RESUMO
Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(-)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (-)-FTC, (-)-ß-D-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (-)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >10(4) copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred rapidly following cessation of any treatment. The enhanced HAART (four- or five-drug combination) showed significant improvement in viral suppression compared to the three-drug combination, but no combination was sufficient to eliminate viral reservoirs.
Assuntos
Terapia Antirretroviral de Alta Atividade/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Animais , Fármacos Anti-HIV/farmacocinética , Fármacos Anti-HIV/uso terapêutico , Combinação de Medicamentos , Cinética , Macaca mulatta , RNA Viral/sangue , Recidiva , Vírus da Imunodeficiência Símia , Carga ViralRESUMO
Understanding the cellular pharmacology of antiretroviral agents in macrophages and subsequent correlation with antiviral potency provides a sentinel foundation for definition of the dynamics between antiretroviral agents and viral reservoirs across multiple cell types, with the goal of eradication of HIV-1 from these cells. Various clinically relevant nucleoside antiviral agents, and the integrase inhibitor raltegravir, were selected for this study. The intracellular concentrations of the active metabolites of the nucleoside analogs were found to be 5- to 140-fold lower in macrophages than in lymphocytes, and their antiviral potency was significantly lower in macrophages constitutively activated with macrophage colony-stimulating factor (M-CSF) during acute infection than in resting macrophages (EC(50), 0.4 to 9.42 µM versus 0.03 to 0.4 µM, respectively). Although tenofovir-treated cells displayed significantly lower intracellular drug levels than cells treated with its prodrug, tenofovir disoproxil fumarate, the levels of tenofovir-diphosphate for tenofovir-treated cells were similar in lymphocytes and macrophages. Raltegravir also displayed significantly lower intracellular concentrations in macrophages than in lymphocytes, independent of the activation state, but had similar potencies in resting and activated macrophages. These data underscore the importance of delivering adequate levels of drug to macrophages to reduce and eradicate HIV-1 infection.
Assuntos
Adenina/análogos & derivados , Adenina/farmacologia , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Organofosfonatos/farmacologia , Pirrolidinonas/farmacologia , Transporte Biológico , Inibidores de Integrase de HIV/farmacologia , HIV-1/enzimologia , HIV-1/crescimento & desenvolvimento , Humanos , Linfócitos/virologia , Ativação de Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/virologia , Especificidade de Órgãos , Cultura Primária de Células , Raltegravir Potássico , Inibidores da Transcriptase Reversa/farmacologia , TenofovirRESUMO
Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low-level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently infected cells, such as resting CD4(+) T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells, and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, preamplification, and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine, and tenofovir. Plasma virus loads at necropsy ranged from 11 to 28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART, in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly the spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of the tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.
Assuntos
Fármacos Anti-HIV , Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Animais , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/virologia , DNA Viral/metabolismo , Modelos Animais de Doenças , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Macaca mulatta , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Distribuição Tecidual , Carga Viral , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251) mutants with a K65R mutation in reverse transcriptase (RT), and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later. RESULTS: The early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia. CONCLUSION: This is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be beneficial, particularly when assisted by antiviral immune responses.
Assuntos
Adenina/análogos & derivados , Substituição de Aminoácidos , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/genética , Organofosfonatos/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Depleção Linfocítica , Macaca , Mutação de Sentido Incorreto , Organofosfonatos/uso terapêutico , RNA Viral/sangue , Seleção Genética , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Tenofovir , Carga Viral , ViremiaRESUMO
Integration of HIV-1 viral DNA into the host genome is carried out by HIV-integrase (IN) and is a critical step in viral replication. Although several classes of compounds have been reported to inhibit IN in enzymatic assays, inhibition is not always correlated with antiviral activity. Moreover, potent antiviral IN inhibitors such as the chicoric acids do not act upon the intended enzymatic target but behave as entry inhibitors instead. The charged nature of the chicoric acids contributes to poor cellular uptake, and these compounds are further plagued by rapid ester hydrolysis in vivo. To address these critical deficiencies, we designed neutral, nonhydrolyzable analogues of the chicoric acids. Herein, we report the synthesis, enzyme inhibition studies, and cellular antiviral data for a series of geminal disulfones. Of the 10 compounds evaluated, 8 showed moderate to high inhibition of IN in purified enzyme assays. The purified enzyme data correlated with antiviral assays for all but two compounds, suggesting alternative modes of inhibition. Time-of-addition studies were performed on these analogues, and the results indicate that they inhibit an early stage in the replication process, perhaps entry. In contrast, the most potent member of the correlative group shows behavior consistent with IN being the cellular target.
