RESUMEN
The DNA polymerase (Pol) δ of Saccharomyces cerevisiae (S.c.) is composed of the catalytic subunit Pol3 along with two regulatory subunits, Pol31 and Pol32. Pol δ binds to proliferating cell nuclear antigen (PCNA) and functions in genome replication, repair, and recombination. Unique among DNA polymerases, the Pol3 catalytic subunit contains a 4Fe-4S cluster that may sense the cellular redox state. Here we report the 3.2-Šcryo-EM structure of S.c. Pol δ in complex with primed DNA, an incoming ddTTP, and the PCNA clamp. Unexpectedly, Pol δ binds only one subunit of the PCNA trimer. This singular yet extensive interaction holds DNA such that the 2-nm-wide DNA threads through the center of the 3-nm interior channel of the clamp without directly contacting the protein. Thus, a water-mediated clamp and DNA interface enables the PCNA clamp to "waterskate" along the duplex with minimum drag. Pol31 and Pol32 are positioned off to the side of the catalytic Pol3-PCNA-DNA axis. We show here that Pol31-Pol32 binds single-stranded DNA that we propose underlies polymerase recycling during lagging strand synthesis, in analogy to Escherichia coli replicase. Interestingly, the 4Fe-4S cluster in the C-terminal CysB domain of Pol3 forms the central interface to Pol31-Pol32, and this strategic location may explain the regulation of the oxidation state on Pol δ activity, possibly useful during cellular oxidative stress. Importantly, human cancer and other disease mutations map to nearly every domain of Pol3, suggesting that all aspects of Pol δ replication are important to human health and disease.
Asunto(s)
ADN Polimerasa III/química , ADN Polimerasa III/metabolismo , ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Microscopía por Crioelectrón , ADN/química , ADN Polimerasa III/ultraestructura , Didesoxinucleótidos/química , Didesoxinucleótidos/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/metabolismo , Modelos Moleculares , Mutación/genética , Neoplasias/genética , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismoRESUMEN
BACKGROUND: Antiretroviral therapy (ART) has significantly reduced HIV-related morbidity and mortality. However, therapeutic benefit of ART is often limited by delayed drug-associated toxicity. Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of ART regimens. NRTIs compete with endogenous deoxyribonucleotide triphosphates (dNTPs) in incorporation into elongating DNA chain resulting in their cytotoxic or antiviral effect. Thus, the efficacy of NRTIs could be affected by direct competition with endogenous dNTPs and/or feedback inhibition of their metabolic enzymes. In this paper, we assessed whether the levels of ribonucleotides (RN) and dNTP pool sizes can be used as biomarkers in distinguishing between HIV-infected patients with ART-induced mitochondrial toxicity and HIV-infected patients without toxicity. METHODS: We used data collected through a case-control study from 50 subjects. Cases were defined as HIV-infected individuals with clinical and/or laboratory evidence of mitochondrial toxicity. Each case was age, gender, and race matched with an HIV-positive without evidence of toxicity. We used a range of machine learning procedures to distinguish between patients with and without toxicity. Using resampling methods like Monte Carlo k-fold cross validation, we compared the accuracy of several machine learning algorithms applied to our data. We used the algorithm with highest classification accuracy rate in evaluating the diagnostic performance of 12 RN and 14 dNTP pool sizes as biomarkers of mitochondrial toxicity. RESULTS: We used eight classification algorithms to assess the diagnostic performance of RN and dNTP pool sizes distinguishing HIV patients with and without NRTI-associated mitochondrial toxicity. The algorithms resulted in cross-validated classification rates of 0.65-0.76 for dNTP and 0.72-0.83 for RN, following reduction of the dimensionality of the input data. The reduction of input variables improved the classification performance of the algorithms, with the most pronounced improvement for RN. Complex tree-based methods worked the best for both the deoxyribose dataset (Random Forest) and the ribose dataset (Classification Tree and AdaBoost), but it is worth noting that simple methods such as Linear Discriminant Analysis and Logistic Regression were very competitive in terms of classification performance. CONCLUSIONS: Our finding of changes in RN and dNTP pools in participants with mitochondrial toxicity validates the importance of dNTP pools in mitochondrial function. Hence, levels of RN and dNTP pools can be used as biomarkers of ART-induced mitochondrial toxicity.
