RESUMO
Although the approved nucleoside reverse transcriptase (RT) inhibitors (NRTI) are integral components of therapy for human immunodeficiency virus type 1 (HIV-1) infection, they can have significant limitations, including the selection of NRTI-resistant HIV-1 and cellular toxicity. Accordingly, there is a critical need to develop new NRTI that have excellent activity and safety profiles and exhibit little or no cross-resistance with existing drugs. In this study, we report that the 3'-azido-2',3'-dideoxypurine nucleosides (ADPNs) 3'-azido-2',3'-dideoxyadenosine (3'-azido-ddA) and 3'-azido-2',3'-dideoxyguanosine (3'-azido-ddG) exert potent antiviral activity in primary human lymphocytes and HeLa and T-cell lines (50% inhibitory concentrations [IC50s] range from 0.19 to 2.1 microM for 3'-azido-ddG and from 0.36 to 10 microM for 3'-azido-ddA) and that their triphosphate forms are incorporated as efficiently as the natural dGTP or dATP substrates by HIV-1 RT. Importantly, both 3'-azido-ddA and 3'-azido-ddG retain activity against viruses containing K65R, L74V, or M184V (IC50 change of <2.0-fold) and against those containing three or more thymidine analog mutations (IC50 change of <3.5-fold). In addition, 3'-azido-ddG does not exhibit cytotoxicity in primary lymphocytes or epithelial or T-cell lines and does not decrease the mitochondrial DNA content of HepG2 cells. Furthermore, 3'-azido-ddG is efficiently phosphorylated to 3'-azido-ddGTP in human lymphocytes, with an intracellular half-life of the nucleoside triphosphate of 9 h. The present data suggest that additional preclinical studies are warranted to assess the potential of ADPNs for treatment of HIV-1 infection.
Assuntos
Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/farmacologia , Didesoxinucleosídeos/efeitos adversos , Didesoxinucleosídeos/farmacologia , HIV-1/efeitos dos fármacos , Inibidores da Transcriptase Reversa/efeitos adversos , Inibidores da Transcriptase Reversa/farmacologia , Fármacos Anti-HIV/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Didesoxinucleosídeos/química , Didesoxinucleosídeos/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Estrutura Molecular , Inibidores da Transcriptase Reversa/químicaRESUMO
The function of the p53 tumor suppressor to inhibit proliferation or initiate apoptosis is often abrogated in tumor cells. Mdm2 and its homolog, Mdm4, are critical inhibitors of p53 that are often overexpressed in human tumors. In mice, loss of Mdm2 or Mdm4 leads to embryonic lethal phenotypes that are completely rescued by concomitant loss of p53. To examine the role of Mdm2 and Mdm4 in a temporal and tissue-specific manner and to determine the relationships of these inhibitors to each other, we generated conditional alleles. We deleted Mdm2 and Mdm4 in cardiomyocytes, since proliferation and apoptosis are important processes in heart development. Mice lacking Mdm2 in the heart were embryonic lethal and showed defects at the time recombination occurred. A critical number of cardiomyocytes were lost by embryonic day 13.5, resulting in heart failure. This phenotype was completely rescued by deletion of p53. Mice lacking Mdm4 in the heart were born at the correct ratio and appeared to be normal. Our studies provide the first direct evidence that Mdm2 can function in the absence of Mdm4 to regulate p53 activity in a tissue-specific manner. Moreover, Mdm4 cannot compensate for the loss of Mdm2 in heart development.
