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1.
Biomed Khim ; 65(5): 398-402, 2019 Aug.
Artigo em Russo | MEDLINE | ID: mdl-31666412

RESUMO

The experimental study of the cardioprotective effect of uridine, the metabolic precursor of the endogenous activator of mitochondrial ATP-dependent K+-channels (mitoKATP-channels), was performed using the model of myocardial ischemia/reperfusion (I/RP) in rats. Ischemia for 30 min followed by reperfusion for 120 min resulted in a significant decrease in ATP and phosphocreatine (PC) content, intensification of lipid peroxidation (LPO), and inhibition of the antioxidant system (AOS) in cardiomyocytes. Uridine in a dose of 30 mg/kg, administered intravenously prior to reperfusion, had a protective effect on myocardial metabolism in the I/RP zone. It prevented the decrease of ATP and PC, limited the LPO processes, evaluated by the content of lipid hydroperoxides and conjugated dienes, and improved the AOS state by, preventing the decrease of superoxide dismutase (SOD) activity and increasing the content of reduced glutathione (GSH). The mitoKATP-channel blocker 5-hydroxydecanoate (5-HD, 5 mg/kg) eliminated the ability of uridine to maintain the ATP level and to exhibit its positive effect on the intensity of the LPO and activity of AOS. The obtained data allow us to conclude that activation of mitoKATP-channels play an important role in the mechanism of the cardioprotective effect of uridine in I/RP damage of myocardium.


Assuntos
Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Uridina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Ácidos Decanoicos , Glutationa/metabolismo , Hidroxiácidos , Peroxidação de Lipídeos , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio , Substâncias Protetoras/farmacologia , Ratos , Superóxido Dismutase/metabolismo
2.
Nucleic Acids Res ; 47(19): 10296-10312, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31495891

RESUMO

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA/química , Transcrição Genética/efeitos dos fármacos , Uridina/análogos & derivados , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Oxigênio/química , Pseudomonas/química , RNA/genética , Clivagem do RNA/efeitos dos fármacos , Streptomyces/química , Especificidade por Substrato , Timidina/química , Timidina/genética , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética , Uracila/química , Uridina/síntese química , Uridina/química , Uridina/farmacologia
3.
Pestic Biochem Physiol ; 158: 112-120, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378345

RESUMO

Cytochrome P450s (P450s) confer resistance against herbicides, and this is increasingly becoming a concern for weed control. As a widespread Gramineae weed in paddy fields, Echinocloa glabrescens has become resistant to the acetolactate synthase (ALS)-inhibiting triazolopyrimidine herbicide penoxsulam. In this study, we found that the GR50 of the resistant population (SHQP-R) decreased substantially from 25.6 to 5.0 and 6.2 g a.i. ha-1 after treatment with the P450 inhibitors piperonyl butoxide (PBO) and malathion, respectively. However, P450 inhibitors almost had no effects on the susceptibility of the sensitive population (JYJD-S) to penoxsulam. To investigate the mechanisms of metabolic resistance, transcriptome sequencing analysis was performed to find candidate genes that may confer resistance to penoxsulam in E. glabrescens. A total of 233 P450 differentially expressed genes (DEGs) were identified by transcriptome sequencing. We found that the metabolic process and metabolic pathways were the most highly enriched in DEGs. Further, twenty-seven candidate P450 DEGs were selected for qPCR validation analyses. After penoxsulam treatment, the relative expression levels were significantly higher in SHQP-R than in JYJD-S. Among these, the relative expression of twenty-three P450 DEGs (eighteen from the CYP72A-71C-74A-96A-734A subfamily; five from CYP81E1-94C1-94B3-714C1-714C2) were upregulated and four P450 DEGs (from CYP724B1-711A1-707A7-97B2) were downregulated. Changes in the expression of these candidate P450 genes in E. glabrescens were in response to penoxsulam, which provides preliminary evidence for the role of P450s in herbicide metabolism in E. glabrescens. However, further functional studies on metabolic resistance to penoxsulam in a resistant E. glabrescens population are required.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Echinochloa/efeitos dos fármacos , Echinochloa/metabolismo , Perfilação da Expressão Gênica/métodos , Sulfonamidas/farmacologia , Uridina/análogos & derivados , Sistema Enzimático do Citocromo P-450/genética , Echinochloa/genética , Resistência a Herbicidas/genética , Malation/farmacologia , Butóxido de Piperonila/farmacologia , Uridina/farmacologia
4.
J Agric Food Chem ; 67(29): 8085-8095, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31265279

