RESUMO
Azidothymidine and ribavirin both inhibit replication of human immunodeficiency virus in vitro and show promise of clinical utility in patients infected with this virus. In this study, the possible interactions of these drugs were examined in vitro, and a reproducible antagonism between azidothymidine and ribavirin was found to occur under a variety of experimental conditions. The mechanism responsible for this antagonism appeared to be inhibition of azidothymidine phosphorylation by ribavirin. Because similar effects may occur in vivo, clinical trials of these two drugs in combination must be performed only under carefully controlled conditions.
Assuntos
HIV/efeitos dos fármacos , Ribavirina/farmacologia , Ribonucleosídeos/farmacologia , Timidina/análogos & derivados , Linhagem Celular , HIV/fisiologia , Humanos , Linfócitos/microbiologia , Monócitos/microbiologia , Fosforilação , Fito-Hemaglutininas/farmacologia , DNA Polimerase Dirigida por RNA/metabolismo , Timidina/antagonistas & inibidores , Timidina/farmacologia , Replicação Viral/efeitos dos fármacos , ZidovudinaRESUMO
This review describes each of the steps in the HIV-1 reverse transcriptase catalytic cycle and evaluates each of these steps as a potential point of inhibition of the enzyme and consequently viral replication. To date, two classes of approved drugs act on the reverse transcriptase. They are: (1) the nucleoside reverse transcriptase inhibitors which either directly inhibit the enzyme or serve as alternative substrates for catalysis (resulting in chain termination) and (2) the non-nucleoside reverse transcriptase inhibitors which bind to an allosteric site and adversely affect the function of the enzyme by slowing the rate of chemical catalysis. In order to provide the best possible analysis of the potential of each of the steps in the catalytic cycle as a site of inhibition, the molecular forces which determine the intrinsic binding affinities and specificity of natural components of the catalytic complex will be described in as much detail as possible.
Assuntos
Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Animais , Fármacos Anti-HIV/síntese química , HIV-1/enzimologia , Humanos , Inibidores da Transcriptase Reversa/síntese químicaRESUMO
Zidovudine is a potent in vitro inhibitor of human immunodeficiency virus (HIV) with varying efficacy against other retroviruses. With the exception of Epstein-Barr virus, all non-retroviruses tested so far have been insensitive to inhibition by zidovudine. In vivo, efficacy of zidovudine was demonstrated against Rauscher murine leukemia virus and feline leukemia virus. In both experimental models, infections completely resolved in animals when the drug was administered soon after infection. These results suggest that prompt initiation of zidovudine therapy, following a known exposure to HIV, should be considered. Mechanism studies show that zidovudine is phosphorylated to the monophosphate and diphosphate derivatives by the host cell cytosolic thymidine kinase and thymidylate kinase, respectively. The identity of the enzyme that phosphorylates zidovudine diphosphate is not known, but is believed to be the cellular nucleoside diphosphate kinase. The triphosphate of zidovudine appears to be the active form of the drug. Zidovudine triphosphate competes well with thymidine 5'-triphosphate for binding to the HIV reverse transcriptase and also functions as an alternative substrate. Incorporation of zidovudine monophosphate results in chain termination. However, it is not clear which mechanism, chain termination or competition with thymidine 5'-triphosphate, or a combination of both, is responsible for the inhibition of HIV replication.
Assuntos
Antivirais/farmacologia , HIV/efeitos dos fármacos , Timidina/análogos & derivados , Animais , Humanos , Retroviridae/efeitos dos fármacos , Timidina/farmacologia , Replicação Viral/efeitos dos fármacos , ZidovudinaRESUMO
The effect of acyclovir on DNA synthesized in HSV-1-infected cells was examined. Viral DNA synthesis was significantly reduced in the presence of 10 microM acyclovir monitored by cRNA-DNA hybridization and isopycnic centrifugation in cesium chloride. DNA synthesized in infected cells in the presence of acyclovir appeared to be chain-terminated when evaluated by rate zonal centrifugation in alkaline sucrose. Cellular DNA synthesis in actively replicating uninfected cells in the presence of acyclovir was evaluated by isopycnic centrifugation. Cellular DNA synthesis appeared unaffected at concentrations of acyclovir up to 100 microM.
