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1.
Biochem Biophys Res Commun ; 525(3): 720-725, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32143826

RESUMO

Microbacterium hydrocarbonoxydans was isolated, using hydrazide compounds as its sole carbon source. The key enzyme that metabolizes these compounds was identified as hydrazidase, and the operon containing the gene coding for the enzyme, was revealed by genome sequencing. The operon also contained genes coding for an ATP-binding cassette transporter (ABC transporter), which was expected to transport the hydrazide compounds. Substrate binding protein (SBP), a component subunit of the transporter, plays an important role in recognizing the correct substrates for transport. Therefore, to elucidate the mechanism of recognition of the unnatural hydrazide compounds, we determined the crystal structures of the SBP, obtained from M. hydrocarbonoxydans (Mh-SBP), complexed with and without the hydrazide compound, at 2.2 Å and 1.75 Å resolutions, respectively. The overall structures of Mh-SBP were similar to those of the SBP in oligopeptide transporters such as OppA. On comparison, the liganded and unliganded structures of Mh-SBP showed an open - close conformation change. Interestingly, the binding mode of the compound to Mh-SBP was almost identical to that of the compound to hydrazidase, suggesting that the ABC transporter served transporting these compounds. Furthermore, based on the hydrazide complex structure, paraben, the other putative substrate of the protein, was successfully used with Mh-SBP to obtain the paraben complex structure.


Assuntos
Actinobacteria/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrazinas/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Ligantes , Modelos Moleculares , Parabenos/química , Parabenos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato
2.
J Med Chem ; 63(9): 4655-4684, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32118427

RESUMO

A high-throughput screen designed to discover new inhibitors of histone acetyltransferase KAT6A uncovered CTX-0124143 (1), a unique aryl acylsulfonohydrazide with an IC50 of 1.0 µM. Using this acylsulfonohydrazide as a template, we herein disclose the results of our extensive structure-activity relationship investigations, which resulted in the discovery of advanced compounds such as 55 and 80. These two compounds represent significant improvements on our recently reported prototypical lead WM-8014 (3) as they are not only equivalently potent as inhibitors of KAT6A but are less lipophilic and significantly more stable to microsomal degradation. Furthermore, during this process, we discovered a distinct structural subclass that contains key 2-fluorobenzenesulfonyl and phenylpyridine motifs, culminating in the discovery of WM-1119 (4). This compound is a highly potent KAT6A inhibitor (IC50 = 6.3 nM; KD = 0.002 µM), competes with Ac-CoA by binding to the Ac-CoA binding site, and has an oral bioavailability of 56% in rats.


Assuntos
Antineoplásicos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Hidrazinas/farmacologia , Sulfonamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Descoberta de Drogas , Estabilidade de Medicamentos , Humanos , Hidrazinas/síntese química , Hidrazinas/metabolismo , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo
3.
Carbohydr Polym ; 232: 115802, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952601

RESUMO

A series of biocompatible and non- toxic polysaccharide molecules have been successfully fabricated and explored their potential application for scavenging the carbonyl species in vitro. These macromolecules were dextrans with different hydrazide substitution ratios determined by TNBS assay, NMR and FTIR characterization. The colorimetric assay had demonstrated that these macromolecules could effectively scavenge acrolein, oxidized bovine serum albumin (BSA) in buffer solutions as well as carbonyl proteins from serum. The scavengers could achieve twice more scavenging effects for modified dextrans with high molecular weight (Mw = 100,000) than those of low ones (Mw = 40,000) with the same substitution ratio. Protein gel electrophoresis confirmed that the formation of the complex between carbonyls and modified dextrans resulted in appearance of slower bands. It also revealed that such macromolecules could protect cultured cells against the toxicity of acrolein or its derivatives. The proposed macromolecules indicated a very promising capability as scavengers for oxidative stress plus its derivatives without side effects.


