Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 153
Filtrar
1.
Int J Mol Sci ; 21(22)2020 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-33233525

RESUMO

We investigated the synthesis of N-docosahexaenoylethanolamine (synaptamide) in neuronal cells from unesterified docosahexaenoic acid (DHA) or DHA-lysophosphatidylcholine (DHA-lysoPC), the two major lipid forms that deliver DHA to the brain, in order to understand the formation of this neurotrophic and neuroprotective metabolite of DHA in the brain. Both substrates were taken up in Neuro2A cells and metabolized to N-docosahexaenoylphosphatidylethanolamine (NDoPE) and synaptamide in a time- and concentration-dependent manner, but unesterified DHA was 1.5 to 2.4 times more effective than DHA-lysoPC at equimolar concentrations. The plasmalogen NDoPE (pNDoPE) amounted more than 80% of NDoPE produced from DHA or DHA-lysoPC, with 16-carbon-pNDoPE being the most abundant species. Inhibition of N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) by hexachlorophene or bithionol significantly decreased the synaptamide production, indicating that synaptamide synthesis is mediated at least in part via NDoPE hydrolysis. NDoPE formation occurred much more rapidly than synaptamide production, indicating a precursor-product relationship. Although NDoPE is an intermediate for synaptamide biosynthesis, only about 1% of newly synthesized NDoPE was converted to synaptamide, possibly suggesting additional biological function of NDoPE, particularly for pNDoPE, which is the major form of NDoPE produced.


Assuntos
Ácidos Araquidônicos/biossíntese , Ácidos Docosa-Hexaenoicos/metabolismo , Endocanabinoides/biossíntese , Etanolaminas/metabolismo , Lisofosfatidilcolinas/metabolismo , Neurônios/metabolismo , Animais , Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/isolamento & purificação , Bitionol/farmacologia , Isótopos de Carbono , Linhagem Celular Tumoral , Cromatografia Líquida , Endocanabinoides/antagonistas & inibidores , Endocanabinoides/isolamento & purificação , Etanolaminas/antagonistas & inibidores , Etanolaminas/isolamento & purificação , Hexaclorofeno/farmacologia , Cinética , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Plasmalogênios/antagonistas & inibidores , Plasmalogênios/biossíntese , Plasmalogênios/isolamento & purificação , Alcamidas Poli-Insaturadas/antagonistas & inibidores , Alcamidas Poli-Insaturadas/isolamento & purificação , Espectrometria de Massas em Tandem
2.
J Biol Chem ; 295(21): 7289-7300, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32284327

RESUMO

N-Acyl-phosphatidylethanolamine phospholipase D (NAPE-PLD) (EC 3.1.4.4) catalyzes the final step in the biosynthesis of N-acyl-ethanolamides. Reduced NAPE-PLD expression and activity may contribute to obesity and inflammation, but a lack of effective NAPE-PLD inhibitors has been a major obstacle to elucidating the role of NAPE-PLD and N-acyl-ethanolamide biosynthesis in these processes. The endogenous bile acid lithocholic acid (LCA) inhibits NAPE-PLD activity (with an IC50 of 68 µm), but LCA is also a highly potent ligand for TGR5 (EC50 0.52 µm). Recently, the first selective small-molecule inhibitor of NAPE-PLD, ARN19874, has been reported (having an IC50 of 34 µm). To identify more potent inhibitors of NAPE-PLD, here we used a quenched fluorescent NAPE analog, PED-A1, as a substrate for recombinant mouse Nape-pld to screen a panel of bile acids and a library of experimental compounds (the Spectrum Collection). Muricholic acids and several other bile acids inhibited Nape-pld with potency similar to that of LCA. We identified 14 potent Nape-pld inhibitors in the Spectrum Collection, with the two most potent (IC50 = ∼2 µm) being symmetrically substituted dichlorophenes, i.e. hexachlorophene and bithionol. Structure-activity relationship assays using additional substituted dichlorophenes identified key moieties needed for Nape-pld inhibition. Both hexachlorophene and bithionol exhibited significant selectivity for Nape-pld compared with nontarget lipase activities such as Streptomyces chromofuscus PLD or serum lipase. Both also effectively inhibited NAPE-PLD activity in cultured HEK293 cells. We conclude that symmetrically substituted dichlorophenes potently inhibit NAPE-PLD in cultured cells and have significant selectivity for NAPE-PLD versus other tissue-associated lipases.


