findings indicate that reducing brain insulin-like growth factor I receptor (IGF-IR) activity promotes ample neuroprotection. We now examined a possible action of IGF-IR on brain glucose transport to explain its wide protective activity, as energy availability is crucial for healthy tissue function. Using (18) FGlucose PET we found that shRNA interference of IGF-IR in mouse somatosensory cortex significantly increased glucose uptake upon sensory stimulation. In vivo microscopy using astrocyte specific staining showed that after IGF-IR shRNA injection in somatosensory cortex, astrocytes displayed greater increases in glucose uptake as compared to astrocytes in the scramble-injected side. Further, mice with the IGF-IR knock down in astrocytes showed increased glucose uptake in somatosensory cortex upon sensory stimulation. Analysis of underlying mechanisms indicated that IGF-IR interacts with glucose transporter 1 (GLUT1), the main facilitative glucose transporter in astrocytes, through a mechanism involving interactions with the scaffolding protein GIPC and the multicargo transporter LRP1 to retain GLUT1 inside the cell. These findings identify IGF-IR as a key modulator of brain glucose metabolism through its inhibitory action on astrocytic GLUT1 activity. GLIA 2016;64:1962-1971.


Assuntos
Astrócitos/metabolismo , Glucose/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Animais Recém-Nascidos , Biotinilação , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Células Cultivadas , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glucosamina/análogos & derivados , Glucosamina/farmacologia , Transportador de Glucose Tipo 1/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Física , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Transfecção , Vibrissas/fisiologia
8.
Nat Chem Biol ; 12(8): 608-13, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27294322

RESUMO

Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Óxidos N-Cíclicos/análise , Óxidos N-Cíclicos/química , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Radicais Livres/análise , Peroxidação de Lipídeos , Lipídeos/química , 4-Cloro-7-nitrobenzofurazano/análise , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/farmacologia , 4-Cloro-7-nitrobenzofurazano/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Óxidos N-Cíclicos/farmacologia , Óxidos N-Cíclicos/uso terapêutico , Dietilnitrosamina , Modelos Animais de Doenças , Corantes Fluorescentes/farmacologia , Corantes Fluorescentes/uso terapêutico , Depuradores de Radicais Livres/análise , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/farmacologia , Depuradores de Radicais Livres/uso terapêutico , Radicais Livres/química , Radicais Livres/metabolismo , Inflamação/prevenção & controle , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/química , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espectrometria de Fluorescência
9.
Biochim Biophys Acta ; 1858(8): 1812-20, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27131444

RESUMO

We examined how hydrophobic peptide-accelerated transleaflet lipid movement (flip-flop) was affected by peptide sequence and vesicle composition and properties. A peptide with a completely hydrophobic sequence had little if any effect upon flip-flop. While peptides with a somewhat less hydrophobic sequence accelerated flip-flop, the half-time remained slow (hours) with substantial (0.5mol%) peptide in the membranes. It appears that peptide-accelerated lipid flip-flop involves a rare event that may reflect a rare state of the peptide or lipid bilayer. There was no simple relationship between peptide overall hydrophobicity and flip-flop. In addition, flip-flop was not closely linked to whether the peptides were in a transmembrane or non-transmembrane (interfacial) inserted state. Flip-flop was also not associated with peptide-induced pore formation. We found that peptide-accelerated flip-flop is initially faster in small (highly curved) unilamellar vesicles relative to that in large unilamellar vesicles. Peptide-accelerated flip-flop was also affected by lipid composition, being slowed in vesicles with thick bilayers or those containing 30% cholesterol. Interestingly, these factors also slow spontaneous lipid flip-flop in the absence of peptide. Combined with previous studies, the results are most consistent with acceleration of lipid flip-flop by peptide-induced thinning of bilayer width.


