Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 430
Filtrar
1.
Pharmacol Res Perspect ; 8(6): e00674, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33124786

RESUMO

SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , /antagonistas & inibidores , Betacoronavirus/enzimologia , Carbamatos/farmacologia , Infecções por Coronavirus/virologia , Didesoxinucleotídeos/farmacologia , Combinação de Medicamentos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Pandemias , Pneumonia Viral/virologia , Sofosbuvir/farmacologia , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia
2.
Cell Metab ; 29(4): 871-885.e5, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30853213

RESUMO

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies.


Assuntos
Inflamação/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sirtuínas/metabolismo , Fatores Etários , Animais , Didesoxinucleotídeos/administração & dosagem , Didesoxinucleotídeos/farmacologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas de Ligação a RNA/antagonistas & inibidores , Sirtuínas/deficiência , Estavudina/administração & dosagem , Estavudina/farmacologia , Nucleotídeos de Timina/administração & dosagem , Nucleotídeos de Timina/farmacologia , Zidovudina/administração & dosagem , Zidovudina/análogos & derivados , Zidovudina/farmacologia
3.
Bioorg Med Chem Lett ; 27(16): 3925-3930, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28676274

RESUMO

We report on the synthesis and properties of a new multimodal theranostic conjugate based on an anticancer fluorinated nucleotide conjugated with a dual-labeled albumin. A fluorine-labeled homocysteine thiolactone has been used as functional handle to synthesize the fluorinated albumin and couple it with a chemotherapeutic agent 5-trifluoromethyl-2'-deoxyuridine 5'-monophosphate (pTFT). The conjugate allows for direct optical and 19F magnetic resonance cancer imaging and release of the drug upon addition of glutathione. Interestingly, the pTFT release from albumin conjugate could only be promoted by the increased acidity (pH 5.4). The in vitro study and primary in vivo investigations showed stronger antitumor activity than free pTFT.


Assuntos
Antineoplásicos/farmacologia , Nucleotídeos/química , Albumina Sérica/química , Nucleotídeos de Timina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Oxirredução , Relação Estrutura-Atividade , Nucleotídeos de Timina/química
4.
Cancer Res ; 77(12): 3306-3316, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28428278

RESUMO

SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). In this study, we show that hepatocyte-specific SND1 transgenic mice (Alb/SND1 mice) develop spontaneous HCC with partial penetrance and exhibit more highly aggressive HCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibited a relative increase in inflammatory markers and spheroid-generating tumor-initiating cells (TIC). Mechanistic investigations defined roles for Akt and NF-κB signaling pathways in promoting TIC formation in Alb/SND1 mice. In human xenograft models of subcutaneous or orthotopic HCC, administration of the selective SND1 inhibitor 3', 5'-deoxythymidine bisphosphate (pdTp), inhibited tumor formation without effects on body weight or liver function. Our work establishes an oncogenic role for SND1 in promoting TIC formation and highlights pdTp as a highly selective SND1 inhibitor as a candidate therapeutic lead to treat advanced HCC. Cancer Res; 77(12); 3306-16. ©2017 AACR.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Animais , Antineoplásicos/farmacologia , Western Blotting , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Progressão da Doença , Endonucleases , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Nucleotídeos de Timina/farmacologia
5.
J Biol Chem ; 292(16): 6695-6702, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28255091

RESUMO

Recent studies have demonstrated the dominant role of induced fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking, and equilibrium thermodynamic methods are of no avail because the key parameters can only be determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37 °C, and the raw data were fit globally to derive a single set of rate constants at 37 °C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding (Kd , ΔG, ΔH, and ΔS at 37 °C) and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Inibidores da Transcriptase Reversa/farmacologia , Nucleotídeos de Timina/farmacologia , Zidovudina/farmacologia , Simulação por Computador , Cumarínicos/farmacologia , DNA/química , Relação Dose-Resposta a Droga , Infecções por HIV/tratamento farmacológico , Íons , Cinética , Magnésio/química , Simulação de Dinâmica Molecular , Mutação , Nucleotídeos/química , Nucleotídeos/genética , Conformação Proteica , Software , Especificidade por Substrato , Temperatura , Termodinâmica
6.
Biochemistry ; 56(1): 33-46, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27936595

RESUMO

Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.