Assuntos
Dextranos/metabolismo , Depuradores de Radicais Livres/metabolismo , Hidrazinas/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Dextranos/química , Depuradores de Radicais Livres/química , Hidrazinas/química , Estrutura Molecular , Carbonilação Proteica , Soroalbumina Bovina/química
4.
J Anal Toxicol ; 44(5): 470-481, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-31897465

RESUMO

We developed a robust analytical method for quantification of malondialdehyde (MDA) in urine and serum samples using dansylhydrazine (DH) as a derivatizing reagent. The derivatization procedure was partially carried out using an autosampler injection program to minimize errors associated with the low-volume addition of reagents and was optimized to yield a stable hydrazone derivative of MDA and its labeled d2-MDA analogue. The target MDA-DH derivatives were separated on an Agilent Zorbax Eclipse Plus Phenyl-Hexyl (3.0 × 100 mm, 3.5 µm) column. The mass-to-charge ratios of the target derivatives [(M+H)+ of 302 and 304 for MDA-DH and d2-MDA-DH, respectively] were analyzed in single ion monitoring mode using a single quadrupole mass spectrometer operated under positive electrospray ionization. The method limits of quantification were 5.63 nM (or 0.405 ng/mL) for urine analysis and 5.68 nM (or 0.409 ng/mL) for serum analysis. The quantification range for urine analysis was 5.63-500 nM (0.405-36.0 ng/mL) while the quantification range for serum analysis was 5.68-341 nM (0.409-24.6 ng/mL). The method showed good relative recoveries (98-103%), good accuracies (92-98%), and acceptable precisions (relative standard deviations 1.8-7.3% for inter-day precision; 1.8-6.1% for intra-day precision) as observed from the repeat analysis of quality control samples prepared at different concentrations. The method was used to measure MDA in individual urine samples (n = 287) and de-identified archived serum samples (n = 22) to assess the overall performance of the method. The results demonstrated that our method is capable of measuring urinary and serum levels of MDA, allowing its future application in epidemiologic investigations.


Assuntos
Compostos de Dansil/metabolismo , Hidrazinas/metabolismo , Malondialdeído/metabolismo , Líquidos Corporais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Humanos , Hidrazinas/sangue , Hidrazinas/urina , Limite de Detecção , Malondialdeído/sangue , Malondialdeído/urina , Espectrometria de Massas em Tandem
5.
Environ Microbiol ; 22(2): 525-536, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31867834

RESUMO

When looking back and wonder how we did it, I became even more aware of how my wanderings in microbiology are all linked, from the start of my PhD with Hans Veldkamp on sulphur-oxidizing bacteria in chemostats. My interests broadened from obligate chemolithoautotrophic bacteria to facultative organisms and the question about the ecological niches of these different metabolic types. The sulphide oxidizing bacteria also may be used to produce elemental sulphur, which can easily be removed from wastewater. This fitted in a long-standing collaboration with Dimitry Sorokin on the ecophysiology and application of alkaliphilic sulphur bacteria. Then came the denitrifying sulphur-oxidizing bacteria and their application to remove sulphide from wastewater, which lead to our interest in nitrate, nitrite and ammonium removal in general. The big surprise was the serendipitous discovery of the 'anammox'-process, whereby ammonium is anaerobically oxidized to dinitrogen gas with nitrite as electron acceptor. The early days of our anammox research are the main focus of this article, which describes the struggle of growing and identifying the most peculiar bacteria we ever came across. A specialized organelle, the anammoxosome was shown to be responsible for the key ammonium oxidation, whereby a rocket fuel, hydrazine, turned out to be an intermediate. Soon after we became aware that anammox is everywhere and in the marine environment makes up a major portion of the nitrogen cycle. The intense scientific collaboration with Mike Jetten and Mark van Loosdrecht and colleagues led to our further understanding and application of this fascinating process, which is briefly summarized in this article. My broader interest in environmental microbiology and microbial ecology has been a regularly returning theme, taking me all over the world to great collaborations lasting to this very day.


