RESUMEN
People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase ß (Pol ß) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol ß or the treatment with a Pol ß pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol ß as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.
Asunto(s)
Antineoplásicos/farmacología , Proteína BRCA1/deficiencia , ADN Polimerasa beta/antagonistas & inhibidores , Animales , Línea Celular Tumoral , ADN Polimerasa beta/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen/efectos de los fármacos , Humanos , Ratones , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Nucleótidos de Timina/farmacologíaRESUMEN
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.
Asunto(s)
Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Betacoronavirus/enzimología , COVID-19 , Carbamatos/farmacología , Infecciones por Coronavirus/virología , Didesoxinucleótidos/farmacología , Combinación de Medicamentos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Pandemias , Neumonía Viral/virología , SARS-CoV-2 , Sofosbuvir/farmacología , Nucleótidos de Timina/farmacología , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Inflamación/metabolismo , Proteínas de Unión al ARN/metabolismo , Sirtuinas/metabolismo , Factores de Edad , Animales , Didesoxinucleótidos/administración & dosificación , Didesoxinucleótidos/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas de Unión al ARN/antagonistas & inhibidores , Sirtuinas/deficiencia , Estavudina/administración & dosificación , Estavudina/farmacología , Nucleótidos de Timina/administración & dosificación , Nucleótidos de Timina/farmacología , Zidovudina/administración & dosificación , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Nucleótidos/química , Albúmina Sérica/química , Nucleótidos de Timina/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración de Iones de Hidrógeno , Estructura Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Nucleótidos de Timina/químicaRESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Células Madre Neoplásicas/patología , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Antineoplásicos/farmacología , Western Blotting , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endonucleasas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Nucleótidos de Timina/farmacologíaRESUMEN
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.
Asunto(s)
Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Timina/farmacología , Zidovudina/farmacología , Simulación por Computador , Cumarinas/farmacología , ADN/química , Relación Dosis-Respuesta a Droga , Infecciones por VIH/tratamiento farmacológico , Iones , Cinética , Magnesio/química , Simulación de Dinámica Molecular , Mutación , Nucleótidos/química , Nucleótidos/genética , Conformación Proteica , Programas Informáticos , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
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.
Asunto(s)
Didesoxinucleótidos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Magnesio/farmacología , Nucleósidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Desoxicitosina/farmacología , Nucleótidos de Desoxiguanina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Humanos , Cinética , Mutación , Nucleótidos de Timina/farmacología , Zalcitabina/farmacología , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH/efectos de los fármacos , Nucleósidos/farmacología , Polifosfatos/farmacología , Profármacos/farmacología , Nucleótidos de Timina/farmacología , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacocinética , Linfocitos T CD4-Positivos/virología , Línea Celular , Permeabilidad de la Membrana Celular , Esterasas/metabolismo , Infecciones por VIH/tratamiento farmacológico , Humanos , Nucleósidos/química , Nucleósidos/metabolismo , Nucleósidos/farmacocinética , Polifosfatos/química , Polifosfatos/metabolismo , Polifosfatos/farmacocinética , Profármacos/química , Profármacos/metabolismo , Profármacos/farmacocinética , Porcinos , Nucleótidos de Timina/síntesis química , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismoRESUMEN
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.
Asunto(s)
Farmacorresistencia Viral Múltiple/genética , Inhibidores Enzimáticos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Clonación Molecular , Didesoxinucleótidos/química , Didesoxinucleótidos/farmacología , Inhibidores Enzimáticos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Genotipo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/genética , Ribonucleasa H del Virus de la Inmunodeficiencia Humana/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Timina/farmacología , Zidovudina/análogos & derivados , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Hidroliasas/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Antituberculosos/química , Bioensayo , Inhibidores Enzimáticos/química , Glucosa/análogos & derivados , Glucosa/química , Glucosa/farmacología , Hidroliasas/metabolismo , Cinética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Nucleótidos de Timina/química , Nucleótidos de Timina/farmacologíaRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , Infecciones por VIH/virología , Profármacos/farmacología , Nucleótidos de Timina/farmacología , Fármacos Anti-VIH/química , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Humanos , Estructura Molecular , Profármacos/química , Linfocitos T/virología , Nucleótidos de Timina/químicaRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Citidina Trifosfato/análogos & derivados , Didesoxinucleótidos/farmacocinética , Lamivudine/análogos & derivados , Leucocitos Mononucleares/metabolismo , Modelos Estadísticos , Semen/metabolismo , Nucleótidos de Timina/farmacocinética , Zidovudina/análogos & derivados , Adulto , Fármacos Anti-VIH/farmacología , Disponibilidad Biológica , Transporte Biológico , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/metabolismo , Células Sanguíneas/patología , Células Sanguíneas/virología , Simulación por Computador , Citidina Trifosfato/farmacocinética , Citidina Trifosfato/farmacología , Didesoxinucleótidos/farmacología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Lamivudine/farmacocinética , Lamivudine/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Masculino , Semen/citología , Semen/efectos de los fármacos , Semen/virología , Nucleótidos de Timina/farmacología , Factores de Tiempo , Zidovudina/farmacocinética , Zidovudina/farmacologíaRESUMEN
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 SiO2â¼dNTP conjugates using the 'click'-reaction between AZTTP and premodified nanoparticles containing the alkyne groups. The substrate properties of SiO2â¼AZT*TP were tested using Klenow fragment and HIV reverse transcriptase. The 3'-triazole derivatives of thymidine triphosphate being a part of the SiO2â¼AZT*TP nanocomposites were shown to be incorporated into the growing DNA chain. It was shown by confocal microscopy that the proposed SiO2â¼AZT*TP nanocomposites penetrate into cells. These nanocomposites were shown to inhibit the reproduction of POX and Herpes viruses at nontoxic concentrations.