Assuntos
Diclorofeno , Inibidores Enzimáticos , Fosfolipase D , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bitionol/química , Bitionol/farmacologia , Diclorofeno/química , Diclorofeno/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Hexaclorofeno/química , Hexaclorofeno/farmacologia , Humanos , Camundongos , Fosfolipase D/antagonistas & inibidores , Fosfolipase D/química , Fosfolipase D/metabolismo , Quinazolinas/química , Quinazolinas/farmacologia , Streptomyces/enzimologia , Sulfonamidas/química , Sulfonamidas/farmacologia
3.
Bioorg Med Chem Lett ; 30(9): 127099, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32171615

RESUMO

Our labs have demonstrated the activity of bithionol and synthetic retinoids against methicillin-resistant Staphylococcus aureus (MRSA), as well as their membrane-acting mechanism of action. However, the compounds lack activity in gram-negative species. Herein, we apply a known strategy for converting gram-positive agents into broad-spectrum therapies: addition of an alkylamine. By appending an alkylamine to the phenols of these known membrane disruptors, we test whether this approach is applicable to our compounds. Ultimately, biological testing in four MRSA strains and three gram-negative species showed abolished or diminished activity in all our analogs compared to their parent compounds and no gram-negative activity. Thus, we find that alkylamines would not elicit broad-spectrum activity from bithionol or CD437 derivatives.


Assuntos
Antibacterianos/farmacologia , Bitionol/química , Etilaminas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Fenóis/química , Retinoides/química , Antibacterianos/síntese química , Antibacterianos/química , Estrutura Molecular
4.
Biochemistry ; 58(41): 4195-4206, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31577135

RESUMO

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.


Assuntos
Sítio Alostérico/genética , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Modelos Moleculares , Difosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Bitionol/farmacologia , Quimera/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinética , Leucina/farmacologia , Plasmídeos/genética , Ligação Proteica , Células Sf9 , Spodoptera , Transfecção
5.
Proc Natl Acad Sci U S A ; 116(33): 16529-16534, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358625

RESUMO

Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Animais , Bitionol/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/química , Modelos Animais de Doenças , Sinergismo Farmacológico , Gentamicinas/farmacologia , Bicamadas Lipídicas/química , Fluidez de Membrana/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Relação Estrutura-Atividade , Lipossomas Unilamelares
6.
Chemosphere ; 224: 519-526, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30831504

RESUMO

The batch experiments were conducted to understand sorption process of bithionol (BIT) in yellow soil (YS) and red soil (RS), while column leaching experiments were performed to evaluate the leaching behavior of BIT and levamisole (LEV) in the tested soils. The adsorption and desorption data fitted well with the Freundlich isotherms (R2 ≥ 0.94). The distribution coefficient of BIT in the YS and RS were 104 and 98.3 L/kg, respectively. Hysteresis was observed for bithionol desorption in the YS and RS, with hysteresis coefficient of 0.917 and 0.928, respectively. Dissolved organic matter (DOM) addition and acid condition enhanced the adsorption of BIT in the soil. Both BIT and LEV showed poor leaching potential in the tested soils. More than 80% of BIT and LEV remained in the surface soil layer and the amount of the two target compounds in the leachates accounted for less than 1% of overall recovery. DOM showed little influence on the concentration of BIT and LEV in the leachates collected at different time. The results could fill the gap on the behavior of BIT and LEV in soil under laboratory conditions.