Assuntos
Fármacos Anti-HIV/síntese química , Inibidores de Integrase de HIV/síntese química , Integrase de HIV/metabolismo , Sulfonas/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Ácidos Cafeicos/síntese química , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Relação Dose-Resposta a Droga , Integrase de HIV/química , Inibidores de Integrase de HIV/química , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Relação Estrutura-Atividade , Succinatos/síntese química , Succinatos/química , Succinatos/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Fatores de TempoRESUMO
16alpha-Bromo-epiandrosterone (epiBr), a synthetic derivative of the natural hormone dehyroepiandrosterone (DHEA), was evaluated for its effects on feline immunodeficiency virus (FIV) infection in experimental cats. The rationale for this study was based on the ability of DHEA to significantly reduce the mortality to viral infections in mice. DHEA and epiBr also have demonstrable in vitro anti-viral activity for both HIV-1 and FIV. Preliminary pharmacokinetic studies in cats demonstrated that subcutaneously injected epiBr was rapidly absorbed, completely metabolized, and nontoxic. Metabolites were excreted in both urine and feces, with the latter having the most complex pattern of breakdown products. Cats were then divided into four groups; two groups were infected with FIV and two uninfected. Two groups, one infected and one uninfected were treated on 5 consecutive days of weeks 0, 4, 8, 12 and 16 with epiBr. The remaining two groups were mock treated with the drug vehicle alone. Treatment started 1 week prior to infection and extended for 4 weeks after infection. Cats were observed for 20 weeks post-FIV infection. Infected cats had identical decreases in blood neutrophil and lymphocyte counts following, regardless of whether they were treated with epiBr or vehicle alone. The CD4/CD8 T-cell ratio was decreased following FIV exposure, but was significantly more decreased for the epiBr treated animals from week 2 post-infection onward. CD4+ T cells were decreased in FIV-infected cats treated with epiBr compared to their untreated cohort, while CD8+ T cells tended to be higher in treated animals. FIV infected cats that were treated with epiBr had over one-log higher virus loads at week 2 post-infection than non-epiBr treated cohorts. In spite of this enhanced initial viremia, the subsequent levels of virus in the blood were significantly lower in epiBr treated versus untreated animals. EpiBr treated cats had significantly higher FIV-p24 antibody responses than control cats receiving vehicle alone, although primary and secondary antibody responses to a T-cell dependent non-FIV antigen, keyhole limpet hemocyanin (KLH), were unaffected. EpiBr treatment significantly decreased the expected FIV-induced suppression of IL-12 p40 mRNA levels in peripheral blood mononuclear cells (PBMCs) observed at weeks 4, 5, 8, 9 and 16 post-infection, but had no influence on FIV-induced changes in IL-4, IL-6, IL-10, IFN-gamma, MIP-1alpha and RANTES.