Assuntos
Compostos de Amônio/metabolismo , Bactérias/metabolismo , Reatores Biológicos/microbiologia , Ciclo do Nitrogênio/fisiologia , Anaerobiose , Microbiologia Ambiental , Hidrazinas/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Nitrogênio/metabolismo , Organelas/metabolismo , Oxirredução , Enxofre/metabolismo , Águas Residuárias/microbiologia
6.
Comput Biol Chem ; 80: 512-523, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31185422

RESUMO

A new series of N'-(substituted phenyl)-5-chloro/iodo-3-phenyl-1H-indole-2-carbohydrazide (5, 6) and N-[2-(substituted phenyl)-4-oxo-1,3-thiazolidin-3-yl]-5-iodo/chloro-3-phenyl-1H-indole-2-carboxamide (7, 8) derivatives were synthesized and evaluated for their anticancer properties. Compounds 5a and 6b, selected as prototypes by the National Cancer Institute for screening against the full panel of 60 human tumor cell lines at a minimum of five concentrations at 10-fold dilutions, demonstrated remarkable antiproliferative activity against leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) cancer, melanoma, ovarian cancer, renal cancer, and breast cancer (MCF-7) cell lines with GI50 values < 0.4 µM. A subset of the compounds was then tested for their potential to inhibit tubulin polymerization. Compounds 6f and 6g showed significant cytotoxicity at the nM level on MCF-7 cells and exhibited significant inhibitory activity on tubulin assembly and colchicine binding at about the same level as combretastatin A-4. Finally, docking calculations were performed to identify the binding mode of these compounds. Group 5 and 6 compounds interacted with the colchicine binding site through hydrophobic interactions similar to those of colchicine. These compounds with antiproliferative activity at high nanomolar concentration can serve as scaffolds for the design of novel microtubule targeting agents.


Assuntos
Antineoplásicos/farmacologia , Hidrazinas/farmacologia , Indóis/farmacologia , Tiazolidinas/farmacologia , Moduladores de Tubulina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrazinas/síntese química , Hidrazinas/química , Hidrazinas/metabolismo , Indóis/síntese química , Indóis/química , Indóis/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Ligação Proteica , Tiazolidinas/síntese química , Tiazolidinas/química , Tiazolidinas/metabolismo , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo
7.
Chem Commun (Camb) ; 55(49): 7029-7032, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31140482

RESUMO

A traceless thioester-producing protocol featuring carboxypeptidase Y-mediated hydrazinolysis of cysteinyl prolyl leucine-tagged peptides has been developed. The hydrazinolysis followed by thioesterification affords cysteinyl prolyl thioesters. Self-editing of the tag and subsequent trans-thioesterification yields peptide thioesters. The developed protocol was successfully applied to the conversion of recombinant proteins to thioesters.


Assuntos
Carboxipeptidases/metabolismo , Cisteína/metabolismo , Ésteres/metabolismo , Hidrazinas/metabolismo , Compostos de Sulfidrila/metabolismo , Cisteína/química , Ésteres/química , Hidrazinas/química , Conformação Molecular , Compostos de Sulfidrila/química
8.
Chem Biodivers ; 16(6): e1900085, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30990952

