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1.
Viruses ; 12(8)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32752057

RESUMO

Hepatitis B virus (HBV) polymerase seems to be very hard to express and purify sufficiently, which has long hampered the generation of anti-HBV drugs based on the nature of the polymerase. To date, there has been no useful system developed for drug screening against HBV polymerase. In this study, we successfully obtained a highly purified reverse transcriptase (RT) domain of the polymerase, which has a template/primer and substrate binding activity, and established a novel high-throughput screening (HTS) system using purified RT protein for finding novel polymerase inhibitors. To examine whether the assay system provides reliable results, we tested the small scale screening using pharmacologically active compounds. As a result, the pilot screening identified already-known anti-viral polymerase agents. Then, we screened 20,000 chemical compounds and newly identified four hits. Several of these compounds inhibited not only the HBV RT substrate and/ template/primer binding activity, but also Moloney murine leukemia virus RT activity, which has an elongation activity. Finally, these candidates did show to be effective even in the cell-based assay. Our screening system provides a useful tool for searching candidate inhibitors against HBV.


Assuntos
Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Células Hep G2 , Vírus da Hepatite B/enzimologia , Ensaios de Triagem em Larga Escala , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , DNA Polimerase Dirigida por RNA , Bibliotecas de Moléculas Pequenas , Replicação Viral/efeitos dos fármacos
2.
PLoS One ; 13(5): e0197664, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29782545

RESUMO

Current therapeutics for hepatitis B virus (HBV) patients such as nucleoside analogs (NAs) are effective; however, new antiviral drugs against HBV are still desired. Since the interaction between the epsilon (ε) sequence of HBV pregenomic RNA and viral polymerase (Pol) is a key step in the HBV replication cycle, we aimed to identify small compounds for its inhibition, and established a pull-down assay system for the detection of ε-RNA-binding-Pol. Screening showed that 5 out of 3,965 compounds inhibited ε-Pol binding, and we identified rosmarinic acid, which exhibited specificity, as a potential antiviral agent. In order to examine the anti-HBV effects of rosmarinic acid, HBV-infected primary human hepatocytes from a humanized mouse liver were treated with rosmarinic acid. The rosmarinic acid treatment decreased HBV components including the amounts of extracellular HBV DNA with negligible cytotoxicity. We also investigated the combined effects of rosmarinic acid and the NA, lamivudine. rosmarinic acid slightly enhanced the anti-HBV activity of lamivudine, suggesting that the HBV replication step targeted by rosmarinic acid is distinct from that of NA. We analyzed an additional 25 rosmarinic acid derivatives, and found that 5 also inhibited ε-Pol. Structural comparisons between these derivatives implied that the "two phenolic hydroxyl groups at both ends" and the "caffeic acid-like structure" of rosmarinic acid are critical for the inhibition of ε-Pol binding. Collectively, our results demonstrate that rosmarinic acid inhibits HBV replication in HBV-infected cells by specifically targeting ε-Pol binding.


Assuntos
Antivirais/farmacologia , Cinamatos/farmacologia , Depsídeos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Células Cultivadas , Cinamatos/administração & dosagem , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Depsídeos/administração & dosagem , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/metabolismo , Células HEK293 , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Lamivudina/administração & dosagem , Camundongos , Quercetina/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Ácido Rosmarínico
3.
Bioorg Med Chem ; 26(8): 1573-1578, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29459146

RESUMO

In this study, a series of 2-arylthio-5-iodo pyrimidine derivatives, as non-nucleoside hepatitis B virus inhibitors, were evaluated and firstly reported as potential anti-HBV agents. To probe the mechanism of active agents, DHBV polymerase was isolated and a non-radioisotopic assay was established for measuring HBV polymerase. The biological results demonstrated that 2-arylthio-5-iodo pyrimidine derivatives targeted HBV polymerase. In addition, pharmacophore models were constructed for future optimization of lead compounds. Further study will be performed for the development of non-nucleoside anti-HBV agents.