RESUMO

Herbicide resistance identification is essential for effective chemical weed control. In this study, we quantified the differences in growth response between penoxsulam resistant (R) and sensitive (S) Echinochloa crus-galli populations, explored the changes in ALS, and performed genetic analyses to identify metabolic genes that are up-regulated by the application of penoxsulam and other common herbicides. The R population showed a 26.0-fold higher resistance to penoxsulam and varied resistance to most tested herbicides with indices ranging from 4.9 to 145.9. A Trp-574-Arg amino acid mutation in ALS and low penoxsulam ALS sensitivity were the main mechanisms underlying herbicide resistance. The penoxsulam resistance can be significantly reversed by two P450s inhibitors and one GST inhibitor. By RNA-Seq, thirty-six highly expressed contigs were selected, and 30 of them were up-regulated in the R population treated by penoxsulam. Many of these genes were significantly expressed when treated with pyroxsulam, metamifop, and quinclorac. These upregulated genes appear to be complementary for plant resistance to penoxsulam and other common herbicides.


Assuntos
Echinochloa/efeitos dos fármacos , Resistência a Herbicidas , Herbicidas/farmacologia , Sulfonamidas/farmacologia , Uridina/análogos & derivados , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Echinochloa/genética , Echinochloa/crescimento & desenvolvimento , Echinochloa/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Uridina/farmacologia
5.
Biomed Pharmacother ; 116: 108982, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31146110

RESUMO

Foot-and-mouth disease (FMD) is one of the most highly contagious animal disease that affects cloven-hoofed animals. However, the FMD vaccine does not provide effective protection until adaptive immune protection elicited by the vaccination occurs. Therefore, an alternative application of antiviral agents for inhibition of the FMD virus (FMDV) is needed. Here, we demonstrated that brequinar could exhibit antiviral activity in swine kidney cells (IBRS-2 cells) infected with two different FMDV serotypes. Subsequently, in vivo activity of brequinar was confirmed in a mouse model of infection. Specifically, brequinar at a concentration of 50 µg, provided 25% protection for 5 days following FMDV challenge. These results suggested that brequinar could be used as effective antiviral agent against FMD.


Assuntos
Antivirais/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Vírus da Febre Aftosa/fisiologia , Febre Aftosa/tratamento farmacológico , Febre Aftosa/virologia , Animais , Antivirais/química , Antivirais/farmacologia , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Vírus da Febre Aftosa/efeitos dos fármacos , Miocárdio/patologia , Suínos , Uridina/farmacologia
6.
Int J Hematol ; 110(2): 161-169, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31020568

RESUMO

Hypomethylating agents (HMAs), azacitidine and decitabine, are standards of care in higher-risk myelodysplastic syndromes and in acute myeloid leukemia patients ineligible for intensive therapy. Over the last 10 years, research efforts have sought to better understand their mechanism of action, both at the molecular and cellular level. These efforts have yet to robustly identify biomarkers for these agents. The clinical activity of HMAs in myeloid neoplasms has been firmly established now but still remains of limited magnitude. Besides optimized use at different stages of the disease, most of the expected clinical progress with HMAs will come from the development of second-generation compounds orally available and/or with improved pharmacokinetics, and from the search, so far mostly empirical, of HMA-based synergistic drug combinations.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Decitabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Ensaios Clínicos como Assunto , Decitabina/química , Decitabina/farmacologia , Esquema de Medicação , Combinação de Medicamentos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/genética , Síndromes Mielodisplásicas/genética , Uridina/administração & dosagem , Uridina/análogos & derivados , Uridina/farmacologia , Uridina/uso terapêutico
7.
Eur J Med Chem ; 171: 462-474, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30933853

RESUMO

The present status of antibiotic resistant requires an urgent invention of novel agents that act on clinically unexplored antibacterial targets. The enzyme MraY (phospho-MurNAc-pentapeptide translocase), essential for bacterial cell wall synthesis, fulfils this criterion as it has not been explored as a target in a clinical context. Specifically, the enzyme is involved in the lipid-linked cycle of peptidoglycan biosynthesis and is reportedly targeted by naturally-occurring nucleoside antibiotics. The antimicrobial 'caprazamycin' class of nucleoside antibiotics targets Mycobacterium tuberculosis and clinically relevant Gram-negative bacteria such as Pseudomonas aeruginosa besides various drug resistant strains and is therefore an eligible starting point for the development of novel agents. In this review, we aim to summarise the structure-activity relationships of the natural, semi-synthetic as well as synthetic analogues of nucleoside antibiotic caprazamycins. This review highlights caprazamycins as promising lead structures for development of potent and selective antimicrobial agents that target MraY, the bacterial enzyme involved in the first membrane-dependent step in bacterial peptidoglycan assembly.


Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Transferases/antagonistas & inibidores , Uridina/análogos & derivados , Antibacterianos/química , Azepinas/química , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Transferases/metabolismo , Uridina/química , Uridina/farmacologia
8.
Bull Exp Biol Med ; 166(6): 806-810, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31020578

RESUMO

We studied the effects of in vivo modulation of activity of mitochondrial ATP-dependent potassium channel (mitoKATP) by uridine on the morphofunctional state of mitochondria in rat cardiomyocytes under conditions of acute hypoxia. Preinjection of uridine to animals reduced the number of structurally modified mitochondria, but had practically no effect on their morphogenesis after hypoxia. Uridine in vivo stimulated the formation of micromitochondria and their release into the cytoplasm. The number of "maternal" mitochondria containing three and more new micromitochondria, increased as well. The use of mitoKATP blocker 5-hydroxydecanoate in parallel with uridine abolished its protective effect, as it significantly inhibited the formation of micromitochondria in rat cardiomyocytes after acute hypoxic exposure.


Assuntos
Hipóxia/metabolismo , Miocárdio/metabolismo , Canais de Potássio/metabolismo , Uridina/farmacologia , Animais , Hipóxia Celular , Ácidos Decanoicos/antagonistas & inibidores , Ácidos Decanoicos/farmacologia , Hidroxiácidos/antagonistas & inibidores , Hidroxiácidos/farmacologia , Hipóxia/tratamento farmacológico , Hipóxia/patologia , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar
9.
Molecules ; 24(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901934

RESUMO

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.


Assuntos
Antivirais/farmacologia , Desoxiaçúcares/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Uridina/farmacologia , Antivirais/química , Linhagem Celular Tumoral , Células Cultivadas , Desoxiaçúcares/química , Relação Dose-Resposta a Droga , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Uridina/análogos & derivados , Uridina/química , Ensaio de Placa Viral
10.
Emerg Microbes Infect ; 7(1): 187, 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30459406

RESUMO

The genus Flavivirus contains many important pathogens, including dengue virus (DENV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV). AR-12 is a celecoxib-derived anticancer agent that possesses antiviral activity against a broad range of viruses. We pharmacologically exploited this unique activity to develop additional antiviral agents, resulting in the production of the AR-12 derivatives P12-23 and P12-34. At nanomolar concentrations, these compounds were effective in suppressing DENV, ZIKV and JEV replication, exhibiting 10-fold improvements in the efficacy and selectivity indices as compared to AR-12. Regarding the mode of antiviral action, P12-23 and P12-34 inhibited viral RNA replication but had no effect on viral binding, entry or translation. Moreover, these AR-12 derivatives co-localized with mitochondrial markers, and their antiviral activity was lost in mitochondria-depleted cells. Interestingly, exogenous uridine or orotate, the latter being a metabolite of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH), abolished the antiviral activity of AR-12 and its derivatives. As DHODH is a key enzyme in the de novo pyrimidine biosynthesis pathway, these AR-12 derivatives may act by targeting pyrimidine biosynthesis in host cells to inhibit viral replication. Importantly, treatment with P12-34 significantly improved the survival of mice that were subcutaneously challenged with DENV. Thus, P12-34 may warrant further evaluation as a therapeutic to control flaviviral outbreaks.


Assuntos
Flavivirus/fisiologia , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/biossíntese , Sulfonamidas/química , Sulfonamidas/farmacologia , Replicação Viral/efeitos dos fármacos , Células A549 , Animais , Antivirais/farmacologia , Vias Biossintéticas , Linhagem Celular , Vírus da Dengue/efeitos dos fármacos , Flavivirus/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Uridina/farmacologia , Zika virus/efeitos dos fármacos
11.
Arch Pharm (Weinheim) ; 351(11): e1800204, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30276854