Assuntos
Bitionol/química , Levamisol/química , Poluentes do Solo/química , Solo/química , Adsorção
7.
Biochem Pharmacol ; 155: 92-101, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29940175

RESUMO

Soluble adenylate cyclase (sAC) is a non-plasma membrane-bound isoform of the adenylate cyclases signaling via the canonical second messenger, 3',5'-cyclic AMP (cAMP). sAC is involved in key physiological processes such as insulin release, sperm motility, and energy metabolism. Thus, sAC has attracted interest as a putative drug target and attempts have been made to develop selective inhibitors. Since sAC has a binding constant for its substrate, ATP, in the millimolar range, reductions in mitochondrial ATP production may be part of the mechanism-of-action of sAC inhibitors and the potential of these compounds to study the physiological outcomes of inhibition of sAC might be severely hampered by this. Here, we evaluate the effects of two commonly employed inhibitors, 2-OHE and KH7, on mitochondrial ATP production and energy metabolism. For comparison, we included a recently identified inhibitor of sAC, bithionol. Employing mitochondria isolated from mouse brain, we show that all three compounds are able to curb ATP production albeit via distinct mechanisms. Bithionol and KH7 mainly inhibit ATP production by working as a classical uncoupler whereas 2-OHE mainly works by decreasing mitochondrial respiration. These findings were corroborated by investigating energy metabolism in acute brain slices from mice. Since all three sAC inhibitors are shown to curb mitochondrial ATP production and affect energy metabolism, caution should be exercised when employed to study the physiological roles of sAC or for validating sAC as a drug target.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Inibidores de Adenilil Ciclases/farmacologia , Bitionol/farmacologia , Estradiol/análogos & derivados , Mitocôndrias/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Inibidores de Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Animais , Bitionol/química , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Estradiol/química , Estradiol/farmacologia , Feminino , Camundongos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia
8.
Artigo em Inglês | MEDLINE | ID: mdl-28918317

RESUMO

Herein, we developed a simple analytical procedure for the quantitation of bithionol residues in animal-derived food products such as porcine muscle, eggs, milk, eel, flatfish, and shrimp using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI+/MS-MS). Samples were extracted with 0.1% solution of formic acid in acetonitrile and the extract was purified using a C18 sorbent. Separation was performed on a Waters XBridge™ C18 reversed-phase analytical column using 0.1% solution of formic acid/acetonitrile as the mobile phase. Six-point matrix-matched calibration indicated good linearity, with the calculated coefficients of determination (R2) being≥0.9813. Intra- and inter-day recoveries (determined at spiking levels equivalent to 1×and 2×the limit of quantitation (0.25µg/kg)) ranged between 80.0 and 94.0%, with the corresponding relative standard deviations (RSDs) being≤8.2%. The developed experimental protocol was applied to different samples purchased from local markets in Seoul, which were tested negative for bithionol residues. In conclusion, the proposed method proved to be versatile and precise, being ideally suited for the routine detection of bithionol residues in animal-derived food products with various protein and fat contents.


Assuntos
Bitionol/análise , Cromatografia Líquida/métodos , Resíduos de Drogas/análise , Contaminação de Alimentos/análise , Espectrometria de Massas em Tandem/métodos , Animais , Bitionol/química , Bitionol/isolamento & purificação , Fracionamento Químico/métodos , Resíduos de Drogas/química , Resíduos de Drogas/isolamento & purificação , Ovos/análise , Limite de Detecção , Modelos Lineares , Leite/química , Reprodutibilidade dos Testes , Alimentos Marinhos/análise
9.
PLoS One ; 12(9): e0185111, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28931042