Assuntos
Antivirais/farmacologia , DNA Viral/biossíntese , Guanina/análogos & derivados , Simplexvirus/efeitos dos fármacos , Aciclovir , Animais , Linhagem Celular , Cricetinae , DNA/biossíntese , Relação Dose-Resposta a Droga , Guanina/farmacologia , Simplexvirus/metabolismoRESUMO
The effect of acyclovir on the deoxyribonucleoside triphosphate pools of Vero cells infected with herpes simplex virus type 1 was examined. Deoxyguanosine triphosphate and deoxyadenosine triphosphate pool levels in infected cells treated with acyclovir increased dramatically compared with pool levels in untreated infected cels. The increases were due, at least in part, to inhibition of viral DNA polymerase activity which resulted in reduced utilization of the deoxyribonucleoside triphosphates. Differences of as much as 26 times were detected in the sensitivity of herpes simplex virus type 1 to inhibition by acyclovir with different Vero cell cultures. These results were due to differences in acyclovir triphosphate levels, not to differences in deoxyguanosine triphosphate levels.
Assuntos
Antivirais/farmacologia , Desoxirribonucleotídeos/metabolismo , Guanina/análogos & derivados , Simplexvirus/crescimento & desenvolvimento , Aciclovir , Animais , Linhagem Celular , Chlorocebus aethiops , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Guanina/metabolismo , Guanina/farmacologia , Ácido Fosfonoacéticos/farmacologia , Nucleotídeos de Timina/metabolismoRESUMO
The following paper is intended to review those nucleoside analogs currently licensed for the treatment of herpetic diseases. Potentially efficacious congeners also will be considered. The mechanism of action, preclinical and clinical efficacy, and the problems associated with the use of each nucleoside analog will be described.
Assuntos
Herpes Simples/tratamento farmacológico , Aciclovir/análogos & derivados , Aciclovir/uso terapêutico , Ganciclovir , Humanos , Nucleosídeos de Purina/uso terapêutico , Nucleosídeos de Pirimidina/uso terapêuticoRESUMO
Nuclear Overhauser effect experiments were used to characterize the protein environment and conformations of dTTP, dATP and AZTTP bound to HIV-RT in the ground state. The results show the initial binding sites for the nucleotides overlap but are not completely coincident. All of the bound nucleotides assume the same anti C4'-exo conformation.
Assuntos
Fármacos Anti-HIV/química , Nucleotídeos de Desoxiadenina/química , Transcriptase Reversa do HIV/química , Nucleotídeos de Timina/química , Zidovudina/análogos & derivados , Fármacos Anti-HIV/metabolismo , Ligação Competitiva , Nucleotídeos de Desoxiadenina/metabolismo , Didesoxinucleotídeos , Eletroforese em Gel de Poliacrilamida , Transcriptase Reversa do HIV/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Nucleotídeos de Timina/metabolismo , Zidovudina/química , Zidovudina/metabolismoAssuntos
Antivirais/farmacologia , Genes Virais , Guanina/análogos & derivados , Simplexvirus/efeitos dos fármacos , Aciclovir , DNA Polimerase Dirigida por DNA/genética , Resistência Microbiana a Medicamentos , Guanina/metabolismo , Guanina/farmacologia , Mutação , Ácido Fosfonoacéticos/farmacologia , Fosforilação , Simplexvirus/genética , Timidina Quinase/genética , Vidarabina/farmacologiaAssuntos
Antivirais/metabolismo , Nucleosídeos/metabolismo , Simplexvirus/metabolismo , Animais , Chlorocebus aethiops , Cinética , Nucleosídeos/farmacologia , Fosforilação , Simplexvirus/efeitos dos fármacos , Relação Estrutura-Atividade , Especificidade por Substrato , Timidina Quinase/análise , Replicação Viral/efeitos dos fármacosRESUMO
The reverse transcriptase of Equine Infectious Anemia Virus (EIAV) was partially purified from virus particles and appeared to be a heterodimer with subunit molecular masses of 70 kdal and 59 kdal. The polymerase activity of this enzyme had an absolute requirement for a divalent cation, preferring Mg++ over Mn++. Addition of a monovalent cation to the reaction mixture enhanced, but was not required for enzyme activity. Kinetically, the reverse transcriptase of EIAV is similar to the reverse transcriptase of Human Imunodeficiency Virus Type 1 (HIV-1). Both enzymes have similar Km values for 2'-deoxynucleoside-5'-triphosphates on the synthetic template/primers tested, both exhibit substrate inhibition, and both are inhibited to similar extents by most nucleoside-triphosphate analogs. The results of this study suggest that the reverse transcriptase of EIAV may be a good model for studying structure/function relationships of retroviral reverse transcriptases.