Assuntos
Sequência de Aminoácidos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Peptídeos/farmacologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Transporte Biológico , Difusão , Fluidez de Membrana , Modelos Químicos , Pressão Osmótica , Fosfatidilcolinas/farmacologia , Fosfolipídeos/química , Estrutura Secundária de Proteína , Lipossomas Unilamelares/química
10.
Phytochemistry ; 124: 99-107, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26854130

RESUMO

We have reported earlier, an orally active insulin-like protein (ILP) from Costus igneus having potent hypoglycemic property in STZ-induced diabetic Swiss mice. The blood glucose level was reduced significantly within two hours after feeding ILP orally in an oral glucose tolerance test. The present study elucidates the mechanism underlying the hypoglycemic action of ILP. Mechanism of action of ILP was studied in differentiated L6 myotubes. 2-NBDG uptake stimulated by ILP was studied in differentiated L6 myotubes under normoglycemic, hyperglycemic and induced insulin resistant conditions. ILP treatment significantly increased 2-NBDG uptake in differentiated L6 myotubes. The levels of insulin signaling molecules IRS-1 and GLUT-4 were assessed in ILP treated L6 myotubes by immunoblot analysis of cytoplasmic and plasma membrane fractions respectively. Immunoblot analysis revealed an increase in cytoplasmic IRS-1 with a concomitant increase in GLUT-4 translocation to the plasma membrane in a time dependent manner. Toxicity studies of ILP were performed on normal as well as diabetic Swiss albino mice. ILP did not show any toxicity in the acute and sub-chronic toxicity studies in normal as well as diabetic Swiss albino mice. Mass spectrometry was carried out to identify ILP. MALDI TOF/TOF MS analysis of ILP revealed sequence homology with the predicted protein from Physcomitrella patens. Our study reveals that ILP acts via insulin signaling pathway and can be used as oral insulin mimetic.


Assuntos
Costus/química , Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Insulina/farmacologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Glicemia/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Diabetes Mellitus Experimental/sangue , Modelos Animais de Doenças , Teste de Tolerância a Glucose , Hipoglicemiantes/química , Camundongos , Músculo Esquelético/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fator de Necrose Tumoral alfa/farmacologia
11.
J Enzyme Inhib Med Chem ; 31(6): 924-30, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26329912

RESUMO

CONTEXT: The inhibition of glutathione S-transferase P1-1 (GSTP1-1) is a sound strategy to overcome drug resistance in oncology practice. OBJECTIVE: The nitrobenzoxadiazolyl (NBD) S-conjugate of glutathione and the corresponding γ-oxa-glutamyl isostere (compounds 1 and 5, respectively) have been disclosed as GST inhibitors. The rationale of their design is discussed in juxtaposition to non-peptide NBD thioethers. MATERIALS AND METHODS: Synthesis of derivatives 1 and 5 and in vitro evaluation on human GSTP1-1 and M2-2 are reported. RESULTS: Conjugates 1 and 5 were found to be low micromolar inhibitors of both isoforms. Furthermore, they display a threefold reduction in selectivity for GSTM2-2 over the P1-1 isozyme in comparison with the potent non-peptide inhibitor nitrobenzoxadiazolyl-thiohexanol (NBDHEX). DISCUSSION AND CONCLUSIONS: Spectroscopic data are congruent with the formation of a stable sigma-complex between GSH and the inhibitors in the protein active site. Conjugate 5 is suitable for in vivo modulation of GST activity in cancer treatment.


Assuntos
4-Cloro-7-nitrobenzofurazano/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glutationa S-Transferase pi/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Glutationa/farmacologia , 4-Cloro-7-nitrobenzofurazano/síntese química , 4-Cloro-7-nitrobenzofurazano/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Glutationa/química , Glutationa S-Transferase pi/metabolismo , Glutationa Transferase/metabolismo , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
12.
Purinergic Signal ; 11(4): 561-9, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26446689