Asunto(s)
Antirretrovirales/efectos adversos , Desoxirribonucleótidos/metabolismo , Didesoxinucleótidos/metabolismo , Infecciones por VIH/tratamiento farmacológico , Aprendizaje Automático , Ribonucleótidos/metabolismo , Algoritmos , Biomarcadores/metabolismo , Estudios de Casos y Controles , Infecciones por VIH/diagnóstico , HumanosRESUMEN
The glycolytic PFKFB3 enzyme is widely overexpressed in cancer cells and an emerging anti-cancer target. Here, we identify PFKFB3 as a critical factor in homologous recombination (HR) repair of DNA double-strand breaks. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an MRN-ATM-γH2AX-MDC1-dependent manner and co-localizes with DNA damage and HR repair proteins. PFKFB3 relocalization is critical for recruitment of HR proteins, HR activity, and cell survival upon IR. We develop KAN0438757, a small molecule inhibitor that potently targets PFKFB3. Pharmacological PFKFB3 inhibition impairs recruitment of ribonucleotide reductase M2 and deoxynucleotide incorporation upon DNA repair, and reduces dNTP levels. Importantly, KAN0438757 induces radiosensitization in transformed cells while leaving non-transformed cells unaffected. In summary, we identify a key role for PFKFB3 enzymatic activity in HR repair and present KAN0438757, a selective PFKFB3 inhibitor that could potentially be used as a strategy for the treatment of cancer.
Asunto(s)
Antineoplásicos/farmacología , Compuestos de Bifenilo/farmacología , Inhibidores Enzimáticos/farmacología , Hidroxibenzoatos/farmacología , Neoplasias/terapia , Fosfofructoquinasa-2/antagonistas & inhibidores , Sulfonas/farmacología , Antineoplásicos/uso terapéutico , Compuestos de Bifenilo/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Quimioradioterapia/métodos , Roturas del ADN de Doble Cadena/efectos de la radiación , Didesoxinucleótidos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Humanos , Hidroxibenzoatos/uso terapéutico , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , ARN Interferente Pequeño/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Radiación Ionizante , Reparación del ADN por Recombinación/efectos de los fármacos , Reparación del ADN por Recombinación/efectos de la radiación , Sulfonas/uso terapéuticoRESUMEN
In this study, we report that the tetraspanin CD81 enhances human immunodeficiency virus (HIV)-1 reverse transcription in HIV-1-infected cells. This is enabled by the direct interaction of CD81 with the deoxynucleoside triphosphate phosphohydrolase SAMHD1. This interaction prevents endosomal accumulation and favours the proteasome-dependent degradation of SAMHD1. Consequently, CD81 depletion results in SAMHD1 increased expression, decreasing the availability of deoxynucleoside triphosphates (dNTP) and thus HIV-1 reverse transcription. Conversely, CD81 overexpression, but not the expression of a CD81 carboxy (C)-terminal deletion mutant, increases cellular dNTP content and HIV-1 reverse transcription. Our results demonstrate that the interaction of CD81 with SAMHD1 controls the metabolic rate of HIV-1 replication by tuning the availability of building blocks for reverse transcription, namely dNTPs. Together with its role in HIV-1 entry and budding into host cells, the data herein indicate that HIV-1 uses CD81 as a rheostat that controls different stages of the infection.