Assuntos
Vírus da Anemia Infecciosa Equina/enzimologia , DNA Polimerase Dirigida por RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Cátions Bivalentes , Células Cultivadas , Cromatografia por Troca Iônica , Cavalos , Cinética , Pele , Especificidade por Substrato , Moldes GenéticosRESUMO
Preclinical screening for the toxicity of antiviral drugs has thus far proven quite successful. Twenty-three antivirals spanning a variety of chemical classes and including a combination product, have been safely developed and are listed in the 1999 Physician's Desk Reference. Several of these antivirals have been administered for many years and in various combinations and we are currently unaware of any being withdrawn for safety-related reasons. Progress in protecting this record includes advances in rational drug design, the development of in vitro and in vivo models available to study both efficacy and safety, and improved bioanalytical capabilities. Coupled with approximately 25 years of experience since vidarabine was approved for the treatment of herpes encephalitis, these advances set the stage for even more precise and focused development of pharmaceuticals for the treatment of life-threatening viral infections, including those caused by HIV, HBV and emerging viruses. Experience to date suggests that each antiviral, particularly the nucleoside analogs, may have unique attributes of efficacy and safety. This brief review is an attempt to highlight a combination of established and recent methods that may be helpful in screening antivirals for potential toxicity. The earliest phases of drug development are mentioned in the introduction, followed by contributions provided by in vitro and in vivo testing in models of viral disease. Finally, current and new methods applicable to screening for toxicity are discussed.
RESUMO
RNA-dependent DNA polymerase activity was found in peparations of a mutant of Newcastle disease virus. The enzyme activity was not found in wild-type virus preparations.
Assuntos
Células L/microbiologia , Mutação , Vírus da Doença de Newcastle/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Técnicas de Cultura , DNA Viral/análise , DNA Viral/biossíntese , Testes de Hemaglutinação , Imunodifusão , Camundongos , Vírus da Doença de Newcastle/análise , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus da Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Viral/análise , Nucleotídeos de Timina/metabolismo , TrítioRESUMO
The effect of the antiviral agent 9-(2-hydroxyethoxymethyl)guanine (acyclovir) on herpes simplex virus type 1 protein synthesis during virus replication was examined. Treatment of infected cells with acyclovir markedly affected the amounts of the four major glycosylated and certain non-glycosylated viral polypeptides synthesized; other viral polypeptides were made in normal amounts. The reduced amount of late protein synthesis was most likely due to the inhibition of progeny viral DNA synthesis by acyclovir.
Assuntos
Aciclovir/farmacologia , Simplexvirus/efeitos dos fármacos , Proteínas Virais/biossíntese , DNA Viral/biossíntese , Glicoproteínas/biossíntese , Simplexvirus/metabolismoRESUMO
The triphosphate form of 9-[(2-hydroxyethoxy)-methyl]guanine (acyclovir), ACVTP, inactivates the herpes simplex virus type 1 DNA polymerase. ACVTP does not innately inactivate resting polymerase, but becomes an inactivator only while being processed as an alternative substrate. Pseudo first-order rates of inactivation were measured at varying concentrations of ACVTP and fixed concentrations of the natural substrate, deoxyguanosine triphosphate. These studies indicated that a reversible enzyme-ACVTP (Michaelis-type) complex is formed at the active site prior to inactivation. The formation of this complex was competitively retarded by deoxyguanosine triphosphate. An apparent dissociation constant (KD) of 3.6 +/- 0.2 (S.D.) nM was determined for ACVTP from this reversible complex. A second method for the estimation of the KD which used the extrapolated initial velocities produced a value of 5.9 +/- 0.4 (S.D.) nM. The rate of conversion of the reversible complex to the inactivated complex, at saturating ACVTP, was calculated to be 0.24 min-1. No reactivation of enzyme activity was detected following isolation of the inactivated complex by rapid desalting on Sephadex G-25. Under these conditions, an overall reactivation rate of 1.5 X 10(-5) min-1 could have been easily detected. Therefore, the overall inhibition constant must have been less than 3 pM. In contrast, when host DNA polymerase alpha was incubated with 14 microM ACVTP, only 60% inhibition of enzyme activity was observed, but inactivation was not detected. These data indicate that ACVTP functions as a suicide inactivator of the herpes simplex virus type 1 DNA polymerase, and is only a weak reversible inhibitor of DNA polymerase alpha.
Assuntos
Aciclovir/análogos & derivados , Inibidores da Síntese de Ácido Nucleico , Simplexvirus/enzimologia , Aciclovir/farmacologia , DNA/metabolismo , Células HeLa/enzimologia , Humanos , Matemática , Simplexvirus/efeitos dos fármacos , Fatores de TempoRESUMO
The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.