RESUMO

ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A2B receptor (A(2B)R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A(2B)R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A(2B)R antagonist, MRS1754 (200 nM), and was also absent in A(2B)R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased (3)H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37 °C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A(2B)R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A(2B)R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Agonistas do Receptor A2 de Adenosina/farmacologia , Desoxiglucose/análogos & derivados , Glucose/metabolismo , Prosencéfalo/metabolismo , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Desoxiglucose/metabolismo , Desoxiglucose/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Prosencéfalo/efeitos dos fármacos , Receptor A2B de Adenosina/genética
13.
ChemMedChem ; 10(10): 1635-40, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26287271

RESUMO

A series of novel fluorine-containing cyclooxygenase-2 (COX-2) inhibitors was designed and synthesized based on the previously reported fluorescent COX-2 imaging agent celecoxib-NBD (3; NBD=7-nitrobenzofurazan). In vitro COX-1/COX-2 inhibitory data show that N-(4-fluorobenzyl)-4-(5-p-tolyl-3-trifluoromethylpyrazol-1-yl)benzenesulfonamide (5; IC50 =0.36 µM, SI>277) and N-fluoromethyl-4-(5-p-tolyl-3-trifluoromethylpyrazol-1-yl)benzenesulfonamide (6; IC50 =0.24 µM, SI>416) are potent and selective COX-2 inhibitors. Compound 5 was selected for radiolabeling with the short-lived positron emitter fluorine-18 ((18) F) and evaluated as a positron emission tomography (PET) imaging agent. Radiotracer [(18) F]5 was analyzed in vitro and in vivo using human colorectal cancer model HCA-7. Although radiotracer uptake into COX-2-expressing HCA-7 cells was high, no evidence for COX-2-specific binding was found. Radiotracer uptake into HCA-7 tumors in vivo was low and similar to that of muscle, used as reference tissue.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Celecoxib/análogos & derivados , Celecoxib/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Ciclo-Oxigenase 2/metabolismo , Desenho de Drogas , Radioisótopos de Flúor/química , Tomografia por Emissão de Pósitrons , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Celecoxib/química , Linhagem Celular Tumoral , Inibidores de Ciclo-Oxigenase 2/síntese química , Inibidores de Ciclo-Oxigenase 2/química , Humanos , Camundongos , Camundongos Endogâmicos , Modelos Moleculares , Estrutura Molecular , Traçadores Radioativos , Distribuição Tecidual
14.
Biol Pharm Bull ; 38(9): 1255-64, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26119425

RESUMO

A simple and rapid assay method for analysis of the metabolic activity of viable but non-culturable (VBNC) Salmonella was established. An environmental isolate of Salmonella Enteritidis (SE), grown to the logarithmic phase, rapidly lost its culturability during incubation with 1-10 mM H2O2 in Luria-Bertani (LB) medium. To assess the viability of the bacteria, we measured 3 different metabolic activities: Respiratory activity by 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) reduction, glucose uptake assessed with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG), and DNA synthesis activity evaluated by 5-ethynyl-2'-deoxyuridine (EdU) incorporation. These activities were analyzed by both confocal laser-scanning microscopy and flow cytometry, together with colony-formation assays on LB-agar plates. The results showed that some of the H2O2-treated SE cells were in the VBNC state and that the extent of H2O2-induced decrease in each metabolic activity varied according to the activity. That is, glucose-uptake activity was not markedly changed, being kept at the highest level; whereas the respiratory activity was less than that of the glucose-uptake, and DNA synthesis activity was the lowest among them. These results suggest that the VBNC state might be characterized by different metabolic activities that vary and correspond to the kind and strength of the stress, threatening bacterial survival in an adverse environment.