RESUMO

Previously, Bithionol (BT) was shown to enhance the chemosensitivity of ovarian cancer cell lines to cisplatin treatment. In the present study, we focused on the anti-tumor potential of the BT-paclitaxel combination when added to a panel of ovarian cancer cell lines. This in vitro study aimed to 1) determine the optimum schedule for combination of BT and paclitaxel and 2) assess the nature and mechanism(s) underlying BT-paclitaxel interactions. The cytotoxic effects of both drugs either alone or in combination were assessed by presto-blue cell viability assay using six human ovarian cancer cell lines. Inhibitory concentrations to achieve 50% cell death (IC50) were determined for BT and paclitaxel in each cell line. Changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27 were determined via immunoblot. Luminescent and colorimetric assays were used to determine caspases 3/7 and autotaxin (ATX) activity. Cellular reactive oxygen species (ROS) were measured by flow cytometry. Our results show that the efficacy of the BT-paclitaxel combination depends upon the concentrations and sequence of addition of paclitaxel and BT. Pretreatment with BT followed by paclitaxel resulted in antagonistic interactions whereas synergistic interactions were observed when both drugs were added simultaneously or when cells were pretreated with paclitaxel followed by BT. Synergistic interactions between BT and paclitaxel were attributed to increased ROS generation and enhanced apoptosis. Decreased expression of pro-survival factors (XIAP, bcl-2, bcl-xL) and increased expression of pro-apoptotic factors (caspases 3/7, PARP cleavage) was observed. Additionally, increased expression of key cell cycle regulators p21 and p27 was observed. These results show that BT and paclitaxel interacted synergistically at most drug ratios which, however, was highly dependent on the sequence of the addition of drugs. Our results suggest that BT-paclitaxel combination therapy may be effective in sensitizing ovarian cancer cells to paclitaxel treatment, thus mitigating some of the toxic effects associated with high doses of paclitaxel.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Bitionol/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Concentração Inibidora 50 , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/administração & dosagem , Diester Fosfórico Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
10.
BMC Cancer ; 17(1): 49, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28086831

RESUMO

BACKGROUND: Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination. METHODS: The cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity. RESULTS: The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27. CONCLUSION: In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bitionol/farmacologia , Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
11.
Cell Rep ; 16(9): 2281-8, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545877

RESUMO

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.


Assuntos
Membrana Celular/metabolismo , Proteína do X Frágil de Retardo Mental/genética , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Canais de Potássio/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Técnicas Biossensoriais , Bitionol/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Proteína do X Frágil de Retardo Mental/antagonistas & inibidores , Proteína do X Frágil de Retardo Mental/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Mutação , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tiazolidinas/farmacologia , Xenopus laevis
12.
mBio ; 7(4)2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27486194

RESUMO

UNLABELLED: Cryptococcus neoformans is a pathogenic fungus that is responsible for up to half a million cases of meningitis globally, especially in immunocompromised individuals. Common fungistatic drugs, such as fluconazole, are less toxic for patients but have low efficacy for initial therapy of the disease. Effective therapy against the disease is provided by the fungicidal drug amphotericin B; however, due to its high toxicity and the difficulty in administering its intravenous formulation, it is imperative to find new therapies targeting the fungus. The antiparasitic drug bithionol has been recently identified as having potent fungicidal activity. In this study, we used a combined gene dosing and drug affinity responsive target stability (GD-DARTS) screen as well as protein modeling to identify a common drug binding site of bithionol within multiple NAD-dependent dehydrogenase drug targets. This combination genetic and proteomic method thus provides a powerful method for identifying novel fungicidal drug targets for further development. IMPORTANCE: Cryptococcosis is a neglected fungal meningitis that causes approximately half a million deaths annually. The most effective antifungal agent, amphotericin B, was developed in the 1950s, and no effective medicine has been developed for this disease since that time. A key aspect of amphotericin B's effectiveness is thought to be because of its ability to kill the fungus (fungicidal activity), rather than just stop or slow its growth. The present study utilized a recently identified fungicidal agent, bithionol, to identify potential fungicidal drug targets that can be used in developing modern fungicidal agents. A combined protein and genetic analysis approach was used to identify a class of enzymes, dehydrogenases, that the fungus uses to maintain homeostasis with regard to sugar nutrients. Similarities in the drug target site were found that resulted in simultaneous inhibition and killing of the fungus by bithionol. These studies thus identify a common, multitarget site for antifungal development.