RESUMO

We report herein a simple and efficient synthesis of a new series of antibacterial uridine nucleosides. The strategy involved a sequential silylation/N-glycosylation/N-propargylation procedure of uracil 1 for preparing the dipolarophile 5 in good yield. A series of novel uridine-[1,2,3]triazole nucleosides 6a-j were efficiently synthesized via the copper-catalyzed azide-alkyne cycloaddition (CuAAC) from dipolarophile 5 with different selected azides. The reactions were carried out under both conventional and ultrasonic irradiation conditions. In general, improvements were observed when reactions were carried out under sonication. Their antibacterial potential has been evaluated by means of a micro-dilution assay against either Gram-positive or Gram-negative bacteria. Compounds 6i and 6j have shown significant bactericidal activity against Staphylococcus aureus (MIC = 10 and 6 µM, respectively), and 6h against Escherichia coli (MIC = 8 µM). Moreover, antibacterial kinetic assays showed that 6i and 6j significantly reduced the S. aureus growth rate at the MIC concentration, after 6 h, compared to their deprotected analogs, 6k and 6l, respectively. Compound 6h also significantly reduced the growth of E. coli. These antibacterial effects may be related to the penetrating properties of these compounds, as revealed by the leakage of nucleic acids from the sensitive strains.


Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nucleosídeos/farmacologia , Uridina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Relação Estrutura-Atividade , Uridina/análogos & derivados , Uridina/química
12.
Molecules ; 23(9)2018 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-30227644

RESUMO

A novel 2'-F,4'-C-OMe⁻arabinouridine (araU) was successfully synthesized and introduced into oligonucleotides. The oligonucleotide containing 2'-F,4'-C-OMe⁻araU exhibited improved nuclease resistance and RNA hybridizing selective ability relative to 2'-F⁻araU. In particular, when 2'-F,4'-C-OMe⁻araU inserted into C⁻H⋯F⁻C bonding-favorable 5'⁻uridine⁻purine⁻3' steps, the modified oligonucleotide showed remarkable binding affinity and selectivity to RNA complements. Thus, 2'-F,4'-C-OMe⁻araU has valuable antisense properties and can be used as novel chemical modification for antisense therapeutic strategy.


Assuntos
Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacologia , Uridina/síntese química , Uridina/farmacologia , Estabilidade Enzimática , Desnaturação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Diester Fosfórico Hidrolases/metabolismo , Venenos de Serpentes/enzimologia , Uridina/química
13.
Chem Biol Interact ; 295: 97-108, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30170108

RESUMO

To overcome the obstacles inflicted by the BBB in Glioblastoma multiforme (GBM) we investigated the use of Multifunctional nanoparticles that designed with a Nano-graphene oxide (NGO) sheet functionalized with magnetic poly (lactic-co-glycolic acid) (PLGA) and was used for glioma targeting delivery of radiosensitizing 5-iodo-2-deoxyuridine (IUdR). In vitro biocompatibility of nanocomposite has been studied by the MTT assay. In vivo efficacy of magnetic targeting on the amount and selectivity of magnetic nanoparticles accumulation in glioma-bearing rats under an external magnetic field (EMF) density of 0.5 T was easily monitored with MRI. IUdR-loaded magnetic NGO/PLGA with a diameter of 71.8 nm, a zeta potential of -33.07 ± 0.07 mV, and a drug loading content of 3.04 ± 0.46% presented superior superparamagnetic properties with a saturation magnetization (Ms) of 15.98 emu/g. Furthermore, Prussian blue staining showed effective magnetic targeting, leading to remarkably improved tumor inhibitory efficiency of IUdR. The tumor volume of rats after treatment with IUdR/NGO/SPION/PLGA + MF was decreased significantly compared to the rats treated with buffer saline, IUdR and SPION/IUdR/NGO/PLGA. Most importantly, our data demonstrate that IUdR/NGO/SPION/PLGA at the present magnetic field prolongs the median survival time of animals bearing gliomas (38 days, p < 0.01). Nanoparticles also had high thermal sensitivities under the alternating magnetic field. In conclusion, we developed magnetic IUdR/NGO/PLGA, which not only achieved to high accumulation at the targeted tumor site by magnetic targeting but also indicated significantly enhanced therapeutic efficiency and toxicity for glioma both in vitro and in vivo. This innovation increases the possibility of improving clinical efficiency of IUdR as a radiosensitizer, or lowering the total drug dose to decrease systemic toxicity.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Glioma/tratamento farmacológico , Imagem Óptica , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Glioma/diagnóstico por imagem , Glioma/patologia , Grafite/química , Grafite/farmacologia , Imagem por Ressonância Magnética , Nanopartículas de Magnetita/química , Masculino , Óxidos/química , Óxidos/farmacologia , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacologia , Ratos , Ratos Wistar , Células Tumorais Cultivadas , Uridina/análogos & derivados , Uridina/química , Uridina/farmacologia
14.
Biochem J ; 475(20): 3293-3309, 2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30254099

RESUMO

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.