Assuntos
Cardiopatias Congênitas/genética , Coração/embriologia , Proteínas Proto-Oncogênicas c-mdm2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Ubiquitina-Proteína Ligases/fisiologia , Alelos , Animais , Apoptose , Proliferação de Células , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Deleção de Genes , Genes Letais , Cardiopatias Congênitas/embriologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
A series of (-)-beta-D-(2R,4R)-dioxolane-thymine-5'-O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1(LAI) in human peripheral blood mononuclear (PBM) cells as well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was observed for the compound 2-4 (5'-O-aliphatic acid esters) without increase in cytotoxicity in comparison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0 and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the half-lives from 12.3 to 48.0 min.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Dioxolanos/farmacologia , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Timina/análogos & derivados , Animais , Fármacos Anti-HIV/toxicidade , Linhagem Celular , Chlorocebus aethiops , Dioxolanos/química , Dioxolanos/toxicidade , Estabilidade de Medicamentos , Meia-Vida , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Pró-Fármacos/toxicidade , Estereoisomerismo , Timina/química , Timina/farmacologia , Timina/toxicidade , Células VeroRESUMO
BACKGROUND: Hepatitis C virus (HCV) polymerase is an essential enzyme for HCV replication and has multiple inhibitor binding sites making it a major target for antiviral intervention. It is apparent that no single drug can inhibit HCV replication in humans. Hence, combinations of nucleoside analogues beta-D-2'-C-methylcytidine (2'-C-MeC; NM-107) or beta-D-2'-deoxy-2'-fluoro-2'-C-methyleytidine (2'-F-C-MeC; PSI-6130) with interferon-alpha2b (IFN-alpha2b) or triple combination with ribavirin (RBV) were evaluated. METHODS: Huh-7 cells containing the self-replicating subgenomic HCV replicon (Clone B) were used for drug combination studies. After drug treatment for 5 days, total cellular RNA was then extracted and both ribosomal RNA and HCV replicon RNA were amplified in a single-step multiplex real-time PCR assay. Drug interaction analyses were performed using the CalcuSyn program. RESULTS: Double combinations of 2'-C-MeC or 2'-F-C-MeC with IFN-alpha2b at all ratios tested had weighted average combination index (Cl(wt)) values <1 indicating synergistic inhibition of HCV replication in the replicon system. For the triple combinations of IFN-alpha2b plus RBV with either 2'-C-MeC or 2'-F-C-MeC, the Cl(wt) values at 1:1:1 ratio tested were 0.5 and 0.8, respectively, indicating synergistic antiviral effects. No apparent cytotoxicity effects were observed with any of the combinations tested. CONCLUSION: These promising in vitro data warrant clinical investigation of the nucleosides analogues such as 2'-C-MeC or 2'-F-C-MeC in their prodrug forms, together with IFN-alphac2b and RBV, for successful treatment of HCV infections.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Hepacivirus/efeitos dos fármacos , Interferon-alfa/farmacologia , Ribavirina/farmacologia , Células Cultivadas , Simulação por Computador , Citidina/análogos & derivados , Desoxicitidina/farmacologia , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Interferon alfa-2 , Modelos Químicos , Método de Monte Carlo , Reação em Cadeia da Polimerase , RNA Ribossômico/análise , RNA Viral/análise , Proteínas Recombinantes , RepliconRESUMO
beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a cytidine analogue with potent and selective anti-hepatitis C virus (HCV) activity in the subgenomic HCV replicon assay, 90% effective concentration (EC90)=4.6 +/- 2.0 microM. The spectrum of activity and cytotoxicity profile of PSI-6130 was evaluated against a diverse panel of viruses and cell types, and against two additional HCV-1b replicons. The S282T mutation, which confers resistance to 2'-C-methyl adenosine and other 2'-methylated nucleosides, showed only a 6.5-fold increase in EC90. When assayed for activity against bovine diarrhoea virus (BVDV), which is typically used as a surrogate assay to identify compounds active against HCV, PSI-6130 showed no anti-BVDV activity. Weak antiviral activity was noted against other flaviviruses, including West Nile virus, Dengue type 2, and yellow fever virus. These results indicate that PSI-6130 is a specific inhibitor of HCV. PSI-6130 showed little or no cytotoxicity against various cell types, including human peripheral blood mononuclear and human bone marrow progenitor cells. No mitochondrial toxicity was observed with PSI-6130. The reduced activity against the RdRp S282T mutant suggests that PSI-6130 is an inhibitor of replicon RNA synthesis. Finally, the no-effect dose for mice treated intraperitoneally with PSI-6130 for six consecutive days was > or =100 mg/kg per day.
Assuntos
Antivirais/farmacologia , Desoxicitidina/análogos & derivados , Hepacivirus/genética , RNA Viral/antagonistas & inibidores , Replicon/genética , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/toxicidade , Linhagem Celular , Desoxicitidina/farmacologia , Desoxicitidina/toxicidade , Hepacivirus/fisiologia , Humanos , Camundongos , RNA Viral/biossínteseRESUMO
A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.