Assuntos
Didesoxinucleotídeos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Magnésio/farmacologia , Nucleosídeos/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Nucleotídeos de Desoxicitosina/farmacologia , Nucleotídeos de Desoxiguanina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletroforese em Gel de Poliacrilamida , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Humanos , Cinética , Mutação , Nucleotídeos de Timina/farmacologia , Zalcitabina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/farmacologia
7.
Angew Chem Int Ed Engl ; 55(17): 5255-8, 2016 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-27008042

RESUMO

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV/efeitos dos fármacos , Nucleosídeos/farmacologia , Polifosfatos/farmacologia , Pró-Fármacos/farmacologia , Nucleotídeos de Timina/farmacologia , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Permeabilidade da Membrana Celular , Esterases/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Nucleosídeos/química , Nucleosídeos/metabolismo , Nucleosídeos/farmacocinética , Polifosfatos/química , Polifosfatos/metabolismo , Polifosfatos/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Suínos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
8.
Nucleic Acids Res ; 44(5): 2310-22, 2016 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-26850643

RESUMO

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.


Assuntos
Farmacorresistência Viral Múltipla/genética , Inibidores Enzimáticos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Ribonuclease H do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Sequência de Aminoácidos , Substituição de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Clonagem Molecular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Inibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H do Vírus da Imunodeficiência Humana/genética , Ribonuclease H do Vírus da Imunodeficiência Humana/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Zidovudina/química , Zidovudina/farmacologia
9.
Anal Biochem ; 498: 53-8, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26778528

RESUMO

Mycobacterium tuberculosis dTDP-d-glucose 4,6-dehydratase (RmlB) is the second enzyme for the biosynthesis of dTDP-l-rhamnose, which is a sugar donor to the synthesis of the cell wall linker, d-N-acetylglucosamine-l-rhamnose. RmlB is essential to mycobacterial growth and is not found in humans; therefore, it is a potential target for developing new anti-tuberculosis drugs. So far, there has been no suitable method for high-throughput screening of RmlB inhibitors. Here, the recombinant M. tuberculosis RmlB was purified and an absorbance-based microtiter plate assay was developed for RmlB activity. It could be used for high-throughput screening of RmlB inhibitors. The kinetic properties of M. tuberculosis RmlB, including optimal pH, optimal temperature, the effect of metal ions, and the kinetic parameters, were determined with this assay. The inhibitory effects of dTTP and dTDP on M. tuberculosis RmlB were also studied with the assay.


Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Hidroliases/antagonistas & inibidores , Mycobacterium tuberculosis/enzimologia , Antituberculosos/química , Bioensaio , Inibidores Enzimáticos/química , Glucose/análogos & derivados , Glucose/química , Glucose/farmacologia , Hidroliases/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia
10.
Nat Commun ; 6: 8716, 2015 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-26503889

RESUMO

The antiviral activity of nucleoside reverse transcriptase inhibitors is often limited by ineffective phosphorylation. We report on a nucleoside triphosphate (NTP) prodrug approach in which the γ-phosphate of NTPs is bioreversibly modified. A series of TriPPPro-compounds bearing two lipophilic masking units at the γ-phosphate and d4T as a nucleoside analogue are synthesized. Successful delivery of d4TTP is demonstrated in human CD4(+) T-lymphocyte cell extracts by an enzyme-triggered mechanism with high selectivity. In antiviral assays, the compounds are potent inhibitors of HIV-1 and HIV-2 in CD4(+) T-cell (CEM) cultures. Highly lipophilic acyl residues lead to higher membrane permeability that results in intracellular delivery of phosphorylated metabolites in thymidine kinase-deficient CEM/TK(-) cells with higher antiviral activity than the parent nucleoside.


Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/virologia , Pró-Fármacos/farmacologia , Nucleotídeos de Timina/farmacologia , Fármacos Anti-HIV/química , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Estrutura Molecular , Pró-Fármacos/química , Linfócitos T/virologia , Nucleotídeos de Timina/química
11.
Antimicrob Agents Chemother ; 59(10): 6395-401, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26239974

RESUMO

The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.


Assuntos
Fármacos Anti-HIV/farmacocinética , Citidina Trifosfato/análogos & derivados , Didesoxinucleotídeos/farmacocinética , Lamivudina/análogos & derivados , Leucócitos Mononucleares/metabolismo , Modelos Estatísticos , Sêmen/metabolismo , Nucleotídeos de Timina/farmacocinética , Zidovudina/análogos & derivados , Adulto , Fármacos Anti-HIV/farmacologia , Disponibilidade Biológica , Transporte Biológico , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Células Sanguíneas/patologia , Células Sanguíneas/virologia , Simulação por Computador , Citidina Trifosfato/farmacocinética , Citidina Trifosfato/farmacologia , Didesoxinucleotídeos/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Lamivudina/farmacocinética , Lamivudina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Masculino , Sêmen/citologia , Sêmen/efeitos dos fármacos , Sêmen/virologia , Nucleotídeos de Timina/farmacologia , Fatores de Tempo , Zidovudina/farmacocinética , Zidovudina/farmacologia
12.
Bioorg Med Chem ; 23(9): 2168-75, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25801161

RESUMO

A system for delivery of analogues of AZT-triphosphates (AZT*TP) based on SiO2 nanoparticles was proposed. For this purpose, a simple and versatile method was developed for the preparation of SiO2∼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2∼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO2∼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2∼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.