Assuntos
Adjuvantes Imunológicos/farmacologia , Androsterona/análogos & derivados , Androsterona/farmacologia , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Vírus da Imunodeficiência Felina/imunologia , Adjuvantes Imunológicos/farmacocinética , Adjuvantes Imunológicos/urina , Androsterona/farmacocinética , Androsterona/urina , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Relação CD4-CD8/veterinária , Gatos , Citocinas/biossíntese , Citocinas/sangue , Citocinas/genética , Ensaio de Imunoadsorção Enzimática/veterinária , Fezes/química , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Síndrome de Imunodeficiência Adquirida Felina/metabolismo , Síndrome de Imunodeficiência Adquirida Felina/virologia , Vírus da Imunodeficiência Felina/genética , Vírus da Imunodeficiência Felina/metabolismo , Interferon gama/genética , Interferon gama/imunologia , Masculino , Reação em Cadeia da Polimerase/veterinária , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Organismos Livres de Patógenos Específicos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/veterináriaRESUMO
Highly active antiretroviral therapy (HAART) can reduce levels of human immunodeficiency virus type 1 (HIV-1) to undetectable levels in infected individuals, but the virus is not eradicated. The mechanisms of viral persistence during HAART are poorly defined, but some reservoirs have been identified, such as latently infected resting memory CD4⺠T cells. During latency, in addition to blocks at the initiation and elongation steps of viral transcription, there is a block in the export of viral RNA (vRNA), leading to the accumulation of multiply-spliced transcripts in the nucleus. Two of the genes encoded by the multiply-spliced transcripts are Tat and Rev, which are essential early in the viral replication cycle and might indicate the state of infection in a given population of cells. Here, the levels of multiply-spliced transcripts were compared to the levels of gag-containing RNA in tissue samples from RT-SHIV-infected rhesus macaques treated with HAART. Splice site sequence variation was identified during development of a TaqMan PCR assay. Multiply-spliced transcripts were detected in gastrointestinal and lymphatic tissues, but not the thymus. Levels of multiply-spliced transcripts were lower than levels of gag RNA, and both correlated with plasma virus loads. The ratio of multiply-spliced to gag RNA was greatest in the gastrointestinal samples from macaques with plasma virus loads <50 vRNA copies per mL at necropsy. Levels of gag RNA and multiply-spliced mRNA in tissues from RT-SHIV-infected macaques correlate with plasma virus load.
Assuntos
Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos , Infecções por HIV/sangue , HIV-1/fisiologia , RNA Mensageiro/sangue , RNA Viral/sangue , Replicação Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Humanos , Macaca mulatta , Masculino , Carga ViralRESUMO
RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.
Assuntos
Transcriptase Reversa do HIV/genética , Mutação , Proteínas Recombinantes de Fusão/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/genética , Substituição de Aminoácidos , Animais , Terapia Antirretroviral de Alta Atividade , Células Cultivadas , Feminino , Genoma Viral/genética , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Macaca mulatta , Masculino , Modelos Moleculares , Estrutura Terciária de Proteína , RNA Viral/sangue , RNA Viral/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Inibidores da Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/enzimologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/enzimologia , Carga Viral/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral/genéticaRESUMO
PURPOSE OF REVIEW: This review will focus on recent developments in several nonhuman primate models of AIDS. These models are being used to address viral latency and persistence during antiretroviral therapy in studies that are not feasible in humans. RECENT FINDINGS: Further characterization of the various macaque models of AIDS has demonstrated that several aspects of viral persistence during antiretroviral therapy model HIV-1 infection in humans, including viral decay kinetics. Widespread distribution of viral RNA and viral DNA has been detected in many tissue organs. In addition, the brain has been identified as a site of persistent viral DNA. SUMMARY: The macaque models of AIDS are well suited for addressing viral persistence during antiretroviral therapy, including viral latency, residual replication, and tissue organ distribution.
Assuntos
Macaca/virologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Latência Viral , Animais , Modelos Animais de Doenças , Infecções por HIV/virologia , HumanosRESUMO
To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2-58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans.
Assuntos
Terapia Antirretroviral de Alta Atividade/métodos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/fisiologia , Alcinos , Animais , Benzoxazinas/uso terapêutico , Cromatografia Líquida de Alta Pressão , Ciclopropanos , Transcriptase Reversa do HIV/genética , HIV-1/genética , Macaca , Macaca mulatta , Inibidores da Transcriptase Reversa/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Viremia/patologiaRESUMO
With more than 40 million people living with human immunodeficiency virus (HIV), there is an urgent need to develop drugs that can be used in the form of a topical microbicide to prevent infection through sexual transmission. DCM205 is a recently discovered small-molecule inhibitor of HIV type 1 (HIV-1) that is able to directly inactivate HIV-1 in the absence of a cellular target. DCM205 is active against CXCR4-, CCR5-, and dual-tropic laboratory-adapted and primary strains of HIV-1. DCM205 binds to the HIV-1 envelope glycoprotein, and competition studies map the DCM205 binding at or near the V3 loop of gp120. Binding to this site interferes with the soluble CD4 interaction. With its ability to disable the virus particle, DCM205 represents a promising new class of HIV entry inhibitor that can be used as a strategy in the prevention of HIV-1/AIDS.