Assuntos
Antivirais/farmacologia , Zalcitabina/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/síntese química , Antivirais/química , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Células Vero , Zalcitabina/análogos & derivados , Zalcitabina/síntese química , Zalcitabina/químicaRESUMO
BACKGROUND: 2'-C-methyl and 4'-azido nucleosides have previously demonstrated inhibition of hepatitis C virus (HCV) replication by targeting the RNA-dependent RNA polymerase NS5B. In an effort to discover new and more potent anti-HCV agents, we envisioned synthesizing nucleoside analogues by combining the 2'-C-methyl-moiety with the 4'-azido-moiety into one molecule. METHODS: 2'-C-methyl-4'-azido pyrimidine nucleosides were synthesized by first converting 2'-C-methyl ribonucleosides to the corresponding 4'-exocyclic methylene nucleosides. Treatment with iodine azide, benzoylation of the 2'- and 3'-hydroxy groups, oxidative displacement of the 5'-iodo group with meta-chloroperoxybenzoic acid, and debenzoylation gave the desired 2'-C-methyl-4'-azido uridine and thymidine analogues in good yield. Standard conversion of uridine to cytidine via the 4-triazole yielded 2'-C-methyl-4'-azido cytidine. In addition, 5'-phosphoramidate derivatives of 2'-C-methyl-4'-azido uridine and cytidine were synthesized to bypass the initial phosphorylation step. RESULTS: The prepared nucleosides and their 5'-monophosphate prodrugs were evaluated for their ability to inhibit replication of the hepatitis C virus in a subgenomic replicon cell based assay. Cytotoxicity in Huh7 cells was determined simultaneously with anti-HCV activity by extraction and amplification of both HCV RNA and ribosomal RNA. Among the newly synthesized compounds, only the 5'-monophosphate nucleoside prodrugs had modest and selective anti-HCV activity. All prepared pyrimidine nucleosides and 5'-monophosphate nucleoside prodrugs displayed no evidence of cytotoxicity at high concentrations. CONCLUSIONS: This work is the first example of both inactive uridine and cytidine analogues of a nucleoside being converted to active anti-HCV nucleosides via 5'-monophosphate prodrugs.
Assuntos
Antivirais/síntese química , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Amidas , Antivirais/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citidina/análogos & derivados , Humanos , Ácidos Fosfóricos , Pró-Fármacos/uso terapêutico , Nucleosídeos de Pirimidina/uso terapêutico , Uridina/análogos & derivados , Replicação Viral/efeitos dos fármacosRESUMO
Studies on cellular drug interactions with antiretroviral agents prior to clinical trials are critical to detect possible drug interactions. Herein, we demonstrated that two 2'-deoxycytidine antiretroviral agents, dexelvucitabine (known as beta-d-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine, DFC, d-d4FC, or RVT) and lamivudine (3TC), combined in primary human peripheral blood mononuclear (PBM) cells infected with human immunodeficiency virus 1 strain LAI (HIV-1(LAI)), resulted in additive-to-synergistic effects. The cellular metabolism of DFC and 3TC was studied in human T-cell lymphoma (CEM) and in primary human PBM cells to determine whether this combination caused any reduction in active nucleoside triphosphate (NTP) levels, which could decrease with their antiviral potency. Competition studies were conducted by coincubation of either radiolabeled DFC with different concentrations of 3TC or radiolabeled 3TC with different concentrations of DFC. Coincubation of radiolabeled 3TC with DFC at concentrations up to 33.3 microM did not cause any marked reduction in 3TC-triphosphate (TP) or any 3TC metabolites. However, a reduction in the level of DFC metabolites was noted at high concentrations of 3TC with radiolabeled DFC. DFC-TP levels in CEM and primary human PBM cells decreased by 88% and 94%, respectively, when high concentrations of 3TC (33.3 and 100 microM) were added, which may influence the effectiveness of DFC-5'-TP on the HIV-1 polymerase. The NTP levels remained well above the median (50%) inhibitory concentration for HIV-1 reverse transcriptase. These results suggest that both beta-d- and beta-l-2'-deoxycytidine analogs, DFC and 3TC, respectively, substrates of 2'-deoxycytidine kinase, could be used in a combined therapeutic modality. However, it may be necessary to decrease the dose of 3TC for this combination to prove effective.