Assuntos
Antifúngicos/farmacologia , Bitionol/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/enzimologia , Oxirredutases/antagonistas & inibidores , Citosol/química , Compensação de Dosagem (Genética) , Simulação de Acoplamento Molecular
13.
Macromol Rapid Commun ; 37(13): 1066-73, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27174683

RESUMO

A new broad bandgap and 2D-conjugated D-A copolymer, PBDTBTz-T, based on bithienyl-benzodithiophene donor unit and bithiazole (BTz) acceptor unit, is designed and synthesized for the application as donor material in polymer solar cells (PSCs). The polymer possesses highly coplanar and crystalline structure with a higher hole mobility and lower HOMO energy level which is beneficial to achieve higher open circuit voltage (Voc ) of the PSCs with the polymer as donor. The PSCs based on PBDTBTz-T:PC71 BM blend film with a lower PC71 BM content of 40% demonstrate a power conversion efficiency (PCE) of 6.09% with a relatively higher Voc of 0.92 V. These results indicate that the lower HOMO energy level of the BTz-based D-A copolymer is beneficial to a high Voc of the PSCs. The polymer, with highly coplanar and crystalline structure, can effectively reduce the content of fullerene acceptor in the active layer and can enhance the absorption and PCE of the PSCs.


Assuntos
Bitionol/química , Fulerenos/química , Polímeros/química , Energia Solar , Estrutura Molecular , Tiofenos/química
14.
Anticancer Drugs ; 27(6): 547-59, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27058706

RESUMO

In terms of the concept of 'drug repurposing', we focused on pharmaceutical-grade Bithionol (BT) as a therapeutic agent against ovarian cancer. Our recent in-vitro study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer. BT was shown to cause cell death by caspases-mediated apoptosis. The present preliminary study further explores the antitumor potential of pharmaceutical-grade BT in an in-vivo xenograft model of human ovarian cancer. Nude Foxn1 mice bearing SKOV-3 human ovarian tumor xenografts were treated with titrated doses of BT and the therapeutic efficacy of pharmaceutical BT was determined using bioluminescence imaging. BT-induced changes in cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay. The effect of BT on autotaxin levels in serum, ascitic fluid, and tumor tissue was assessed by colorimetric and western blot techniques. BT treatment did not show antitumor potential or enhanced survival time at any of the doses tested. No apparent signs of toxicity were observed with any of the doses tested. Immunohistological analysis of tumor sections did not indicate a significant decrease in cellular proliferation (Ki-67 assay). An increase in apoptosis (by TUNEL assay) was observed in all BT-treated mice compared with vehicle-treated mice. Although BT did not show significant antitumor activity in the present study, the ability of BT to induce apoptosis still makes it a promising therapeutic agent. Further confirmatory and optimization studies are essential to enhance the therapeutic effects of BT.


Assuntos
Antineoplásicos/farmacologia , Bitionol/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Animais , Antineoplásicos/efeitos adversos , Apoptose/efeitos dos fármacos , Bitionol/efeitos adversos , Proliferação de Células/efeitos dos fármacos , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Estimativa de Kaplan-Meier , Camundongos Nus , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
J Biol Chem ; 291(18): 9776-84, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-26961873

RESUMO

The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.


Assuntos
Trifosfato de Adenosina/química , Adenilil Ciclases/química , Bicarbonatos/química , Bitionol/química , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , Humanos
16.
Neurobiol Dis ; 74: 144-57, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25173807