Assuntos
Fármacos Anti-HIV/farmacologia , Anti-Infecciosos Locais/farmacologia , HIV-1/efeitos dos fármacos , Pirogalol/análogos & derivados , Sulfonas/farmacologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Síndrome da Imunodeficiência Adquirida/transmissão , Antígenos CD4/efeitos dos fármacos , Produtos do Gene env/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Células HeLa , Humanos , Pirogalol/farmacologia , Produtos do Gene env do Vírus da Imunodeficiência HumanaRESUMO
In a previous study, we prepared a small library of chicoric acid analogs that possessed both potent anti-integrase and antiviral activity. It was also shown that active compounds fell into one of two groups: those that inhibited an early stage in viral replication and those that inhibited at a later stage. In this study, a series of vinyl geminal disulfone-containing compounds possessing a range of ring substituents has been synthesized to probe the impact of structure on inhibitory mechanisms. Four active compounds were identified using HIV drug susceptibility assays. Three of the inhibitors possessing either no substituents or electron-withdrawing substituents on the aromatic rings led to high levels of cytotoxicity and antiviral activity. Intrigued by the potential implications of electronic effects on activity, we probed whether the active compounds could be nonspecifically reacting via 1,4-addition. To investigate this hypothesis, the compounds were incubated with glutathione and upon LC/MS analysis, molecular ion peaks corresponding to both mono and double addition adducts were identified. Second, we synthesized analogs lacking the ability to participate in 1,4-addition and tested them for antiviral activity and cytotoxicity, and found the compounds inactive for both activities. Taken together, the studies reported herein suggest that compounds lacking electron-donating substituents on the aromatic ring are promiscuous acceptors of biological nucleophiles, whereas compounds possessing electron-donating substituents seem to resist addition or at least be more selective and significantly less toxic.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Inibidores de Integrase de HIV/síntese química , Inibidores de Integrase de HIV/farmacologia , HIV-1/efeitos dos fármacos , Sulfonas/síntese química , Sulfonas/farmacologia , Compostos de Vinila/síntese química , Compostos de Vinila/farmacologia , Fármacos Anti-HIV/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Integrase de HIV/química , Humanos , Imunoensaio , Indicadores e Reagentes , Espectrometria de Massas , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-AtividadeRESUMO
RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.
Assuntos
Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , RNA Viral/genética , RNA/metabolismo , Replicação Viral , Pareamento de Bases , Linhagem Celular , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/genética , Cinética , Regiões Promotoras Genéticas/genética , RNA/química , RNA/genética , RNA Nuclear Pequeno/genética , RNA Viral/química , RNA Viral/metabolismo , Fatores de Tempo , Transcrição Gênica/genéticaRESUMO
We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.
Assuntos
Terapia Antirretroviral de Alta Atividade , Modelos Animais de Doenças , Transcriptase Reversa do HIV , HIV-1 , Recombinação Genética , Vírus da Imunodeficiência Símia , Animais , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-1/genética , HIV-1/fisiologia , Humanos , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Vírus da Imunodeficiência Símia/enzimologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Resultado do Tratamento , Carga ViralRESUMO
Rhesus and human cytomegalovirus (RhCMV and HCMV, respectively) exhibit comparable inhibition by benzimidazole nucleosides, including 2,5,6-trichloro-(1-beta-d-ribofuranosyl)benzimidazole (TCRB), and pyrrolo[2,3-d]pyrimidines. The two HCMV protein targets of TCRB, UL89 and UL56, are highly conserved with their RhCMV homologues. These data indicate that infection of rhesus macaques with RhCMV represents a useful model to test novel anti-HCMV drugs.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Citomegalovirus/efeitos dos fármacos , Nucleosídeos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Sequência Conservada , Fibroblastos , Humanos , Macaca mulatta , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Ensaio de Placa ViralRESUMO
Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.
Assuntos
DNA Viral/metabolismo , HIV-1/genética , Transcrição Gênica , Uracila/metabolismo , Sequência de Bases , Sistema Livre de Células , Primers do DNA , Ribonuclease H/metabolismo , Moldes GenéticosRESUMO
We used a focal infectivity assay with HeLa H1-JC.37 cells to directly compare susceptibilities of simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) to protease inhibitors. SIVmac239 was inhibited by indinavir, saquinavir, and ritonavir, with 50% effective concentrations (means +/- standard deviations) of 39 +/- 8, 55 +/- 3, and 13 +/- 5 nM, respectively. The corresponding values for inhibition of HIV-1 were 66 +/- 4, 47 +/- 10, and 25 +/- 14 nM, respectively.
