RESUMEN
Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Animales , Afidicolina/farmacología , Células Cultivadas , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleótidos de Desoxiadenina/farmacología , Didesoxinucleótidos/farmacología , Femenino , Fase G2 , Indoles/farmacología , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Oocitos , Cultivo Primario de Células , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/metabolismo , Tetrahidroisoquinolinas/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
Background: The purpose of the present study was to examine the chemopreventive effect of stampidine, an aryl phosphate derivative of stavudine, in side by side comparison with the standard anti-breast cancer drug paclitaxel in the well-established 7,12-dimethylbenz(a)anthracene (DMBA)-induced murine breast cancer model.Methods: Groups of 20 female mice were challenged with the DMBA. DMBA-challenged mice were assigned to various chemoprevention treatments, including stampidine, paclitaxel, and stampidine plus paclitaxel according to the same treatment schedules for 25 weeks.Results: Stampidine resulted in substantially reduced numbers of tumors, tumor weight as well as tumor size in DMBA-treated mice. Stampidine was as effective as paclitaxel in the model and their combination exhibited greater chemopreventive activity, as measured by reduced tumor incidence and improved tumor-free survival as well as overall survival of DMBA-treated mice. The length of time for the initial tumor to appear in DMBA-challenged mice treated with stampidine was longer than that of mice treated DMBA-challenged control mice. Tumors from mice treated with stampidine or stampidine plus paclitaxel displayed unique changes of a signature protein cassette comprised BRCA1, p21, Bax, and Bcl-2.Conclusion: Stampidine has potent chemopreventive activity and is as effective as the standard chemotherapy drug paclitaxel in the chemical carcinogenesis.
Asunto(s)
Anticarcinógenos/farmacología , Neoplasias de la Mama/prevención & control , Didesoxinucleótidos/farmacología , Estavudina/análogos & derivados , Timidina Monofosfato/análogos & derivados , 9,10-Dimetil-1,2-benzantraceno , Animales , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Supervivencia sin Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Paclitaxel/farmacología , Estavudina/farmacología , Tasa de Supervivencia , Timidina Monofosfato/farmacología , Factores de TiempoRESUMEN
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
A conjugate of triphosphorylated 2',3'-dideoxyuridine (ddU) with SiO2 nanoparticles was obtained via the CuAAC click chemistry between a γ-alkynyl ddU triphosphate and azido-modified SiO2 nanoparticles. Assessment of cytotoxicity in human breast adenocarcinoma MCF7 cells demonstrated that ddU triphosphate conjugated to SiO2 nanoparticles exhibited a 50% decrease in cancer cell growth at a concentration of 183⯱â¯57⯵g/mL, which corresponds to 22⯱â¯7⯵M of the parent nucleotide, whereas the parent nucleoside, nucleotide and alkynyl triphosphate precursor do not show any cytotoxicity. The data provide an example of remarkable potential of novel conjugates of SiO2 nanoparticles with phosphorylated nucleoside analogues, even those, which have not been used previously as therapeutics, for application as new anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Didesoxinucleótidos/farmacología , Nanopartículas/química , Dióxido de Silicio/farmacología , Nucleótidos de Uracilo/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Didesoxinucleótidos/síntesis química , Didesoxinucleótidos/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células MCF-7 , Estructura Molecular , Dióxido de Silicio/química , Relación Estructura-Actividad , Nucleótidos de Uracilo/síntesis química , Nucleótidos de Uracilo/químicaRESUMEN
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
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
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
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
The natural nucleotide adenosine triphosphate (ATP) and nucleotide analogues such as azidothymidine triphosphate (AZT-TP) display important pharmacological activities for the treatment of ischemia and HIV infections, respectively. Their clinical use is, however, limited mostly due to their hydrophilicity, which highly restricts their diffusion into the target cells. Few nanocarriers have been proposed to address the challenge of ATP/AZT-TP cellular delivery, but the loading efficiency, preparation complexity, and efficient cellular delivery remain important barriers to their development. In this study, we propose an original, straightforward and versatile design of nucleotide and nucleotide analogue nanocarriers based on the natural polysaccharide chitosan (CS). We show that the drugs ATP and AZT-TP can induce ionotropic gelation of CS, leading to CS/ATP and CS/AZT-TP nanoparticles with high drug entrapment efficiency and loading rate-up to 44%. Such nanocarriers release ATP and AZT-TP in physiological media and allow an efficient in vitro cellular delivery of these molecules down to the cell cytoplasm.