Asunto(s)
Didesoxinucleótidos/metabolismo , VIH-1/genética , Transcripción Reversa , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Tetraspanina 28/metabolismo , Replicación del ADN , VIH-1/fisiología , Células HeLa , Humanos , Macrófagos/virología , Proteína 1 que Contiene Dominios SAM y HD/genética , Tetraspanina 28/genética , Replicación ViralRESUMEN
Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase ß (Pol ß). However, whether human Pol ß is essential for lentivirus replication in human cells is unclear. Here, we abolished DNA polymerase ß (Pol ß) expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate the role of Pol ß in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol ß-knock-out was confirmed by enhanced sensitivity to methyl methanesulfonate-induced DNA damage. Of note, nuclear extracts from Pol ß-knock-out THP-1 cells prepared from both dividing and nondividing stages displayed significantly reduced capability to repair the gapped HIV-1 integration intermediate DNA substrate in a biochemical simulation. However, nuclear extract from both dividing and nondividing stages of the Pol ß-KO cells had detectable gap repair activity, suggesting that other host DNA polymerases also repair gapped HIV-1 DNA, particularly in dividing cells. Next, when we compared transduction using HIV-1 and simian immunodeficiency virus in control and Pol ß-KO cells, the loss of the Pol ß expression did not affect transduction efficiency of these lentiviruses in both dividing and nondividing stages. Finally, the gap repair assay indicated that limited cellular dNTP pools, but not Pol ß expression, are a primary factor for HIV-1 DNA gap repair, particularly in nondividing cells. These data support the idea that Pol ß polymerase activity is dispensable for HIV-1 infection in both dividing and nondividing stages of human cells targeted by the virus.
Asunto(s)
ADN Polimerasa beta/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Macrófagos/virología , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular , Supervivencia Celular , ADN Polimerasa beta/antagonistas & inhibidores , ADN Polimerasa beta/química , ADN Polimerasa beta/genética , Reparación del ADN , Didesoxinucleótidos/metabolismo , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Infecciones por VIH/virología , VIH-1/genética , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Dominios y Motivos de Interacción de Proteínas , ARN/metabolismo , ARN Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Integración ViralRESUMEN
Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.
Asunto(s)
ADN Nucleotidilexotransferasa/metabolismo , Hibridación Fluorescente in Situ/métodos , Sondas ARN/química , Animales , Biotina , Didesoxinucleótidos/química , Didesoxinucleótidos/metabolismo , Drosophila melanogaster/genética , Femenino , Colorantes Fluorescentes/química , Sondas de Oligonucleótidos/química , Oligonucleótidos/química , Ovario/fisiología , Sondas ARN/metabolismo , Nucleótidos de Uracilo/química , Nucleótidos de Uracilo/metabolismoRESUMEN
Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).
Asunto(s)
Coenzimas/metabolismo , ADN Primasa/metabolismo , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Magnesio/metabolismo , Manganeso/metabolismo , Enzimas Multifuncionales/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Antivirales/metabolismo , Antivirales/uso terapéutico , Trifosfato de Arabinofuranosil Citosina/metabolismo , Trifosfato de Arabinofuranosil Citosina/uso terapéutico , Cationes Bivalentes/metabolismo , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Citidina Trifosfato/uso terapéutico , Desoxirribonucleótidos/metabolismo , Didesoxinucleótidos/metabolismo , Didesoxinucleótidos/uso terapéutico , Emtricitabina/análogos & derivados , Emtricitabina/metabolismo , Emtricitabina/uso terapéutico , Humanos , Cinética , Lamivudine/análogos & derivados , Lamivudine/metabolismo , Lamivudine/uso terapéuticoRESUMEN
MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis.
Asunto(s)
Biotinilación , Didesoxinucleótidos/genética , MicroARNs/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células A549 , Animales , Calibración , ADN Complementario/química , ADN Complementario/genética , Didesoxinucleótidos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , MicroARNs/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
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
Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways.