Assuntos
Antivirais/farmacologia , Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Pirimidinas/farmacologia , Compostos de Sulfidrila/farmacologia , Animais , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Patos , Produtos do Gene pol/metabolismo , Células Hep G2 , Humanos , Fígado/virologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Inibidores da Transcriptase Reversa , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química
4.
Minerva Med ; 108(6): 507-512, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28889724

RESUMO

BACKGROUND: To observe the effect of the combined administration of entecavir and adefovir dipivoxil to improve hepatic fibrosis in hepatitis B patients with interferon resistance. METHODS: This study comprised 90 hepatitis B patients with hepatic fibrosis and interferon (IFN) resistance who were admitted in the hospital's department of infectious disease for diagnosis and treatment between January 2013 and September 2015. They were randomly divided into two groups in accordance with the random number table: the combination treatment group (N.=45) and the entecavir group (N.=45). They were observed for any variations in the indexes of liver function and fibrosis, as well as the Model for end-stage liver disease (MELD) scores, before and after treatment. RESULTS: After treatment, the levels of the indexes in both groups (the combination treatment group vs. the entecavir group) were as follows: bilirubin (67.5±7.7 vs. 82.4±13.5 µmol/L); International Normalized Ratio (INR) (1.21±0.8 vs. 1.14±0.7); creatinine (147.3±12.4 vs. 287.4±21.6 mg/dL); GGT (67.4±23.2 vs. 88.4±23.7 U/L); and ALT (63.4±40.8 vs. 96.5±23.5 U/L). In comparison of the indexes of hepatic fibrosis between the two groups, we found the following differences: PCIII (67.5±7.7 vs. 82.4±13.5 µg/L); IV-C (61.3±18.7 vs. 74.5±17.9 µg/L); HA (147.3±12.4 vs. 87.4±31.6 µg/L); and LN (88.7±13.2 vs 102.5±23.4 µg/L). The results showed that the differences in comparison of the indexes before and after the treatment were statistically significant (P<0.05). After treatment, the MELD score of patients in the combination treatment group was significantly lower than that in the entecavir group (18.7±3.2 vs. 22.5±3.4), with a statistically significant difference (P<0.05). CONCLUSIONS: In the chronic hepatitis B patients with interferon resistance, the combined administration of entecavir and adefovir dipivoxil can significantly improve liver function, hepatic fibrosis and MELD scores. The results highlight the need to promote the benefits of this drug combination in helping chronic hepatitis B patients with interferon resistance, and to promote its application in clinical practices.


Assuntos
Adenina/análogos & derivados , Antivirais/uso terapêutico , Guanina/análogos & derivados , Hepatite B Crônica/complicações , Cirrose Hepática/tratamento farmacológico , Organofosfonatos/uso terapêutico , Inibidores da Transcriptase Reversa/uso terapêutico , Adenina/administração & dosagem , Adenina/uso terapêutico , Idoso , Alanina Transaminase/sangue , Antivirais/administração & dosagem , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Creatinina/sangue , Farmacorresistência Viral , Quimioterapia Combinada , Doença Hepática Terminal/etiologia , Doença Hepática Terminal/prevenção & controle , Feminino , Produtos do Gene pol/antagonistas & inibidores , Guanina/administração & dosagem , Guanina/uso terapêutico , Hepatite B Crônica/tratamento farmacológico , Humanos , Interferons/farmacologia , Interferons/uso terapêutico , Cirrose Hepática/sangue , Cirrose Hepática/etiologia , Masculino , Pessoa de Meia-Idade , Organofosfonatos/administração & dosagem , Inibidores da Transcriptase Reversa/administração & dosagem , Índice de Gravidade de Doença
5.
Artigo em Inglês | MEDLINE | ID: mdl-28396546

RESUMO

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATP (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Didanosina/uso terapêutico , Produtos do Gene pol/antagonistas & inibidores , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Inibidores da Transcriptase Reversa/uso terapêutico , Zalcitabina/uso terapêutico , Zidovudina/uso terapêutico , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , Vírus da Hepatite B/genética , Humanos , Mutação/genética , Conformação Proteica , Estrutura Quaternária de Proteína
7.
Cell Host Microbe ; 17(3): 285-287, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25766287

RESUMO

The pathogen sensor RIG-I recognizes viral RNA and signals to induce an antiviral response. In this issue of Cell Host & Microbe, Weber et al. (2015), along with recent work by Sato et al. (2015), demonstrate that RIG-I directly inhibits viral replication independent of antiviral signaling.