Asunto(s)
Adenosina Trifosfato/farmacología , Didesoxinucleótidos/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Nanopartículas/química , Nucleótidos/química , Zidovudina/análogos & derivados , Adenosina Trifosfato/química , Animales , Supervivencia Celular , Quitosano/química , Didesoxinucleótidos/química , Infecciones por VIH/tratamiento farmacológico , Macrófagos/metabolismo , Ratones , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
A series of d4T di- or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. The steady-state kinetic study of compounds 1-4 in an enzymatic incorporation assay by HIV-1 RT follows Michaelis-Menten profile. In addition, compounds 2-4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells, as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.
Asunto(s)
Fármacos Anti-VIH/química , Didesoxinucleótidos/química , Polifosfatos/química , Estavudina/análogos & derivados , Timidina Monofosfato/síntesis química , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Línea Celular , Didesoxinucleótidos/síntesis química , Didesoxinucleótidos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/metabolismo , Humanos , Cinética , Estavudina/síntesis química , Estavudina/farmacología , Timidina Monofosfato/química , Timidina Monofosfato/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
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.
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Fármacos Anti-VIH/síntesis química , Didesoxinucleótidos/síntesis química , Estavudina/análogos & derivados , Nucleótidos de Timina/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Línea Celular , Didesoxinucleótidos/química , Didesoxinucleótidos/farmacología , Estabilidad de Medicamentos , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Humanos , Hidrólisis , Mutación , Solventes/química , Estavudina/síntesis química , Estavudina/química , Estavudina/farmacología , Estereoisomerismo , Relación Estructura-Actividad , Timidina Quinasa/genética , Nucleótidos de Timina/química , Nucleótidos de Timina/farmacologíaRESUMEN
INTRODUCTION: Pre-exposure prophylaxis (PrEP) is an evolving new approach to prevention of sexually transmitted HIV-1 that employs antiretroviral (ARV) agents prior to potential HIV-1 exposure in an attempt to reduce the likelihood of HIV-1 infection postexposure. The identification of new ARV agents with potent activity against multidrug-resistant HIV remains an unmet and urgent challenge in the field of PrEP. AREAS COVERED: This article reviews the preclinical and early clinical activity and safety profile of stampidine, a novel antiretroviral (ARV) drug candidate that exhibits remarkable subnanomolar to low nanomolar in vitro antiretroviral potency against genotypically and phenotypically nucleoside reverse transcriptase inhibitor (NRTI)-resistant primary clinical HIV isolates, non-nucleoside RT-resistant HIV-1 isolates. Stampidine has a favorable pharmacokinetic profile in mice, rats, dogs and cats with 25 or 50 mg/kg tolerable dose levels yielding micromolar plasma concentrations that are 1000-fold higher than its in vitro IC(50) value against HIV. Stampidine has a favorable, safety profile in mice, rats, dogs and cats and it showed significant in vivo ARV activity in HIV-infected Hu-PBL-SCID mice as well as FIV-infected domestic cats. Furthermore, it did not cause any maternal toxicity, developmental toxicity or teratogenicity in rabbits treated at 10 - 40 mg/kg/day dose levels. In a recently completed first-in-human Phase I clinical trial, stampidine did not cause dose-limiting toxicity at single dose levels ranging from 5 to 25 mg/kg. EXPERT OPINION: The favorable safety and activity profile of stampidine warrants its further development as a promising next-generation PrEP candidate to prevent the sexual transmission of HIV-1. The discovery of stampidine as a potent antiretroviral agent represents a significant step forward in the development of effective therapeutic as well as preventive strategies against HIV/AIDS.
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Antirretrovirales/farmacología , Antirretrovirales/uso terapéutico , Didesoxinucleótidos/farmacología , Didesoxinucleótidos/uso terapéutico , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Estavudina/análogos & derivados , Timidina Monofosfato/análogos & derivados , Animales , Ensayos Clínicos Fase I como Asunto , Ensayos Clínicos Fase III como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Estavudina/farmacología , Estavudina/uso terapéutico , Timidina Monofosfato/farmacología , Timidina Monofosfato/uso terapéuticoRESUMEN
Nucleoside analogs (NAs) are used to treat numerous viral infections and cancer. They compete with endogenous nucleotides (dNTP/NTP) for incorporation into nascent DNA/RNA and inhibit replication by preventing subsequent primer extension. To date, an integrated mathematical model that could allow the analysis of their mechanism of action, of the various resistance mechanisms, and their effect on viral fitness is still lacking. We present the first mechanistic mathematical model of polymerase inhibition by NAs that takes into account the reversibility of polymerase inhibition. Analytical solutions for the model point out the cellular- and kinetic aspects of inhibition. Our model correctly predicts for HIV-1 that resistance against nucleoside analog reverse transcriptase inhibitors (NRTIs) can be conferred by decreasing their incorporation rate, increasing their excision rate, or decreasing their affinity for the polymerase enzyme. For all analyzed NRTIs and their combinations, model-predicted macroscopic parameters (efficacy, fitness and toxicity) were consistent with observations. NRTI efficacy was found to greatly vary between distinct target cells. Surprisingly, target cells with low dNTP/NTP levels may not confer hyper-susceptibility to inhibition, whereas cells with high dNTP/NTP contents are likely to confer natural resistance. Our model also allows quantification of the selective advantage of mutations by integrating their effects on viral fitness and drug susceptibility. For zidovudine triphosphate (AZT-TP), we predict that this selective advantage, as well as the minimal concentration required to select thymidine-associated mutations (TAMs) are highly cell-dependent. The developed model allows studying various resistance mechanisms, inherent fitness effects, selection forces and epistasis based on microscopic kinetic data. It can readily be embedded in extended models of the complete HIV-1 reverse transcription process, or analogous processes in other viruses and help to guide drug development and improve our understanding of the mechanisms of resistance development during treatment.