Asunto(s)
Didesoxinucleótidos/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Línea Celular , Expresión Génica , Genes Reporteros , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Interacciones Huésped-Patógeno , Humanos , Lamivudine/farmacología , Luciferasas/genética , Luciferasas/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/virología , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Monoméricas/genética , Organofosfonatos/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína 1 que Contiene Dominios SAM y HD , Estavudina/farmacología , Tenofovir , Replicación Viral/efectos de los fármacos , Zidovudina/farmacologíaRESUMEN
Alzheimer's disease (AD) is a senile dementia with increased incidence in older subjects (age >65 years). One of the earliest markers of AD is oxidative DNA damage. Recently, it has been reported that preclinical AD patient brains show elevated levels of oxidative damage in both nuclear and mitochondrial nucleic acids. Moreover, different oxidative lesions in mitochondrial DNA are between 5- and 10-fold higher than in nuclear DNA in both control and AD postmortem brains. We previously showed that there is a significant loss of base excision repair (BER) components in whole tissue extracts of AD and mild cognitive impairment subjects relative to matched control subjects. However, comprehensive analysis of specific steps in BER levels in mitochondrial extracts of AD patient brains is not available. In this study, we mainly investigated various components of BER in mitochondrial extracts of AD and matched control postmortem brain samples. We found that the 5-hydroxyuracil incision and ligase activities are significantly lower in AD brains, whereas the uracil incision, abasic site cleavage, and deoxyribonucleotide triphosphate incorporation activities are normal in these samples.
Asunto(s)
Enfermedad de Alzheimer/genética , Daño del ADN/genética , Reparación del ADN/genética , ADN Mitocondrial/genética , Mitocondrias/genética , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Encéfalo/ultraestructura , ADN Ligasas/metabolismo , Didesoxinucleótidos/metabolismo , Humanos , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo/genética , Uracilo/análogos & derivados , Uracilo/metabolismoRESUMEN
Direct sequencing of RNA by primer extension is a fast and accurate method that is useful in a variety of situations. It is a valuable technique for determining the faithfulness of in vitro processing reactions, such as splicing or RNA editing. It is often used as an alternative to reverse transcription PCR (RT-PCR) followed by cloning and DNA sequencing. In the primer extension reaction, a radiolabeled probe (almost always a 5'-end-labeled DNA oligonucleotide) is annealed to the target RNA of interest. After hybridization, cDNA synthesis by reverse transcription proceeds from the 3' end of the primer in the presence of chain-terminating dideoxynucleotide triphosphates. The cDNA products are fractionated on denaturing polyacrylamide gels and analyzed by phosphorimaging or autoradiography.
Asunto(s)
ARN/química , ARN/genética , Análisis de Secuencia de ARN/métodos , Autorradiografía , Cartilla de ADN/metabolismo , ADN Complementario/biosíntesis , Electroforesis en Gel de Gradiente Desnaturalizante , Didesoxinucleótidos/metabolismo , Electroforesis en Gel de Poliacrilamida , Hibridación de Ácido Nucleico , ARN/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
Terminal deoxynucletidyl transferase (TdT) is overexpressed in some cancer types, where it might compete with pol µ during the mutagenic repair of double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. Here we report the discovery and characterization of pyrrolyl and indolyl diketo acids that specifically target TdT and behave as nucleotide-competitive inhibitors. These compounds show a selective toxicity toward MOLT-4 compared to HeLa cells that correlate well with in vitro selectivity for TdT. The binding site of two of these inhibitors was determined by cocrystallization with TdT, explaining why these compounds are competitive inhibitors of the deoxynucleotide triphosphate (dNTP). In addition, because of the observed dual localization of the phenyl substituent, these studies open the possibility of rationally designing more potent compounds.