RESUMO

The discovery of J147 represented a significant milestone in the treatment of age-related disorders, which was further augmented by the recent identification of mitochondrial ATP synthase as the therapeutic target. However, the underlying molecular events associated with the modulatory activity of J147 have remained unresolved till date. Herein, we present, for the first time, a dynamical approach to investigate the allosteric regulation of mATP synthase by J147, using a reliable human αÎ³ß protein model. The highlight of our findings is the existence of the J147-bound protein in distinct structural associations at different MD simulation periods coupled with concurrent open↔close transitions of the ß catalytic and α allosteric (ATP5A) sites as defined by Cα distances (d), TriCα (Θ) and dihedral (φ) angular parameters. Firstly, there was an initial pairing of the αγ subunits away from the ß subunit followed by the formation of the 'non-catalytic' αß pair at a distance from the γ subunit. Interestingly, J147-induced structural arrangements were accompanied by the systematic transition of the ß catalytic site from a closed to an open state, while there was a concurrent transition of the allosteric site from an open αE conformation to a closed state. Consequentially, J147 reduced the structural activity of the whole αÎ³ß complex, while the unbound system exhibited high atomistic deviations and structural flexibility. Furthermore, J147 exhibited favorable binding at the allosteric site of mATP synthase with considerable electrostatic energy contributions from Gln215, Gly217, Thr219, Asp312, Asp313, Glu371 and Arg406. These findings provide details on the possible effects of J147 on mitochondrial bioenergetics, which could facilitate the structure-based design of novel small-molecule modulators of mATP synthase in the management of Alzheimer's disease and other neurodegenerative disorders.


Assuntos
Curcumina/análogos & derivados , Hidrazinas/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Regulação Alostérica , Doença de Alzheimer/tratamento farmacológico , Sítios de Ligação , Domínio Catalítico , Curcumina/farmacologia , Humanos , Hidrazinas/metabolismo , Hidrazinas/uso terapêutico , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , Simulação de Acoplamento Molecular , Análise de Componente Principal , Eletricidade Estática , Termodinâmica
9.
Biomed Res Int ; 2019: 2181370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31032337

RESUMO

Introduction: Oxidative stress is a state of imbalance between the production of reactive oxygen species and antioxidant defenses. It results in the oxidation of all cellular elements and, to a large extent, proteins, causing inter alia the formation of carbonyl groups in their structures. The study focused on assessment of changes in the plasma protein-bound carbonyls in police horses after combat training and after rest and the applicability of infrared spectroscopy with a Fourier transform, utilizing the attenuated total reflectance (FTIR-ATR) in detecting plasma protein oxidation. Methods: We evaluated the influence of both the different concentrations of hydrogen peroxide and combat training on protein carbonylation in horse blood plasma. The oxidation of plasma proteins was assessed using a spectrophotometric method based on the carbonyl groups derivatization with 2,4-dinitrophenylhydrazine (DNPH). The measured values were correlated with the carbonyl groups concentrations determined by means of the FTIR-ATR method. Results: The linear correlation between the DNPH and FTIR-ATR methods was shown. The concentration of plasma protein-bound carbonyls significantly deceased in police horses after one-day rest when compared to the values measured directly after the combat training (a drop by 23%, p<0.05 and 29%, p<0.01 measured by DNPH and FTIR-ATR methods, respectively). These results were consistent with the proteins phosphorylation analysis. Conclusion: The FTIR-ATR method may be applied to measure the level of plasma proteins peroxidation.


Assuntos
Antioxidantes/metabolismo , Proteínas Sanguíneas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Animais , Antioxidantes/química , Proteínas Sanguíneas/química , Proteínas Sanguíneas/efeitos dos fármacos , Cavalos , Humanos , Hidrazinas/química , Hidrazinas/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxirredução , Espécies Reativas de Oxigênio/sangue , Espectroscopia de Infravermelho com Transformada de Fourier
10.
Bioorg Med Chem ; 27(9): 1818-1823, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30885567

RESUMO

In view of expanding the structure activity relationship of xanthine oxidase inhibitors, a series of 3-oxo-6-aryl-2,3-dihydropyridazine-4-carbohydrazide/carboxylic acid derivatives were designed by molecular docking and synthesized. All the target compounds were evaluated for their in vitro XO inhibition by using febuxostat and allopurinol as the standard controls. Most of the hydrazide derivatives exhibited potency levels in the micromolar range. From the view of docking study, hydrazide derivatives bind to the active site of XO through a novel interaction mode, which is different from that of febuxostat bearing a carboxyl group. The most promising compound 8b was further subjected to kinetic analysis to deduce their modes of inhibition.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Hidrazinas/química , Xantina Oxidase/antagonistas & inibidores , Animais , Sítios de Ligação , Domínio Catalítico , Bovinos , Inibidores Enzimáticos/metabolismo , Hidrazinas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Xantina Oxidase/metabolismo
11.
Eur J Med Chem ; 166: 432-444, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30739825