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Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Nucleósidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Secuencia de Bases , ADN Viral/química , ADN Viral/metabolismo , Didesoxinucleótidos/farmacología , Farmacorresistencia Viral , 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 , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , ARN Viral/metabolismo , Nucleótidos de Timina/farmacología , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
The first diastereoselective synthesis of aryloxy phosphoramidate prodrugs of 3'-deoxy-2',3'-didehydrothymidine monophosphate (d4TMP) is reported. In our approach, (S)-4-isopropylthiazolidine-2-thione 1 was used as a chiral auxiliary to introduce the stereochemistry at the phosphorus atom. In the last step of the developed reaction sequence, the nucleoside analogue d4T was introduced to a stereochemically pure phosphordiamidate which led to the formation of the almost diastereomerically pure phosphoramidate prodrugs 8a-d (≥95% de). As expected, the individually prepared diastereomers of the phosphoramidate prodrugs showed significant differences in the antiviral activity. Moreover, the difference was strongly dependent on the aryl substituent attached to the phosphoramidate moiety.
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Antivirales/síntesis química , Didesoxinucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , Profármacos/síntesis química , Estavudina/análogos & derivados , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Antivirales/química , Antivirales/farmacología , Línea Celular , Didesoxinucleótidos/química , Didesoxinucleótidos/farmacología , Humanos , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Profármacos/química , Profármacos/farmacología , Estavudina/síntesis química , Estavudina/química , Estavudina/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
3'-Deoxy-3'-fluorothymidine (FLT, alovudine(®)) belongs to the most potent agents inhibiting HIV-1 replication. Its 5'-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5'-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5'-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and ß (pol ß) was investigated. It was shown that 3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate (4-SFLTTP) and 3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with K(i)(app) values of 0.091 and 0.022 µM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with K(i)(app) of 0.174 µM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with K(i)(app) of 0.055 µM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol ß, with K(i)(app) values of 16.84 and 4.04 µM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3'-fluorothymidine 5'-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989; Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT. Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.
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Fármacos Anti-VIH/farmacología , ADN Polimerasa beta/antagonistas & inhibidores , Didesoxinucleótidos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Inhibidores de la Síntesis del Ácido Nucleico , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Timina/farmacología , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , ADN Polimerasa gamma , ADN Polimerasa Dirigida por ADN , Didesoxinucleósidos/farmacología , Didesoxinucleótidos/síntesis química , Didesoxinucleótidos/química , VIH-1/enzimología , Humanos , Imagen por Resonancia Magnética , Conformación Molecular , Polirribonucleótidos/metabolismo , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Relación Estructura-Actividad , Nucleótidos de Timina/síntesis química , Nucleótidos de Timina/químicaRESUMEN
A probable NRTI molecule, viz. 3'-N,N-dimethylamino-2',3'-dideoxythymidine (4) and its 5'-O-carboxyl ester prodrugs - 5'-(N-alpha-BOC-L-phenylalanyl)-3'-N,N-dimethylamino-2',3'-dideoxythymidine (5), 5'-L-phenylalanyl-3'-N,N-dimethylamino-2',3'-dideoxythymidine (6) and 5'-decanoyl-3'-N,N-dimethylamino-2',3'-dideoxythymidine (7) have been synthesized and screened against HIV, HSV-1 and 2, parainfluenza-3, vesicular stomatitis and several other viruses. The compound 6 showed good antiviral activity with EC(50) value 0.03 microM (SI=8) against VSV in Hela and HEL cell lines. However, the lead compound 4 and its derivatives 5, 6 and 7 showed no remarkable activity against HIV-1 and other viruses. Molecular docking studies with HIV-1 RT using DS 2.5 and pymol softwares have shown marked differences in the interaction patterns between the lead compound 4 and AZT.