Asunto(s)
Unión Competitiva , ADN Nucleotidilexotransferasa/antagonistas & inhibidores , ADN Nucleotidilexotransferasa/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Nucleótidos/metabolismo , Apoptosis/efectos de los fármacos , Dominio Catalítico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , ADN Nucleotidilexotransferasa/química , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Didesoxinucleótidos/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/metabolismo , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Ácidos Hexurónicos/farmacología , Humanos , Modelos MolecularesRESUMEN
BACKGROUND: A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4(+) T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects. METHODS: Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests. RESULTS: VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4(+) T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: -1.47 log(10) copies/mL; CD4(+) T cell count: +135 cells/mm(3)) and fewest adverse events. CONCLUSIONS: VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system activation associated with HIV-1 disease. Rapid reductions in markers of immune system hyperactivation and cellular proliferation were obtained despite the fact that VS411 did not attain maximal suppression of HIV RNA, suggesting this effect was due to the HALT component. TRIAL REGISTRATION: ITEudraCT 2007-002460-98.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Didanosina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , ARN Viral/biosíntesis , Urea/análogos & derivados , Adulto , Análisis de Varianza , Fármacos Anti-VIH/farmacología , Biomarcadores/metabolismo , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Preparaciones de Acción Retardada , Nucleótidos de Desoxiadenina/metabolismo , Didanosina/farmacología , Didesoxinucleótidos/metabolismo , Método Doble Ciego , Esquema de Medicación , Combinación de Medicamentos , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Humanos , Inmunomodulación/efectos de los fármacos , Masculino , Placebos , ARN Viral/efectos de los fármacos , Urea/farmacología , Urea/uso terapéutico , Carga ViralRESUMEN
Treatment of HIV-1 with nucleoside reverse transcription inhibitors leads to the emergence of resistance mutations in the reverse transcriptase (RT) gene. Resistance to 3'-azido-3'-deoxythymidine (AZT) and to a lesser extent to 2'-3'-didehydro-2'-3'-dideoxythymidine is mediated by phosphorolytic excision of the chain terminator. Wild-type RT excises AZT by pyrophosphorolysis, while thymidine-associated resistance mutations in RT (TAMs) favour ATP as the donor substrate. However, in vitro, resistant RT still uses pyrophosphate more efficiently than ATP. We performed in vitro (-) strong-stop DNA synthesis experiments, with wild-type and AZT-resistant HIV-1 RTs, in the presence of physiologically relevant pyrophosphate and/or ATP concentrations and found that in the presence of pyrophosphate, ATP and AZTTP, TAMs do not enhance in vitro (-) strong-stop DNA synthesis. We hypothesized that utilisation of ATP in vivo is driven by intrinsic low pyrophosphate concentrations within the reverse transcription complex, which could be explained by the packaging of a cellular pyrophosphatase. We showed that over-expressed flagged-pyrophosphatase was associated with HIV-1 viral-like particles. In addition, we demonstrated that when HIV-1 particles were purified in order to avoid cellular microvesicle contamination, a pyrophosphatase activity was specifically associated to them. The presence of a pyrophosphatase activity in close proximity to the reverse transcription complex is most likely advantageous to the virus, even in the absence of any drug pressure.
Asunto(s)
Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Pirofosfatasas/metabolismo , Virión/enzimología , Adenosina Trifosfato/metabolismo , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , ADN Viral/genética , ADN Viral/metabolismo , Didesoxinucleótidos/metabolismo , Difosfatos/metabolismo , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/genética , Cinética , Mutación , Pirofosfatasas/genética , Estavudina/metabolismo , Estavudina/farmacología , Especificidad por Sustrato , Nucleótidos de Timina/metabolismo , Virión/efectos de los fármacos , Virión/genética , Zidovudina/análogos & derivados , Zidovudina/metabolismo , Zidovudina/farmacologíaRESUMEN
Resistance to nucleoside reverse transcriptase (RT) inhibitors is conferred on human immunodeficiency virus type 1 through thymidine analogue resistance mutations (TAMs) that increase the ability of RT to excise chain-terminating nucleotides after they have been incorporated. The RT mutation M184V is a potent suppressor of TAMs. In RT containing TAMs, the addition of M184V suppressed the excision of 3'-deoxy-3'-azidothymidine monophosphate (AZTMP) to a greater extent on an RNA template than on a DNA template with the same sequence. The catalytically inactive RNase H mutation E478Q abolished this difference. The reduction in excision activity was similar with either ATP or pyrophosphate as the acceptor substrate. Decreased excision of AZTMP was associated with increased cleavage of the RNA template at position -7 relative to the primer terminus, which led to increased primer-template dissociation. Whether M184V was present or not, RT did not initially bind at the -7 cleavage site. Cleavage at the initial site was followed by RT dissociation and rebinding at the -7 cleavage site, and the dissociation and rebinding were enhanced when the M184V mutation was present. In contrast to the effect of M184V, the K65R mutation suppressed the excision activity of RT to the same extent on either an RNA or a DNA template and did not alter the RNase H cleavage pattern. Based on these results, we propose that enhanced RNase H cleavage near the primer terminus plays a role in M184V suppression of AZT resistance, while K65R suppression occurs through a different mechanism.