Assuntos
Didesoxinucleotídeos/administração & dosagem , Didesoxinucleotídeos/química , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Dióxido de Silício/química , Simplexvirus/efeitos dos fármacos , Nucleotídeos de Timina/administração & dosagem , Nucleotídeos de Timina/química , Triazóis/química , Vírus da Varíola/efeitos dos fármacos , Zidovudina/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Chlorocebus aethiops , Química Click , Didesoxinucleotídeos/farmacologia , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Simplexvirus/crescimento & desenvolvimento , Relação Estrutura-Atividade , Nucleotídeos de Timina/farmacologia , Vírus da Varíola/crescimento & desenvolvimento , Células Vero , Zidovudina/administração & dosagem , Zidovudina/química , Zidovudina/farmacologia
13.
Parasitol Res ; 114(4): 1313-26, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25566774

RESUMO

Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids.


Assuntos
Doença de Chagas/parasitologia , DNA Polimerase beta/genética , Regulação Enzimológica da Expressão Gênica , Trypanosoma cruzi/enzimologia , DNA Polimerase beta/efeitos dos fármacos , DNA Polimerase beta/isolamento & purificação , DNA Polimerase beta/metabolismo , DNA de Cinetoplasto/química , DNA de Cinetoplasto/genética , Didesoxinucleotídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Etilmaleimida/farmacologia , Humanos , Fosforilação , Filogenia , Análise de Sequência de DNA , Nucleotídeos de Timina/farmacologia , Trypanosoma cruzi/genética
14.
Antiviral Res ; 109: 125-31, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25010891

RESUMO

Although more-recently developed antivirals target different molecules in the HIV-1 replication cycle, nucleoside reverse transcriptase inhibitors (NRTIs) remain central for HIV-1 therapy. Here, we test the anti-HIV activity of a phosphonate chimera of two well-known NRTIs, namely AZT and 3TC. We show that this newly synthesized compound suppressed HIV-1 infection in lymphoid tissue ex vivo more efficiently than did other phosphonates of NRTIs. Moreover, the new compound was not toxic for tissue cells, thus making the chimeric phosphonate strategy a valid approach for the development of anti HIV-1 compound heterodimers.


Assuntos
Fármacos Anti-HIV/farmacologia , Didesoxinucleotídeos/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Lamivudina/farmacologia , Tonsila Palatina/efeitos dos fármacos , Nucleotídeos de Timina/farmacologia , Zidovudina/análogos & derivados , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Didesoxinucleotídeos/química , Avaliação Pré-Clínica de Medicamentos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Técnicas In Vitro , Lamivudina/química , Tonsila Palatina/virologia , Nucleotídeos de Timina/química , Replicação Viral/efeitos dos fármacos , Zidovudina/química , Zidovudina/farmacologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-24940681

RESUMO

A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation.


Assuntos
Inibidores Enzimáticos/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Lycopersicon esculentum/enzimologia , Timidina Quinase/antagonistas & inibidores , Nucleotídeos de Timina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Fosforilação/efeitos dos fármacos , Timidina Quinase/metabolismo , Zidovudina/metabolismo
16.
J Bacteriol ; 196(15): 2842-50, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24858186

RESUMO

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.


Assuntos
Bacteriófago T7/genética , Didesoxinucleotídeos/farmacologia , Escherichia coli/enzimologia , Inibidores da Síntese de Ácido Nucleico , Inibidores da Síntese de Ácido Nucleico/farmacologia , Nucleotídeos de Timina/farmacologia , Bacteriófago T7/efeitos dos fármacos , Bacteriófago T7/enzimologia , Bacteriófago T7/crescimento & desenvolvimento , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Didesoxinucleotídeos/metabolismo , Escherichia coli/virologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inativação de Genes , Inibidores da Síntese de Ácido Nucleico/metabolismo , Fosforilação , Pirimidina Fosforilases/genética , Pirimidina Fosforilases/metabolismo , Deleção de Sequência , Timidina/metabolismo , Timidina Quinase/genética , Timidina Quinase/metabolismo , Nucleotídeos de Timina/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/genética , Proteínas Virais/metabolismo
17.
Bioorg Med Chem ; 21(7): 1988-91, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23411398