RESUMO

Phenelzine was first employed to design new aryl hydrazine-based LSD1 inhibitors based on the experience-based discovery (EBD) strategy. Among these compounds, D8 potently inhibited LSD1 (IC50 = 882.30 nM) in a reversible manner. Compound D8 was selective to LSD1 over MAO-A/B and showed H3K4me2 competitive binding to LSD1. The interaction between H3K4me2 and LSD1 was also confirmed by the Co-IP assay. In LSD1 overexpressed A549 cells, compound D8 dose-dependently induced accumulation of LSD1 substrates H3K4me1/2 and H3K9me1/2, showed cellular target engagement to LSD1 and significantly inhibited cell migration of A549 cells. Docking studies suggested that compound D8 occupied the peptide binding region and therefore blocked the access of the peptide substrate to the FAD, finally leading to the demethylase activity inhibition of LSD1. The findings indicate that aryl hydrazines are new scaffolds for the design of LSD1 inhibitors, the identification of D8 provides further evidence for our previously proposed general principle that fused heterocycles with an amine group are potentially active toward LSD1 by competitive binding to LSD1 with H3 peptide substrates.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Hidrazinas/química , Hidrazinas/farmacologia , Células A549 , Ligação Competitiva , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Histona Desmetilases/metabolismo , Histonas/metabolismo , Humanos , Hidrazinas/metabolismo , Metilação/efeitos dos fármacos , Relação Estrutura-Atividade
12.
Anal Chim Acta ; 1052: 137-144, 2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30685032

RESUMO

Among organic small molecules, hydrogen peroxide (H2O2) and hydrazine (NH2NH2) often cause concern because they are widely used in biological and chemical industries. Here, we present a novel probe RH-1 for colorimetric detection of NH2NH2 and fluorescent imaging of H2O2. In this probe, rhodamine was used as the main skeleton due to its favorable spectroscopic performance and stable absorption. Importantly, a benzoic acid group is present in the rhodamine skeleton, which can react with NH2NH2 by amidation. The rhodamine skeleton can also be modified with chromogens to detect H2O2. Results showed that RH-1 can be used for colorimetric detection of NH2NH2 and fluorescent monitoring of H2O2 with high selectivity and sensitivity. The detection limits for NH2NH2 and H2O2 were 0.27 and 0.16 µM, respectively. Moreover, RH-1 can fluorescently image H2O2 in living cells with low cytotoxicity.


Assuntos
Colorimetria , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Hidrazinas/metabolismo , Peróxido de Hidrogênio/metabolismo , Imagem Óptica , Animais , Sobrevivência Celular , Concentração de Íons de Hidrogênio , Camundongos , Células RAW 264.7
13.
J Virol ; 93(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30541831