Assuntos
Salmonella enteritidis/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , DNA Bacteriano/metabolismo , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Citometria de Fluxo , Glucose/metabolismo , Peróxido de Hidrogênio/farmacologia , Microscopia Confocal , Salmonella enteritidis/efeitos dos fármacos
15.
Biochim Biophys Acta ; 1848(8): 1656-70, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917957

RESUMO

Insufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox. SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Antibióticos Antineoplásicos/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Galactosilceramidas/farmacologia , Lipídeos de Membrana/farmacologia , Neoplasias/metabolismo , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacologia , Membrana Celular/metabolismo , Cromatografia em Camada Delgada , Química Click , Doxorrubicina/metabolismo , Galactosilceramidas/química , Galactosilceramidas/metabolismo , Células HeLa , Humanos , Bicamadas Lipídicas , Lipossomos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Estrutura Molecular , Polietilenoglicóis/metabolismo , Porosidade , Temperatura Ambiente , Fatores de Tempo
16.
Phytochemistry ; 116: 283-289, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25796090

RESUMO

Withania somnifera, known in India as Asghawhanda, is used traditionally to treat many medical problems including diabetes and has demonstrated therapeutic activity in various animal models as well as in diabetic patients. While much of W. somnifera's therapeutic activity is attributed to withanolides, their role in the anti-diabetic activity of W. somnifera has not been adequately studied. In the present study, we evaluated the anti-diabetic activity of W. somnifera extract and purified withanolides, as well as the effect of various elicitors on this activity. W. somnifera leaf and root extracts increased glucose uptake in myotubes and adipocytes in a dose dependent manner, with the leaf extract more active than the root extract. Leaf but not root extract increased insulin secretion in basal pancreatic beta cells but not in stimulated cells. Six withanolides isolated from W. somnifera were tested for anti-diabetic activity based on glucose uptake in skeletal myotubes. Withaferin A was found to increase glucose uptake, with 10µM producing a 54% increase compared with control, suggesting that withaferin A is at least partially responsible for W. somnifera's anti-diabetic activity. Elicitors applied to the root growing solutions affected the physiological state of the plants, altering membrane leakage or osmotic potential. Methyl salicylate and chitosan increased withaferin A content by 75% and 69% respectively, and extracts from elicited plants increased glucose uptake to a higher extent than non-elicited plants, demonstrating a correlation between increased content of withaferin A and anti-diabetic activity.


Assuntos
Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Withania/química , Vitanolídeos/farmacologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Quitosana/farmacologia , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hipoglicemiantes/química , Índia , Israel , Estrutura Molecular , Salicilatos/farmacologia
17.
Traffic ; 16(5): 476-92, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25615589

RESUMO

The Golgi complex plays a prominent role in the modification and sorting of lipids and proteins, and is a highly dynamic organelle that is dispersed and rearranged before and after mitosis. Several reagents including 4-nitrobenzo-2-oxa-1,3-diazole-labeled C6-ceramide (NBD-C6-ceramide, a ceramide having an NBD-bound C6-N-acyl chain) and Golgi-specific proteins that emit fluorescence are used as Golgi markers. In the present study, we synthesized a new ceramide analog, acetyl-C16-ceramide-NBD (a ceramide having an acetylated C-1 hydroxyl group, C16-N-acyl chain, and NBD-bound C15-sphingosine), and showed that it preferentially accumulated in the Golgi complex without cytotoxicity for over 24 h. Pathways for cellular uptake and interorganelle trafficking of acetyl-C16-ceramide-NBD were investigated. Acetyl-C16-ceramide-NBD was transported to the Golgi complex via ceramide transport proteins. In contrast to NBD-C6-ceramide, acetyl-C16-ceramide-NBD was resistant to ceramide metabolic enzymes such as sphingomyelin synthase and glucosylceramide synthase. Because of its weaker cytotoxicity and resistance to ceramide metabolic enzymes, the localization of the Golgi complex could be observed in acetyl-C16-ceramide-NBD-labeled cells before and after mitosis.


Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Ceramidas/farmacologia , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , 4-Cloro-7-nitrobenzofurazano/química , 4-Cloro-7-nitrobenzofurazano/metabolismo , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Ácido Araquidônico/metabolismo , Transporte Biológico , Células CHO , Proteínas de Transporte/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/química , Ceramidas/metabolismo , Cricetulus , Corantes Fluorescentes , Complexo de Golgi/ultraestrutura , Humanos , Mitose/efeitos dos fármacos , Estrutura Molecular
18.
J Mol Neurosci ; 55(1): 126-130, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25091860

RESUMO

Glucose is a necessary source of energy for sustaining cell activities and homeostasis in the brain. Enhanced glucose uptake protects cells during energy depletion including brain ischemia. Astrocytes enhance their glucose uptake during ischemia to supply substrates to neurons and thus support neuronal survival. Radiolabeled substrates are commonly used for in vitro measurement of glucose uptake in astrocytes. Here we optimized a method to measure glucose uptake by astrocytes during oxygen-glucose deprivation (OGD) using the fluorescent substrate 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Uptake buffers for 2-NBDG were the same as for (14)C-labeled α-methyl-D-glucopyranoside. Cell lysis buffer was optimized for observing the fluorescence of 2-NBDG, and Hoechst 33258 DNA staining was used for normalization of the 2-NBDG concentration. Uptake was performed on cultures of primary astrocytes by incubating the cells at 37 °C in buffer containing 25-200 µM 2-NBDG. Flow cytometry was performed to visualize uptake in intact cells, and a fluorescence microplate reader was used to measure the intracellular concentration of 2-NBDG in cell homogenates. 2-NBDG uptake was concentration dependent in astrocytes that were exposed or not exposed to OGD. OGD significantly increased 2-NBDG uptake by about 1.2 to 2.5 times in astrocytes compared to control cells. These results show that 2-NBDG can be used to detect glucose transport in astrocytes exposed to OGD.


Assuntos
Astrócitos/metabolismo , Glucose/metabolismo , Oxigênio/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Transporte Biológico Ativo , Hipóxia Celular , Células Cultivadas , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Corantes Fluorescentes/farmacologia , Glucose/deficiência , Ratos , Ratos Sprague-Dawley
19.
J Nat Prod ; 76(11): 2080-7, 2013 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-24224843

RESUMO

As part of our ongoing search for new antidiabetic agents from medicinal plants, we found that a methanol extract of Morinda citrifolia showed potential stimulatory effects on glucose uptake in 3T3-L1 adipocyte cells. Bioassay-guided fractionation of this active extract yielded two new lignans (1 and 2) and three new neolignans (9, 10, and 14), as well as 10 known compounds (3-8, 11-13, and 15). The absolute configurations of compounds 9, 10, and 14 were determined by ECD spectra analysis. Compounds 3, 6, 7, and 15 showed inhibitory effects on PTP1B enzyme with IC50 values of 21.86 ± 0.48, 15.01 ± 0.20, 16.82 ± 0.42, and 4.12 ± 0.09 µM, respectively. Furthermore, compounds 3, 6, 7, and 15 showed strong stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. This study indicated the potential of compounds 3, 6, 7, and 15 as lead molecules for antidiabetic agents.


Assuntos
Hipoglicemiantes/isolamento & purificação , Insulina/farmacologia , Lignanas/isolamento & purificação , Lignanas/farmacologia , Morinda/química , Plantas Medicinais/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Células 3T3-L1/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Concentração Inibidora 50 , Lignanas/química , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , República da Coreia
20.
J Cell Physiol ; 228(12): 2399-407, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23757167

RESUMO

Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca(2+) release and protein kinase activation and long-term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-2-deoxyglucose and [(3) H]2-deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin-bound testosterone-induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc-eGFP. Subsequently, testosterone increased GLUT4myc-GFP exposure at the plasma membrane. Inhibition of Akt by the Akt-inhibitor-VIII had no effect. However, inhibition of Ca(2+) /calmodulin protein kinase (CaMKII) (KN-93 and autocamtide-2 related inhibitory peptide II) and AMP-activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc-eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4-dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes.


Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Testosterona/farmacologia , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Desoxiglucose/análogos & derivados , Desoxiglucose/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/metabolismo
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