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Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , Mutación , Nucleótidos/metabolismo , Adenosina Trifosfato/metabolismo , Cartilla de ADN/metabolismo , Didesoxinucleótidos/metabolismo , Farmacorresistencia Viral/genética , Humanos , ARN Viral/biosíntesis , Ribonucleasa H/metabolismo , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismoRESUMEN
The cellular pharmacology of zidovudine (ZDV) and lamivudine (3TC) in vivo is not completely understood. This prospective longitudinal study investigated the relationship between HIV-1 serostatus, sex, race, and time on therapy with intracellular and plasma ZDV and 3TC concentrations. Of 20 HIV-seronegative and 23 HIV-seropositive volunteers enrolled, 16 (8 women) and 21 (5 women) completed all 12 study days, respectively. Volunteers began ZDV-3TC therapy (plus a third active drug in HIV-seropositive volunteers), and steady-state concentrations (C(ss)) were determined after days 1, 3, 7, and 12. A repeated-measures mixed model was utilized. HIV-seronegative status was associated with 22% (95% confidence interval [CI], 0%, 50%) and 37% (15%, 67%) higher C(ss) estimates compared to those of HIV-seropositive individuals for intracellular ZDV-TP and 3TC-TP levels, respectively. African-Americans had 36% (8%, 72%) higher ZDV-TP estimates than non-African-Americans. Sex was not associated with ZDV-TP or 3TC-TP (P > 0.19). Women had 36% (4%, 78%) higher plasma ZDV, but the effect was lessened when normalized by lean body weight (5% [-19%, 38%]; P = 0.68). Plasma 3TC was 19% (0%, 41%) higher in HIV-seropositive volunteers and 22% (0%, 48%) higher in African American volunteers, but these effects were not significant when corrected for creatinine clearance (7% [-9%, 20%] and -5% [-26%, 12%] for HIV serostatus and race, respectively; P > 0.35). These results suggest that HIV-seropositive status decreases and African American race elevates the cellular triphosphates of ZDV and 3TC. This information extends knowledge of ZDV and 3TC cellular pharmacology in vivo and provides new leads for future cellular pharmacology studies aimed at optimizing HIV prevention/treatment with these agents.