Asunto(s)
Didesoxinucleótidos/administración & dosificación , Didesoxinucleótidos/química , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Dióxido de Silicio/química , Simplexvirus/efectos de los fármacos , Nucleótidos de Timina/administración & dosificación , Nucleótidos de Timina/química , Triazoles/química , Virus de la Viruela/efectos de los fármacos , Zidovudina/análogos & derivados , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Chlorocebus aethiops , Química Clic , Didesoxinucleótidos/farmacología , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Simplexvirus/crecimiento & desarrollo , Relación Estructura-Actividad , Nucleótidos de Timina/farmacología , Virus de la Viruela/crecimiento & desarrollo , Células Vero , Zidovudina/administración & dosificación , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Enfermedad de Chagas/parasitología , ADN Polimerasa beta/genética , Regulación Enzimológica de la Expresión Génica , Trypanosoma cruzi/enzimología , ADN Polimerasa beta/efectos de los fármacos , ADN Polimerasa beta/aislamiento & purificación , ADN Polimerasa beta/metabolismo , ADN de Cinetoplasto/química , ADN de Cinetoplasto/genética , Didesoxinucleótidos/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Etilmaleimida/farmacología , Humanos , Fosforilación , Filogenia , Análisis de Secuencia de ADN , Nucleótidos de Timina/farmacología , Trypanosoma cruzi/genéticaRESUMEN
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.
Asunto(s)
Fármacos Anti-VIH/farmacología , Didesoxinucleótidos/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Lamivudine/farmacología , Tonsila Palatina/efectos de los fármacos , Nucleótidos de Timina/farmacología , Zidovudina/análogos & derivados , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Didesoxinucleótidos/química , Evaluación Preclínica de Medicamentos , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Humanos , Técnicas In Vitro , Lamivudine/química , Tonsila Palatina/virología , Nucleótidos de Timina/química , Replicación Viral/efectos de los fármacos , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Retroalimentación Fisiológica/efectos de los fármacos , Solanum lycopersicum/enzimología , Timidina Quinasa/antagonistas & inhibidores , Nucleótidos de Timina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Fosforilación/efectos de los fármacos , Timidina Quinasa/metabolismo , Zidovudina/metabolismoRESUMEN
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.
Asunto(s)
Bacteriófago T7/genética , Didesoxinucleótidos/farmacología , Escherichia coli/enzimología , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Nucleótidos de Timina/farmacología , Bacteriófago T7/efectos de los fármacos , Bacteriófago T7/enzimología , Bacteriófago T7/crecimiento & desarrollo , ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Didesoxinucleótidos/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Inhibidores de la Síntesis del Ácido Nucleico/metabolismo , Fosforilación , Pirimidina Fosforilasas/genética , Pirimidina Fosforilasas/metabolismo , Eliminación de Secuencia , Timidina/metabolismo , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Nucleótidos de Timina/metabolismo , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
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.
Asunto(s)
Bacteriófago T4/enzimología , Diseño de Fármacos , Inhibidores de la Síntesis del Ácido Nucleico , Nucleótidos de Timina/química , Nucleótidos de Timina/farmacología , Bacteriófago T4/química , Sitios de Unión , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Luz , Unión ProteicaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Nucleótidos de Timina/síntesis química , Tuberculosis/microbiología , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Antiinfecciosos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Espectroscopía de Resonancia Magnética , Mycobacterium tuberculosis/enzimología , Nucleótidos de Timina/química , Nucleótidos de Timina/farmacología , Tuberculosis/tratamiento farmacológicoRESUMEN
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.