RESUMO

Aß accumulation plays a central role in the pathogenesis of Alzheimer's disease (AD). Recent studies suggest that the process of Aß nucleated polymerization is essential for Aß fibril formation, pathology spreading and toxicity. Therefore, targeting this process represents an effective therapeutic strategy to slow or block disease progression. To discover compounds that might interfere with the Aß seeding capacity, toxicity and pathology spreading, we screened a focused library of FDA-approved drugs in vitro using a seeding polymerization assay and identified small molecule inhibitors that specifically interfered with Aß seeding-mediated fibril growth and toxicity. Mitoxantrone, bithionol and hexachlorophene were found to be the strongest inhibitors of fibril growth and protected primary cortical neuronal cultures against Aß-induced toxicity. Next, we assessed the effects of these three inhibitors in vivo in the mThy1-APPtg mouse model of AD (8-month-old mice). We found that mitoxantrone and bithionol, but not hexachlorophene, stabilized diffuse amyloid plaques, reduced the levels of Aß42 oligomers and ameliorated synapse loss, neuronal damage and astrogliosis. Together, our findings suggest that targeting fibril growth and Aß seeding capacity constitutes a viable and effective strategy for protecting against neurodegeneration and disease progression in AD.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Degeneração Neural/fisiopatologia , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/efeitos dos fármacos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/toxicidade , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Bitionol/farmacocinética , Bitionol/farmacologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Gliose/tratamento farmacológico , Gliose/patologia , Gliose/fisiopatologia , Hexaclorofeno/farmacocinética , Hexaclorofeno/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitoxantrona/farmacocinética , Mitoxantrona/farmacologia , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacocinética , Fragmentos de Peptídeos/toxicidade , Placa Amiloide/tratamento farmacológico , Placa Amiloide/patologia , Placa Amiloide/fisiopatologia , Ratos
17.
BMC Cancer ; 14: 61, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24495391

RESUMO

BACKGROUND: Drug resistance is a cause of ovarian cancer recurrence and low overall survival rates. There is a need for more effective treatment approaches because the development of new drug is expensive and time consuming. Alternatively, the concept of 'drug repurposing' is promising. We focused on Bithionol (BT), a clinically approved anti-parasitic drug as an anti-ovarian cancer drug. BT has previously been shown to inhibit solid tumor growth in several preclinical cancer models. A better understanding of the anti-tumor effects and mechanism(s) of action of BT in ovarian cancer cells is essential for further exploring its therapeutic potential against ovarian cancer. METHODS: The cytotoxic effects of BT against a panel of ovarian cancer cell lines were determined by Presto Blue cell viability assay. Markers of apoptosis such as caspases 3/7, cPARP induction, nuclear condensation and mitochondrial transmembrane depolarization were assessed using microscopic, FACS and immunoblotting methods. Mechanism(s) of action of BT such as cell cycle arrest, reactive oxygen species (ROS) generation, autotaxin (ATX) inhibition and effects on MAPK and NF-kB signalling were determined by FACS analysis, immunoblotting and colorimetric methods. RESULTS: BT caused dose dependent cytotoxicity against all ovarian cancer cell lines tested with IC50 values ranging from 19 µM - 60 µM. Cisplatin-resistant variants of A2780 and IGROV-1 have shown almost similar IC50 values compared to their sensitive counterparts. Apoptotic cell death was shown by expression of caspases 3/7, cPARP, loss of mitochondrial potential, nuclear condensation, and up-regulation of p38 and reduced expression of pAkt, pNF-κB, pIκBα, XIAP, bcl-2 and bcl-xl. BT treatment resulted in cell cycle arrest at G1/M phase and increased ROS generation. Treatment with ascorbic acid resulted in partial restoration of cell viability. In addition, dose and time dependent inhibition of ATX was observed. CONCLUSIONS: BT exhibits cytotoxic effects on various ovarian cancer cell lines regardless of their sensitivities to cisplatin. Cell death appears to be via caspases mediated apoptosis. The mechanisms of action appear to be partly via cell cycle arrest, ROS generation and inhibition of ATX. The present study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Bitionol/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , NF-kappa B/metabolismo , Neoplasias Ovarianas/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
PLoS One ; 8(8): e70506, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23990907

RESUMO

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.


Assuntos
Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Triazóis/farmacologia , Trifosfato de Adenosina/química , Anfotericina B/química , Antifúngicos/química , Bitionol/química , Linhagem Celular Tumoral , Floxuridina/química , Humanos , Hifas/efeitos dos fármacos , Sepse/tratamento farmacológico , Esporos Fúngicos/efeitos dos fármacos , Tacrolimo/química , Triazóis/química
19.
Lab Invest ; 93(5): 508-19, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23478591