RESUMO

Inhibitors that covalently damage proteins or nucleic acids offer great potency, but are difficult to rationally design and suffer from poor specificity. Here we outline a general concept for constructing covalent inhibitors, called the two-component covalent inhibitor (TCCI). The approach takes advantage of two ligand analogs equipped with pre-reactive groups. Binding of the analogs to the adjacent sites of a target biopolymer brings the pre-reactive groups in close proximity and causes their interaction followed by covalent damage of the target. In the present study we used light-activated pre-reactive groups to inactivate a DNA polymerase. It was found that the efficiency of a traditional single-component inhibitor was greatly reduced in the presence of a non-target protein, while the TCCI was not significantly affected. Our findings suggest that TCCI approach has advantages in inactivation of biopolymers in complex multi-component systems.


Assuntos
Bacteriófago T4/enzimologia , Desenho de Fármacos , Inibidores da Síntese de Ácido Nucleico , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Bacteriófago T4/química , Sítios de Ligação , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Luz , Ligação Proteica
18.
Bioorg Khim ; 39(6): 718-27, 2013.
Artigo em Russo | MEDLINE | ID: mdl-25696933

RESUMO

The interaction of CDI-activated diethyl phosphonoacetate with methyl 4-aminobenzoat or 3,5-difluoromethylphenylamine followed by treatment with Me3SiBr in DMF led to N-aryl aminocarbonylmethyl phosphonates and their ethyl esters. Their coupling with 3'-acetyl-α-thymidine followed by removal of the acetyl groups gave (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl-, aminocarbonyl- and carboxy)phenyl]-aminocarbonylmethyl phosphonates, (α-D-thymidine-5'-il)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. The phosphonates were stable in different conditions, low cytotoxic (in Vero and K562 cells) and were able to penetrate into K562 cells. The only ethyl ester of (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl)phenyl]-aminocarbonylmethyl phosphonate in high concentration (200 µg/mL) inhibited in vitro the growth of laboratory sensitive strain of Mycobacterium tuberculosis H37Rv.


Assuntos
Inibidores Enzimáticos/síntese química , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleotídeos de Timina/síntese química , Tuberculose/microbiologia , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Mycobacterium tuberculosis/enzimologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Tuberculose/tratamento farmacológico
19.
Mol Carcinog ; 52(3): 183-94, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22086658

RESUMO

Pemetrexed, a new-generation antifolate, has demonstrated promising single-agent activity in front- and second-line treatments of non-small cell lung cancer. However, the molecular mechanism of pemetrexed-mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase-2, -3, -8, and -9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase-dependent apoptotic mechanism. The molecular events of pemetrexed-mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53-dependent and -independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S-phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glutamatos/farmacologia , Guanina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Guanina/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Pemetrexede , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Nucleotídeos de Timina/farmacologia
20.
J Med Chem ; 55(16): 7245-52, 2012 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-22827702

RESUMO

Methyl-substituted cycloSal-pronucleotides of d4TMP were synthesized with high diastereoselectivities in satisfying chemical yields. The individual diastereomers were tested against HIV-1 and HIV-2 infected wild-type CEM/0 and HIV-2 infected thymidine kinase deficient CEM cells. All diastereomers tested showed significant antiviral activity in CEM/0 and strong activity in CEM/TK(-) cell cultures. The antiviral activities were strongly dependent on the chirality at the phosphate group and the position of the methyl-group(s) in the cycloSal moiety. In CEM/TK(-) cell cultures the difference in antiviral potency was found to be 7- to 20-fold. The stability of each diastereomer was studied in aqueous phosphate buffer and in CEM/0 cell extracts. Large differences in the half-lives were found. A comparison of the relative lipophilicity of the methyl-substituted cycloSal triesters was performed based on the retention times obtained by reversed phase HPLC. The results obtained clearly confirm the importance of a diastereoselective synthesis of cycloSal-pronucleotides.


Assuntos
Fármacos Anti-HIV/síntese química , Didesoxinucleotídeos/síntese química , Estavudina/análogos & derivados , Nucleotídeos de Timina/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Estabilidade de Medicamentos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Hidrólise , Mutação , Solventes/química , Estavudina/síntese química , Estavudina/química , Estavudina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Timidina Quinase/genética , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...