Assuntos
Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatócitos/fisiologia , Fígado/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Viral/imunologia , Animais , Feminino , Humanos
8.
Immunity ; 42(1): 123-32, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25557055

RESUMO

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.


Assuntos
Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatócitos/fisiologia , Fígado/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Viral/imunologia , Animais , Pré-Escolar , Feminino , Células Hep G2 , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Imunidade Inata , Interferons/metabolismo , Fígado/virologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/imunologia , RNA Viral/genética , Receptores de Superfície Celular , Transgenes/genética , Quimeras de Transplante , Replicação Viral/genética
9.
Antivir Ther ; 20(4): 387-95, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25624410

RESUMO

BACKGROUND: Nucleoside/nucleotide analogue (NA) treatment causes selection pressure for HBV strains carrying mutations conferring NA resistance. Drug-resistance mutations occur in the reverse transcriptase (RT) region of the HBV polymerase gene and spontaneously arise during viral replication. These mutations can also alter the hepatitis B surface (HBs) protein and in some cases reduce binding to HBs antibodies. The spread of NA-resistant HBV may impact the efficacy of antiviral treatment and hepatitis B immunization programmes. In this study, we used direct sequencing to assess the occurrence of HBV carrying known mutations that confer NA resistance in the largest cohort of treatment-naive patients with chronic hepatitis B (CHB) to date. METHODS: HBV DNA samples isolated from 702 patients were sequenced and the RT region subjected to mutational analysis. RESULTS: There was high genetic variability among the HBV samples analysed: A1 (63.7%), D3 (14.5%), A2 (3.3%), A3 (0.1%), B1 (0.1%), B2 (0.1%), C2 (0.9%), D1 (0.9%), D2 (4.6%), D4 (5.1%), D unclassified subgenotype (0.7%), E (0.6%), F2a (4.6%), F4 (0.4%) and G (0.4%). HBV strains harbouring mutations conferring NA resistance alone or combined with compensatory mutations were identified in 1.6% (11/702) of the patients. CONCLUSIONS: HBV strains harbouring resistance mutations can comprise the major population of HBV quasispecies in treatment-naive patients. In Brazil, there is a very low frequency of untreated patients who are infected with these strains. These findings suggest that the spread and natural selection of drug-resistant HBV is an uncommon event and/or most of these strains remain unstable in the absence of NA selective pressure.


Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Produtos do Gene pol/genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/virologia , Mutação , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Brasil , DNA Viral/genética , DNA Viral/imunologia , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/metabolismo , Genótipo , Guanina/análogos & derivados , Guanina/farmacologia , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/imunologia , Humanos , Lamivudina/farmacologia , Testes de Sensibilidade Microbiana , Organofosfonatos/farmacologia , Estudos Retrospectivos , Análise de Sequência de DNA , Tenofovir/farmacologia , Replicação Viral/efeitos dos fármacos
10.
Nat Rev Immunol ; 15(2): 70-1, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25598531
11.
Intervirology ; 57(3-4): 171-80, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25034485

RESUMO

Since the licensing of lamivudine in 1999, the treatment of chronic hepatitis B has been revolutionized by the introduction of oral nucleoside and nucleotide analogues (NAs), which act as inhibitors of the HBV polymerase. The effectiveness of the first of these substances was limited by incomplete response and resistance development in many patients, but today, highly potent substances are available that make a reliable and durable suppression of HBV replication, a reduction of necroinflammatory activity in the liver, and even a reversion of liver fibrosis achievable for almost all patients. Beyond that, NA treatment can prevent the development of hepatocellular carcinoma in many patients. HBeAg seroconversion appears in approximately 50% of all HBeAg-positive patients during NA treatment. However, the ideal treatment endpoint, the serologic loss of HBsAg, remains a rare event almost exclusively achievable for HBeAg-positive patients. After cessation of the treatment, HBV replication tends to relapse in most patients, which is why the duration of NA treatment is indefinite. Future treatment strategies should aim at tailoring individual NA treatment regimens to increase HBs loss rates and optimize treatment duration.