Assuntos
Cisteína/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Etilmaleimida/metabolismo , Animais , Relação Dose-Resposta a Droga , Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Transportador Equilibrativo 1 de Nucleosídeo/genética , Feminino , Humanos , Ligação Proteica/fisiologia , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tioinosina/farmacologia , Uridina/metabolismo , Uridina/farmacologia , Xenopus laevis
15.
Molecules ; 23(8)2018 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-30104510

RESUMO

Herein we present the methodology for obtaining glycosyltransferase inhibitors, analogues of natural enzyme substrates of donor-type: UDP-glucose and UDP-galactose. The synthesis concerned glycoconjugates, nucleoside analogues containing an acyclic ribose mimetic linked to a uracil moiety in their structure. The biological activity of the synthesised compounds was determined on the basis of their ability to inhibit the model enzyme action of ß-1,4-galactosyltransferase from bovine milk. The obtained results allowed to expand and supplement the existing library of synthetic compounds that are able to regulate the biological activity of enzymes from the GT class.


Assuntos
Glicoconjugados/síntese química , Glicoconjugados/farmacologia , Uridina/síntese química , Uridina/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicoconjugados/química , Glicosiltransferases/antagonistas & inibidores , Estrutura Molecular , Uridina/análogos & derivados , Uridina/química
16.
ACS Chem Biol ; 13(9): 2542-2550, 2018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30080379

RESUMO

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here, we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-aminosugar acetyltransferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC50 values ranging from 35 ± 7 µM to 174 ± 21 µM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogs of lead compounds. Finally, the compounds were found to be nontoxic to mammalian cells, further supporting the opportunities for future development.


Assuntos
Bactérias/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Difosfato de Uridina/metabolismo , Uridina/análogos & derivados , Uridina/farmacologia , Acetiltransferases/antagonistas & inibidores , Bactérias/metabolismo , Campylobacter/enzimologia , Campylobacter/metabolismo , Campylobacter jejuni/efeitos dos fármacos , Campylobacter jejuni/enzimologia , Linhagem Celular , Clostridium difficile/enzimologia , Clostridium difficile/metabolismo , Inibidores Enzimáticos/síntese química , Glicosiltransferases/antagonistas & inibidores , Humanos , Modelos Moleculares , Nucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/farmacologia , Técnicas de Síntese em Fase Sólida/métodos , Relação Estrutura-Atividade , Uridina/síntese química
17.
Artigo em Inglês | MEDLINE | ID: mdl-30150466

RESUMO

Inhibition of NS5A has emerged as an attractive strategy to intervene in hepatitis C virus (HCV) replication. Ruzasvir (formerly MK-8408) was developed as a novel NS5A inhibitor to improve upon the potency and barrier to resistance of early compounds. Ruzasvir inhibited HCV RNA replication with 50% effective concentrations (EC50s) of 1 to 4 pM in Huh7 or Huh7.5 cells bearing replicons for HCV genotype 1 (GT1) to GT7. The antiviral activity was modestly (10-fold) reduced in the presence of 40% normal human serum. The picomolar potency in replicon cells extended to sequences of clinical isolates available in public databases that were synthesized and tested as replicons. In GT1a, ruzasvir inhibited common NS5A resistance-associated substitutions (RASs), with the exception of M28G. De novo resistance selection studies identified pathways with certain amino acid substitutions at residues 28, 30, 31, and 93 across genotypes. Substitutions at position 93 were more common in GT1 to -4, while changes at position 31 emerged frequently in GT5 and -6. With the exception of GT4, the reintroduction of selected RASs conferred a ≥100-fold potency reduction in the antiviral activity of ruzasvir. Common RASs from other classes of direct-acting antiviral agents (DAAs) did not confer cross-resistance to ruzasvir. The interaction of ruzasvir with an NS3/4A protease inhibitor (grazoprevir) and an NS5B polymerase prodrug (uprifosbuvir) was additive to synergistic, with no evidence of antagonism or cytotoxicity. The antiviral profile of ruzasvir supported its further evaluation in human trials in combination with grazoprevir and uprifosbuvir.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Pirrolidinas/farmacologia , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Linhagem Celular Tumoral , Farmacorresistência Viral/efeitos dos fármacos , Quimioterapia Combinada/métodos , Genótipo , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/virologia , Humanos , Inibidores de Proteases/farmacologia , Quinoxalinas/farmacologia , Replicon/efeitos dos fármacos , Uridina/análogos & derivados , Uridina/farmacologia
18.
Phytochemistry ; 155: 45-52, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30075391