Assuntos
Inibidores da Protease de HIV/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , Células HeLa , Humanos , Indinavir/farmacologia , Ritonavir/farmacologia , Saquinavir/farmacologiaRESUMO
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2. Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated. We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs. On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT. The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme. Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes. The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased. This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes. Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors.
Assuntos
Resistência Microbiana a Medicamentos/fisiologia , Transcriptase Reversa do HIV/metabolismo , Vírus da Imunodeficiência Felina/enzimologia , Inibidores da Transcriptase Reversa/farmacologia , Sequência de Aminoácidos , Nucleotídeos de Desoxiguanina/química , Nucleotídeos de Desoxiguanina/farmacologia , Didesoxinucleotídeos , Escherichia coli , Foscarnet/química , Foscarnet/farmacologia , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , Humanos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Cinética , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Nevirapina/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Inibidores da Transcriptase Reversa/química , Homologia de Sequência de AminoácidosRESUMO
The methionine-to-valine mutation in codon 184 (M184V) in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) confers resistance to (-)-2'-deoxy-3'-thiacytidine (3TC; lamivudine) and increased sensitivity to 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir). We have used the SIV model to evaluate the effect of the M184V mutation on the emergence of resistance to the combination of 3TC plus PMPA. A site-directed mutant of SIVmac239 containing M184V (SIVmac239-184V) was used to select for resistance to both 3TC and PMPA by serial passage in the presence of increasing concentrations of both drugs. Under these selection conditions, the M184V mutation reverted in the majority of the selections. Variants resistant to both drugs were found to have the lysine-to-arginine mutation at codon 65 (K65R), which has previously been associated with resistance to PMPA in both SIV and HIV. Similarly, in rhesus macaques infected with SIVmac239-184V for 46 weeks and treated daily with (-)-2'-deoxy-5-fluoro-3'-thiacytidine [(-)-FTC], there was no reversion of M184V, but this mutation reverted to 184 M in all three animals within 24 weeks of treatment with (-)-FTC and PMPA. Although the addition of PMPA to the (-)-FTC therapy induced a decrease in virus loads in plasma, these loads eventually returned to pre-PMPA levels in each case. All animals receiving this combination developed the K65R mutation. These results demonstrate that the combination of PMPA with 3TC or (-)-FTC selects for the K65R mutation and against the M184V mutation in SIV RT.
Assuntos
Adenina/análogos & derivados , Adenina/farmacologia , Lamivudina/farmacologia , Mutação , Organofosfonatos , Compostos Organofosforados/farmacologia , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa/farmacologia , Vírus da Imunodeficiência Símia/enzimologia , Sequência de Bases , Linhagem Celular , Primers do DNA , Farmacorresistência Viral , Mutagênese Sítio-Dirigida , Vírus da Imunodeficiência Símia/efeitos dos fármacos , TenofovirRESUMO
The specificity of nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for the RT of human immunodeficiency virus type 1 (HIV-1) has prevented the use of simian immunodeficiency virus (SIV) in the study of NNRTIs and NNRTI-based highly active antiretroviral therapy. However, a SIV-HIV-1 chimera (RT-SHIV), in which the RT from SIVmac239 was replaced with the RT-encoding region from HIV-1, is susceptible to NNRTIs and is infectious to rhesus macaques. We have evaluated the antiviral activity of efavirenz against RT-SHIV and the emergence of efavirenz-resistant mutants in vitro and in vivo. RT-SHIV was susceptible to efavirenz with a mean effective concentration of 5.9 +/- 4.5 nM, and RT-SHIV variants selected with efavirenz in cell culture displayed 600-fold-reduced susceptibility. The efavirenz-resistant mutants of RT-SHIV had mutations in RT similar to those of HIV-1 variants that were selected under similar conditions. Efavirenz monotherapy of RT-SHIV-infected macaques produced a 1.82-log-unit decrease in plasma viral-RNA levels after 1 week. The virus load rebounded within 3 weeks in one treated animal and more slowly in a second animal. Virus isolated from these two animals contained the K103N and Y188C or Y188L mutations. The RT-SHIV-rhesus macaque model may prove useful for studies of antiretroviral drug combinations that include efavirenz.