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of hospitalization of infants and young children, causing considerable respiratory disease and repeat infections that may lead to chronic respiratory conditions such as asthma, wheezing, and bronchitis. RSV causes ∼34 million new episodes of lower respiratory tract illness (LRTI) in children younger than 5 years of age, with >3 million hospitalizations due to severe RSV-associated LRTI. The standard of care is limited to symptomatic relief as there are no approved vaccines and few effective antiviral drugs; thus, a safe and efficacious RSV therapeutic is needed. Therapeutic targeting of host proteins hijacked by RSV to facilitate replication is a promising antiviral strategy as targeting the host reduces the likelihood of developing drug resistance. The nuclear export of the RSV M protein, mediated by the nuclear export protein exportin 1 (XPO1), is crucial for RSV assembly and budding. Inhibition of RSV M protein export by leptomycin B correlated with reduced RSV replication in vitro In this study, we evaluated the anti-RSV efficacy of Verdinexor (KPT-335), a small molecule designed to reversibly inhibit XPO1-mediated nuclear export. KPT-335 inhibited XPO1-mediated transport and reduced RSV replication in vitro KPT-335 was effective against RSV A and B strains and reduced viral replication following prophylactic or therapeutic administration. Inhibition of RSV replication by KPT-335 was due to a combined effect of reduced XPO1 expression, disruption of the nuclear export of RSV M protein, and inactivation of the NF-κB signaling pathway.IMPORTANCE RSV is an important cause of LRTI in infants and young children for which there are no suitable antiviral drugs offered. We evaluated the efficacy of KPT-335 as an anti-RSV drug and show that KPT-335 inhibits XPO1-mediated nuclear export, leading to nuclear accumulation of RSV M protein and reduction in RSV levels. KPT-335 treatment also resulted in inhibition of proinflammatory pathways, which has important implications for its effectiveness in vivo.


Assuntos
Acrilamidas/farmacologia , Hidrazinas/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Células A549 , Acrilamidas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/imunologia , Humanos , Hidrazinas/metabolismo , Carioferinas/efeitos dos fármacos , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/metabolismo , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Células Vero
14.
BMC Res Notes ; 11(1): 863, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518414

RESUMO

OBJECTIVE: The advent of ligand-based receptor capture methodologies, allows the identification of unknown receptor candidates for orphan extracellular ligands. However, further target validation can be tedious, laborious and time-consuming. Here, we present a methodology that provides a fast and cost-efficient alternative for candidate target verification on living cells. RESULTS: In the described methodology a ligand of interest (e.g. transferrin, epidermal growth factor or insulin) was conjugated to a linker (TriCEPS) that carries a biotin. To confirm ligand/receptor interactions, the ligand-TriCEPS conjugates were first added onto living cells and cells were subsequently labeled with a streptavidin-fluorophore and analyzed by flow cytometry (thus referred as Flow-TriCEPS). Flow-TriCEPS was also used to validate identified receptor candidates when combined with a siRNA knock down approach (i.e. reduction of expression levels). This approach is versatile as it can be applied for different classes of ligands (proteins, peptides, antibodies) and different cell lines. Moreover, the method is time-efficient since it takes advantage of the large variety of commercially available (and certified) siRNAs.


Assuntos
Biotina/análogos & derivados , Receptores ErbB/metabolismo , Citometria de Fluxo/métodos , Hidrazinas/metabolismo , Succinimidas/metabolismo , Biotina/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Insulina/metabolismo , Ligantes , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes
15.
Nat Commun ; 9(1): 3687, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206228

RESUMO

Fosfazinomycin and kinamycin are natural products that contain nitrogen-nitrogen (N-N) bonds but that are otherwise structurally unrelated. Despite their considerable structural differences, their biosynthetic gene clusters share a set of genes predicted to facilitate N-N bond formation. In this study, we show that for both compounds, one of the nitrogen atoms in the N-N bond originates from nitrous acid. Furthermore, we show that for both compounds, an acetylhydrazine biosynthetic synthon is generated first and then funneled via a glutamyl carrier into the respective biosynthetic pathways. Therefore, unlike other pathways to N-N bond-containing natural products wherein the N-N bond is formed directly on a biosynthetic intermediate, during the biosyntheses of fosfazinomycin, kinamycin, and related compounds, the N-N bond is made in an independent pathway that forms a branch of a convergent route to structurally complex natural products.