Assuntos
Ribonucleotídeo Redutases/isolamento & purificação , Simplexvirus/enzimologia , Trifosfato de Adenosina/farmacologia , Cistina Difosfato/metabolismo , Cinética , Magnésio/farmacologia , Ribonucleotídeo Redutases/antagonistas & inibidoresRESUMO
Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using [32P]rA12-18.dT10 as a representative template-primer and [alpha-32P]dTTP as a representative 2'-deoxynucleoside-5'-triphosphate. UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme. In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit. These results suggest that the polymerase activity of the heterodimer residues on p66.
Assuntos
HIV-1/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Marcadores de Afinidade/metabolismo , Clonagem Molecular , Reagentes de Ligações Cruzadas , Escherichia coli/genética , HIV-1/fisiologia , Cinética , Substâncias Macromoleculares , Peso Molecular , Ligação Proteica , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Nucleotídeos de Timina/metabolismo , Replicação ViralRESUMO
The Sp-isomer of thymidine 5'-O-(1-thiotriphosphate) is used as substrate by HIV-1 reverse transcriptase. The absolute configuration of the internucleotide linkage in the oligonucleotide product was identified by 31p NMR spectroscopy to be the Rp-isomer, indicating that incorporation of dTTP alpha S into the oligonucleotide proceeded with inversion of configuration at the alpha-phosphorus. The mechanistic implications of these observations are discussed.
Assuntos
DNA/biossíntese , HIV/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Catálise , DNA/ultraestrutura , Técnicas In Vitro , Cinética , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Inibidores da Transcriptase Reversa , EstereoisomerismoRESUMO
Rat embryo fibroblasts (REF) morphologically transformed by herpes simplex virus type 2 (HSV-2) and tumor-derived cells were tested for ability to grow in the presence of 9-(2-hydroxyethoxymethyl) guanine (acyclovir). Results indicated that the effective dose of acyclovir (ACV) required to inhibit HSV-2-transformed and tumor-derived cell growth by 50% (ED50) compared to mock-treated control cells averaged 15 to 75 micrograms/ml. In contrast, the ED50 of acyclovir was more than HEp-2 cells. HSV-2-transformed and tumor-derived cells after both low (less than 30) and high (greater than 30) serial passages expressed detectable levels of the virus-coded thymidine kinase (TK) measured in cell extracts by serum neutralization assay. HSV-2-transformed or tumor-derived cells converted two- to ten-fold more acyclovir to phosphorylated forms than nontransformed REF cells. Preliminary data showed that the drug inhibited tumor development in newborn syngeneic rats inoculated with HSV-2-transformed cells. The inhibitory activity of acyclovir and presence of low levels of HSV-2 TK activity appeared to correlate.
Assuntos
Aciclovir/farmacologia , Transformação Celular Neoplásica , Simplexvirus/efeitos dos fármacos , Infecções Tumorais por Vírus/tratamento farmacológico , Aciclovir/metabolismo , Animais , Antineoplásicos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Fosforilação , Ratos , Simplexvirus/enzimologia , Timidina Quinase/metabolismoRESUMO
We present evidence that mutation frequencies in a mammalian system can vary according to the replication fidelity of the DNA polymerase. We demonstrated previously that several derivatives of herpes simplex virus type 1 that encode polymerases resistant to various antiviral drugs (e.g., nucleotide analogues) also produce reduced numbers of spontaneous mutants. Here we show that the DNA polymerase from one antimutator virus exhibits enhanced replication fidelity. First, the antimutator virus showed a reduced response to known mutagens that promote base mispairing during DNA replication (N-methyl-N'-nitro-N-nitrosoguanidine, 5-bromo-deoxyuridine). Second, purified DNA polymerase from the antimutator produced fewer replication errors in vitro, based on incorporation of mispaired nucleotides or analogues with abnormal sugar rings. We have investigated possible mechanisms for the enhanced fidelity of the antimutator polymerase. We show that the mutant enzyme has altered interactions with nucleoside triphosphates, as indicated by its resistance to nucleotide analogues and elevated Km values for normal nucleoside triphosphates. We present evidence against increased proofreading by an associated 3',5' exonuclease (as seen for T4 bacteriophage antimutator polymerases), based on nuclease levels in the mutant polymerase. We propose that reduced affinity of the polymerase for nucleoside triphosphates accounts for the antimutator phenotype by accentuating differences in base-pair stability, thus facilitating selection of correct nucleotides.