RESUMO

Lysophosphatidic acid (LPA) has a critical role in lymphocyte migration to secondary lymphoid organs. Autotaxin (ATX)/lysophospholipase D, in the vascular endothelium, is the main enzyme involved in LPA production. Whether ATX is involved in pathological lymphocyte migration to the inflamed mucosa has not been studied. We investigated the involvement of ATX in inflammatory bowel disease patients and two murine models of colitis. Tissue samples were obtained by intestinal biopsies from patients with Crohn's disease and those with ulcerative colitis with informed consent. ATX immunoreactivity was colocalized with MAdCAM-1-positive high-endothelial-like vessels, close to sites of lymphocyte infiltration. Enhanced expression of ATX mRNA was observed in the inflamed mucosa from Crohn's disease and ulcerative colitis patients. ATX mRNA expression level was remarkably higher in the actively inflamed mucosa than in the quiescent mucosa in the same patient. In the T-cell-transferred mouse model, ATX mRNA expression level gradually increased as colitis developed. In the dextran sodium sulfate mouse model, the expression level was considerably higher in colonic mucosa of chronically developed colitis than in colonic mucosa of acute colitis. Administration of an ATX inhibitor, bithionol, remarkably decreased lymphocyte migration to the intestine and ameliorated both dextran sodium sulfate-induced colitis and CD4-induced ileocolitis. In transwell assays, administration of bithionol or 1-bromo-3(s)-hydroxy-4-(palmitoyloxy) butylphosphonate (BrP-LPA) significantly decreased transmigration of splenocytes through high-endothelial-like vessels induced by TNF-α. We conclude that enhanced expression of ATX in the active mucosa has been implicated in the pathophysiology of inflammatory bowel disease through enhancing aberrant lymphocyte migration to the inflamed mucosa.


Assuntos
Quimiotaxia de Leucócito/fisiologia , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Mucosa Intestinal/metabolismo , Diester Fosfórico Hidrolases/biossíntese , Análise de Variância , Animais , Bitionol , Antígenos CD4 , Movimento Celular , Colite Ulcerativa/patologia , Colo/metabolismo , Colo/patologia , Doença de Crohn/patologia , Sulfato de Dextrana , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2 , Mucosa Intestinal/irrigação sanguínea , Mucosa Intestinal/patologia , Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo
20.
Mol Biochem Parasitol ; 183(1): 52-62, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22342964

RESUMO

Glutamate dehydrogenases (GDHs) play key roles in cellular redox, amino acid, and energy metabolism, thus representing potential targets for pharmacological interventions. Here we studied the functional network provided by the three known glutamate dehydrogenases of the malaria parasite Plasmodium falciparum. The recombinant production of the previously described PfGDH1 as hexahistidyl-tagged proteins was optimized. Additionally, PfGDH2 was cloned, recombinantly produced, and characterized. Like PfGDH1, PfGDH2 is an NADP(H)-dependent enzyme with a specific activity comparable to PfGDH1 but with slightly higher K(m) values for its substrates. The three-dimensional structure of hexameric PfGDH2 was solved to 3.1 Å resolution. The overall structure shows high similarity with PfGDH1 but with significant differences occurring at the subunit interface. As in mammalian GDH1, in PfGDH2 the subunit-subunit interactions are mainly assisted by hydrogen bonds and hydrophobic interactions, whereas in PfGDH1 these contacts are mediated by networks of salt bridges and hydrogen bonds. In accordance with this, the known bovine GDH inhibitors hexachlorophene, GW5074, and bithionol were more effective on PfGDH2 than on PfGDH1. Subcellular localization was determined for all three plasmodial GDHs by fusion with the green fluorescent protein. Based on our data, PfGDH1 and PfGDH3 are cytosolic proteins whereas PfGDH2 clearly localizes to the apicoplast, a plastid-like organelle specific for apicomplexan parasites. This study provides new insights into the structure and function of GDH isoenzymes of P. falciparum, which represent potential targets for the development of novel antimalarial drugs.


Assuntos
Glutamato Desidrogenase/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Regulação Alostérica , Sequência de Aminoácidos , Bitionol/química , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hexaclorofeno/química , Indóis/química , Cinética , Dados de Sequência Molecular , Fenóis/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...