Assuntos
Vias Biossintéticas , Ácido Glutâmico/metabolismo , Hidrazinas/metabolismo , Compostos Organofosforados/metabolismo , Quinonas/metabolismo , Ácido Glutâmico/química , Hidrazinas/química , Nitritos/metabolismo , Isótopos de Nitrogênio , Compostos Organofosforados/química , Espectroscopia de Prótons por Ressonância Magnética , Quinonas/química , Ácido Succínico/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(37): 9098-9103, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30150407

RESUMO

Ladderane lipids are unique to anaerobic ammonium-oxidizing (anammox) bacteria and are enriched in the membrane of the anammoxosome, an organelle thought to compartmentalize the anammox process, which involves the toxic intermediate hydrazine (N2H4). Due to the slow growth rate of anammox bacteria and difficulty of isolating pure ladderane lipids, experimental evidence of the biological function of ladderanes is lacking. We have synthesized two natural and one unnatural ladderane phosphatidylcholine lipids and compared their thermotropic properties in self-assembled bilayers to distinguish between [3]- and [5]-ladderane function. We developed a hydrazine transmembrane diffusion assay using a water-soluble derivative of a hydrazine sensor and determined that ladderane membranes are as permeable to hydrazine as straight-chain lipid bilayers. However, pH equilibration across ladderane membranes occurs 5-10 times more slowly than across straight-chain lipid membranes. Langmuir monolayer analysis and the rates of fluorescence recovery after photobleaching suggest that dense ladderane packing may preclude formation of proton/hydroxide-conducting water wires. These data support the hypothesis that ladderanes prevent the breakdown of the proton motive force rather than blocking hydrazine transmembrane diffusion in anammox bacteria.


Assuntos
Bactérias/química , Permeabilidade da Membrana Celular , Membrana Celular/química , Hidrazinas/química , Hidróxidos/química , Fosfolipídeos/química , Prótons , Anaerobiose/fisiologia , Bactérias/metabolismo , Membrana Celular/metabolismo , Hidrazinas/metabolismo , Hidróxidos/metabolismo , Fosfolipídeos/metabolismo
17.
Bioorg Chem ; 80: 112-120, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29894890

RESUMO

In search of better α-glucosidase inhibitors, a series of bis-indolylmethane sulfonohydrazides derivatives (1-14) were synthesized and evaluated for their α-glucosidase inhibitory potential. All derivatives exhibited outstanding α-glucosidase inhibition with IC50 values ranging between 0.10 ±â€¯0.05 to 5.1 ±â€¯0.05 µM when compared with standard drug acarbose having IC50 value 856.28 ±â€¯3.15 µM. Among the series, analog 7 (0.10 ±â€¯0.05 µM) with tri-chloro substitution on phenyl ring was identified as the most potent inhibitor of α-glucosidase (∼ 8500 times). The structure activity relationship has been also established. Molecular docking studies were also performed to help understand the binding interaction of the most active analogs with receptors. From the docking studies, it was observed that all the active bis-indolylmethane sulfonohydrazides derivatives showed considerable binding interactions within the active site (acarbose inhibition site) of α-glucosidase. We also evaluated toxicity of all derivatives and found none of them are toxic.


Assuntos
Inibidores de Glicosídeo Hidrolases/síntese química , Hidrazinas/química , alfa-Glucosidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Inibidores de Glicosídeo Hidrolases/metabolismo , Hidrazinas/metabolismo , Ligação de Hidrogênio , Indóis/química , Concentração Inibidora 50 , Metano/química , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , alfa-Glucosidases/química
18.
Int J Mol Med ; 42(1): 615-624, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29693158

RESUMO

Platelet activation and subsequent accumulation at sites of vascular injury are central to thrombus formation, which is considered to be a trigger of several cardiovascular diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicinal herb for promoting blood circulation by removing blood stasis. In our previous study, several compounds extracted from this herb, including luteolin­4'­O­ß­D­glucopyranoside (LGP), were revealed to exert inhibitory effects on adenosine diphosphate (ADP)­induced platelet aggregation. The aim of present study was to confirm these antiplatelet effects and elucidate the potential mechanisms. Using a platelet­aggregation assay, it was revealed that LGP significantly inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid. It was also found that LGP exhibited marked inhibitory effects on the activation of αIIbß3 integrin, the secretion of serotonin from granules, and the synthesis of thromboxane A2. In addition, the results showed that LGP suppressed Ras homolog family member A and phosphoinositide 3­kinase/Akt/glycogen synthase kinase 3ß signal transduction. Data from a radiolabeled ligand­binding assay indicated that LGP exhibited apparent competing effects on thromboxane receptor (TP) and P2Y12 receptors. In conclusion, the data presented here demonstrated that LGP, a natural compound from C. nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual­receptor inhibition on P2Y12 and TP receptors.