RESUMO

Ten uridine derivatives (lepidiumuridine B-K) were isolated from the seeds of Lepidium apetalum Willd. Lepidiumuridine B-J were previously undescribed compounds, and were structurally characterized using analysis of their NMR and MS data. Lepidiumuridine C, D, I, and J increased cell proliferation and expression of ERα in the MCF-7 cell line. In addition, blockage of ERα completely abolished cell proliferation and expression of ERα in MCF-7 cells, suggesting that the proliferation effects of lepidiumuridine C, D, I, and J were ERα-mediated. The uridine derivatives might belong to undescribed phytoestrogens.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Estrogênios/farmacologia , Lepidium/química , Sementes/química , Uridina/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptor alfa de Estrogênio/biossíntese , Estrogênios/química , Estrogênios/isolamento & purificação , Humanos , Células MCF-7 , Conformação Molecular , Relação Estrutura-Atividade , Uridina/química , Uridina/isolamento & purificação
19.
Proc Natl Acad Sci U S A ; 115(7): E1366-E1373, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29378947

RESUMO

Many drugs require extensive metabolism en route to their targets. High-resolution visualization of prodrug metabolism should therefore utilize analogs containing a small modification that does not interfere with its metabolism or mode of action. In addition to serving as mechanistic probes, such analogs provide candidates for theranostics when applied in both therapeutic and diagnostic modalities. Here a traceable mimic of the widely used anticancer prodrug cytarabine (ara-C) was generated by converting a single hydroxyl group to azide, giving "AzC." This compound exhibited the same biological profile as ara-C in cell cultures and zebrafish larvae. Using azide-alkyne "click" reactions, we uncovered an apparent contradiction: drug-resistant cells incorporated relatively large quantities of AzC into their genomes and entered S-phase arrest, whereas drug-sensitive cells incorporated only small quantities of AzC. Fluorescence microscopy was used to elucidate structural features associated with drug resistance by characterizing the architectures of stalled DNA replication foci containing AzC, EdU, γH2AX, and proliferating cell nuclear antigen (PCNA). Three-color superresolution imaging revealed replication foci containing one, two, or three partially resolved replication forks. Upon removing AzC from the media, resumption of DNA synthesis and completion of the cell cycle occurred before complete removal of AzC from genomes in vitro and in vivo. These results revealed an important mechanism for the low toxicity of ara-C toward normal tissues and drug-resistant cancer cells, where its efficient incorporation into DNA gives rise to highly stable, stalled replication forks that limit further incorporation of the drug, yet allow for the resumption of DNA synthesis and cellular division following treatment.


Assuntos
Antineoplásicos/química , Azidas/química , Citarabina/química , Replicação do DNA , DNA/química , Pró-Fármacos/química , Uridina/análogos & derivados , Peixe-Zebra/crescimento & desenvolvimento , Animais , Antineoplásicos/farmacologia , Azidas/farmacologia , Ciclo Celular , Citarabina/farmacologia , Humanos , Pró-Fármacos/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Uridina/química , Uridina/farmacologia
20.
Eur J Med Chem ; 143: 107-113, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29172078

RESUMO

A novel ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-(fluoromethyl)uridine phosphoramidate prodrug (1) has been synthesized. This compound exhibits submicromolar-level antiviral activity in vitro against HCV genotypes 1b, 1a, 2a, and S282T replicons (EC50 = 0.18-1.13 µM) with low cytotoxicity (CC50 > 1000 µM). Administered orally, prodrug 1 is well tolerated at doses of up to 4 g/kg in mice, and produces a high level of the corresponding triphosphate in rat liver.


Assuntos
Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Compostos Organofosforados/farmacologia , Pró-Fármacos/farmacologia , Uridina/análogos & derivados , Administração Oral , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hepacivirus/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/virologia , Camundongos , Camundongos Endogâmicos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/química , Pró-Fármacos/administração & dosagem , Pró-Fármacos/síntese química , Pró-Fármacos/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Uridina/administração & dosagem , Uridina/química , Uridina/farmacologia , Replicação Viral/efeitos dos fármacos
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