Assuntos
Fármacos Anti-HIV , Interações Medicamentosas , HIV-1/efeitos dos fármacos , Lamivudina , Inibidores da Transcriptase Reversa , Zalcitabina/análogos & derivados , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/virologia , Células Cultivadas , Sinergismo Farmacológico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , HIV-1/fisiologia , Humanos , Lamivudina/metabolismo , Lamivudina/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Zalcitabina/metabolismo , Zalcitabina/farmacologiaRESUMO
The emergence of resistance to lamivudine has been one of the major stumbling blocks to successful treatment and control of hepatitis B virus (HBV) infections. The major mechanism of resistance has been attributed to the alteration in the YMDD motif of the HBV polymerase due to an amino acid change of rtM204 to V/I and an accompanying rtL180M conversion. A novel mutation pattern in a patient having clinical breakthrough under lamivudine therapy was discovered. The mutant had a rtL180C/M204I genotype and was detected after 2 years of therapy with lamivudine. To characterize this novel variant, site-directed mutagenesis was performed using a vector construct containing the HBV genome. Transient transfection studies in human hepatoma cells with HBV carrying the new mutant demonstrated that the rtL180C/M204I mutant was resistant to lamivudine up to 10 microM. The resistance profile was comparable to that of the previously reported rtL180 M/M204I-containing virus. These observations were further confirmed by generation of stable cultures transfected with the mutant virus.
Assuntos
Farmacorresistência Viral/genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Lamivudina/farmacologia , Mutação , Inibidores da Transcriptase Reversa/farmacologia , Idoso , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , DNA Viral/análise , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Beta-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine (D-FDOC) is an effective inhibitor of human immunodeficiency virus 1 (HIV-1) and HIV-2, simian immunodeficiency virus, and hepatitis B virus (HBV) in vitro. The purpose of this study was to evaluate the intracellular metabolism of d-FDOC in human hepatoma (HepG2), human T-cell lymphoma (CEM), and primary human peripheral blood mononuclear (PBM) cells by using tritiated compound. By 24 h, the levels of D-FDOC-triphosphate (D-FDOC-TP) were 2.8 +/- 0.4, 6.7 +/- 2.3, and 2.0 +/- 0.1 pmol/10(6) cells in HepG2, CEM, and primary human PBM cells, respectively. Intracellular D-FDOC-TP concentrations remained greater than the 50% inhibitory concentration for HIV-1 reverse transcriptase for up to 24 h after removal of the drug from cell cultures. In addition to d-FDOC-monophosphate (D-FDOC-MP), -diphosphate (D-FDOC-DP), and -TP, D-FDOC-DP-ethanolamine and d-FDOC-DP-choline were detected in all cell extracts as major intracellular metabolites. D-FDOC was not a substrate for Escherichia coli thymidine phosphorylase. No toxicity was observed in mice given D-FDOC intraperitoneally for 6 days up to a dose of 100 mg/kg per day. Pharmacokinetic studies in rhesus monkeys indicated that D-FDOC has a t(1/2) of 2.1 h in plasma and an oral bioavailability of 38%. The nucleoside was excreted unchanged primary in the urine, and no metabolites were detected in plasma or urine. These results suggest that further safety and pharmacological studies are warranted to assess the potential of this nucleoside for the treatment of HIV- and HBV-infected individuals.
Assuntos
Antivirais , Citidina , Citidina/análogos & derivados , Animais , Antivirais/administração & dosagem , Antivirais/metabolismo , Antivirais/farmacocinética , Antivirais/toxicidade , Linhagem Celular Tumoral , Citidina/administração & dosagem , Citidina/metabolismo , Citidina/farmacocinética , Citidina/toxicidade , Escherichia coli/enzimologia , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Macaca mulatta , Masculino , Camundongos , Timidina Fosforilase/metabolismoRESUMO
The antiviral efficacies and cytotoxicities of 2',3'- and 4'-substituted 2',3'-didehydro-2',3'-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC(90)s], 0.14 to 5.2 micro M), but marked increases in EC(90)s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC(90)s, 53 to >100 micro M). The beta-L-enantiomers of both classes of compounds were more potent than the corresponding beta-D-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC(90), 0.25 micro M). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 micro M. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4'-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC(90), 0.08 micro M) and lamivudine-resistant HIV-1 (EC(90) = 0.15 micro M) is markedly reduced by introduction of a 3'-fluorine in the sugar (EC(90)s of compound 2a, 37.5 and 494 micro M, respectively).