Assuntos
Glucosídeos/farmacologia , Luteolina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Receptores Purinérgicos P2Y12/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Ácido Araquidônico/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Ácidos Graxos Insaturados/metabolismo , Feminino , Glucosídeos/química , Glicogênio Sintase Quinase 3 beta/metabolismo , Hidrazinas/metabolismo , Luteolina/química , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Serotonina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tromboxano A2/biossíntese , Trítio , Proteína rhoA de Ligação ao GTP/metabolismo
19.
J Appl Microbiol ; 125(1): 121-132, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29542226

RESUMO

AIMS: The aim of this study was to explore the vertical distribution traits of anaerobic ammonium-oxidizing (anammox) bacterial relative abundance and community composition along the oxic/anoxic sediment profiles in a shallow lake. METHODS AND RESULTS: The Illumina Miseq-based sequencing and quantitative polymerase chain reactions were utilized to analyse relative abundance of anammox hydrazine synthase (hzsB) gene in comparison with bacterial 16S rRNA genes, anammox bacterial relative abundance (the number of anammox sequences divided by total number of sequences), community composition and diversity in sediments. The relative abundance of hzsB gene at the low-nitrogen (LN) site in the lake sediments showed that the vertical distribution of anammox bacteria increased to a peak, then decreased with increasing depth. Moreover, the relative abundance of hzsB gene at the high-nitrogen site was significantly lower than that at the LN site. Additionally, the community composition results showed that Candidatus Brocadia sp. was the dominant genus. In addition, the anammox bacterial diversity was also site specific. Redundancy analysis showed that the total N and the NH4+ -N content might be the most important factors affecting anammox bacterial community composition in the studied sites. CONCLUSIONS: The results revealed the specific vertical variance of anammox bacterial distribution and community composition in oxic/anoxic sediments of a eutrophic shallow lake. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that anammox bacteria displayed the particular distribution in freshwater sediments, which implied a strong response to the anthropogenic eutrophication.


Assuntos
Bactérias/classificação , Biodiversidade , Eutrofização , Sedimentos Geológicos/microbiologia , Lagos/microbiologia , Bactérias/genética , Bactérias/metabolismo , Genes Bacterianos/genética , Sedimentos Geológicos/química , Hidrazinas/metabolismo , Lagos/química , Nitrogênio/análise , Oxirredução , Filogenia , RNA Ribossômico 16S/genética
20.
Spectrochim Acta A Mol Biomol Spectrosc ; 196: 160-167, 2018 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-29444498

RESUMO

A novel dicyanoisophorone based fluorescent probe HP was developed to detect hydrazine. Upon the addition of hydrazine, probe HP displayed turn-on fluorescence in the red region with a large Stokes shift (180nm). This probe exhibited high selectivity and high sensitivity to hydrazine in solution. The detection limit of HP was found to be 3.26ppb, which was lower than the threshold limit value set by USEPA (10ppb). Moreover, the probe was successfully applied to detect hydrazine in different water samples and living cells.


Assuntos
Técnicas Citológicas/métodos , Corantes Fluorescentes/química , Hidrazinas/análise , Isocianatos/química , Cicloexanonas/química , Dimetil Sulfóxido , Estabilidade de Medicamentos , Células HeLa , Humanos , Hidrazinas/metabolismo , Concentração de Íons de Hidrogênio , Microscopia Confocal , Microscopia de Fluorescência
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