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Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Lamivudine/farmacología , Lamivudine/uso terapéutico , Zidovudina/farmacocinética , Zidovudina/uso terapéutico , Adulto , Negro o Afroamericano , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Didesoxinucleótidos/metabolismo , Femenino , Humanos , Lamivudine/análogos & derivados , Lamivudine/metabolismo , Estudios Longitudinales , Masculino , Estudios Prospectivos , Factores Sexuales , Nucleótidos de Timina/metabolismo , Zidovudina/análogos & derivados , Zidovudina/metabolismoRESUMEN
UNLABELLED: Mericitabine (RG7128) is a nucleoside polymerase inhibitor (NPI), which requires intracellular uptake and phosphorylation to two active triphosphates. Mathematical modeling has provided important insights for characterizing hepatitis C virus (HCV) RNA decline and estimating in vivo effectiveness of antiviral agents; however, it has not been used to characterize viral kinetics with NPIs. HCV RNA was frequently measured in 32 treatment-experienced patients infected with HCV genotype 1 during and after mericitabine monotherapy for 14 days with 750 mg or 1500 mg administered once (qd) or twice daily (bid). The initial decline of HCV RNA was typically slower than with interferon-α or protease inhibitors, and 12 patients presented a novel pattern of HCV RNA kinetics characterized by a monophasic viral decline. Viral kinetics could be well fitted by assuming that the effectiveness in blocking viral production gradually increased over time to reach its final value, ε(2), consistent with previous accumulation time estimates of intracellular triphosphates. ε(2) was high with bid dosing (mean 750 mg and 1500 mg: 98.0% and 99.8%, respectively; P = 0.018) and significantly higher than in patients treated qd (mean qd versus bid: 90% versus 99%, P < 10(-7)). Virus rebounded rapidly upon drug discontinuation, which was attributed to the elimination of active drug and the subsequent decline of drug effectiveness, with mean t(1/2) = 13.9 hours in the bid regimens. CONCLUSION: The observed slower initial decline likely represents the time needed to accumulate intracellular triphosphates and is consistent with in vitro data. When administered bid, mericitabine reached a high, dose-dependent, final effectiveness in blocking viral production that rapidly dropped upon treatment cessation. Understanding HCV RNA kinetics with mericitabine could provide valuable insights for combining it with other direct-acting antiviral agents.
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Desoxicitidina/análogos & derivados , Hepacivirus/fisiología , Hepatitis C/virología , Carga Viral/efectos de los fármacos , Carga Viral/fisiología , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiología , Antivirales/farmacología , Antivirales/uso terapéutico , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Didesoxinucleótidos/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Genotipo , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/metabolismo , Humanos , Modelos Teóricos , ARN Viral/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
ß-D-3'-Azido-2',3'-dideoxyguanosine (3'-azido-ddG) is a potent inhibitor of HIV-1 replication with a superior resistance profile to zidovudine. Recently, we identified five novel 6-modified-3'-azido-ddG analogs that exhibit similar or superior anti-HIV-1 activity compared to 3'-azido-ddG in primary cells. To gain insight into their structure-activity-resistance relationships, we synthesized their triphosphate (TP) forms and assessed their ability to inhibit HIV-1 reverse transcriptase (RT). Steady-state and pre-steady-state kinetic experiments show that the 6-modified-3'-azido-ddGTP analogs act as adenosine rather than guanosine mimetics in DNA synthesis reactions. The order of potency of the TP analogs against wild-type RT was: 3'-azido-2,6-diaminopurine >3'-azido-6-chloropurine; 3'-azido-6-N-allylaminopurine > 2-amino-6-N,N-dimethylaminopurine; 2-amino-6-methoxypurine. Molecular modeling studies reveal unique hydrogen-bonding interactions between the nucleotide analogs and the template thymine base in the active site of RT. Surprisingly, the structure-activity relationship of the analogs differed in HIV-1 RT ATP-mediated excision assays of their monophosphate forms, suggesting that it may be possible to rationally design a modified base analog that is efficiently incorporated by RT but serves as a poor substrate for ATP-mediated excision reactions. Overall, these studies identify a promising strategy to design novel nucleoside analogs that exert profound antiviral activity against both WT and drug-resistant HIV-1.
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Fármacos Anti-VIH/química , Didesoxinucleósidos/química , Didesoxinucleótidos/química , Transcriptasa Inversa del VIH/química , Inhibidores de la Transcriptasa Inversa/química , Adenosina/análogos & derivados , Adenosina Trifosfato/química , Fármacos Anti-VIH/metabolismo , Dominio Catalítico , Didesoxinucleótidos/metabolismo , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Modelos Moleculares , Imitación Molecular , Mutación , Inhibidores de la Transcriptasa Inversa/metabolismoRESUMEN
Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.