Assuntos
Antivirais/uso terapêutico , Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Adenina/análogos & derivados , Adenina/uso terapêutico , Arabinofuranosiluracila/análogos & derivados , Arabinofuranosiluracila/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/prevenção & controle , Guanina/análogos & derivados , Guanina/uso terapêutico , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Lamivudina/uso terapêutico , Fígado/patologia , Fígado/virologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/virologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/prevenção & controle , Organofosfonatos/uso terapêutico , DNA Polimerase Dirigida por RNA/efeitos dos fármacos , Telbivudina , Tenofovir , Timidina/análogos & derivados , Timidina/uso terapêutico , Resultado do Tratamento , Replicação Viral/efeitos dos fármacos
12.
PLoS One ; 9(5): e97579, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24827459

RESUMO

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.


Assuntos
Antivirais/metabolismo , Endonucleases/genética , Produtos do Gene pol/genética , Marcação de Genes , Vírus da Hepatite B/genética , Transativadores/genética , Proteínas do Core Viral/genética , Antivirais/química , Sequência de Bases , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Viral/genética , Dependovirus/genética , Endonucleases/química , Endonucleases/metabolismo , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/química , Vetores Genéticos , Células HEK293 , Vírus da Hepatite B/química , Hepatócitos/virologia , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas , Transativadores/antagonistas & inibidores , Transativadores/química , Proteínas do Core Viral/antagonistas & inibidores , Proteínas do Core Viral/química , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genética , Dedos de Zinco/genética
13.
Bing Du Xue Bao ; 30(6): 713-20, 2014 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-25868288

RESUMO

Clinically being applied treatment against chronic hepatitis has three limitations: low response rates, severe adverse effects and a high rate of drug resistance. Hence, novel targets for antiviral therapy need to be developed so as to provide an armory of different strategies. During the replication of hepatitis B virus, the interaction of viral polymerase (P protein, also called P) and epsilonRNA is indispensable for the initiation of reverse transcription via protein priming and the pregenome RNA (pgRNA) packaging. Three strategies are currently developed for blocking P-epsilon interaction: heat shock protein inhibitors, epsilonaptamers and chemical compounds for blocking formation of P-epsilon complex. Previously, our group has for the first time worldwide in vitro screened several aptamers, which are able to interfere with the P-epsilon interaction. A strong inhibition against HBV was observed in vitro and in vivo experiments, respectively. In conclusion, the so far developed chemicals suppressing the P-epsilon interaction may bypass or overcome the viral resistance problems during clinic treatment and represent a highly attractive option for therapeutic intervention.


Assuntos
Produtos do Gene pol/metabolismo , Vírus da Hepatite B/enzimologia , Hepatite B/virologia , RNA Viral/metabolismo , Animais , Regulação Viral da Expressão Gênica , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/genética , Hepatite B/terapia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , RNA Viral/genética , Replicação Viral
14.
Antiviral Res ; 96(2): 234-44, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22921338

RESUMO

Enterovirus 71 (EV 71) and Coxsackievirus A16 (CA 16) are two major causative agents of hand, foot and mouth disease (HFMD). They have been associated with severe neurological and cardiological complications worldwide, and have caused significant mortalities during large-scale outbreaks in China. Currently, there are no effective treatments against EV 71 and CA 16 infections. We now describe the development of a novel minicircle vector based RNA interference (RNAi) system as a therapeutic approach to inhibiting EV 71 and CA 16 replication. Small interfering RNA (siRNA) molecules targeting the conserved regions of the 3C(pro) and 3D(pol) function gene of the EV 71 and CA 16 China strains were designed based on their nucleotide sequences available in GenBank. This RNAi system was found to effectively block the replication and gene expression of these viruses in rhabdomyosarcoma (RD) cells and virus-infected mice model. The inhibitory effects were confirmed by a corresponding decrease in viral RNA, viral protein, and progeny virus production. In addition, no significant adverse off-target silencing or cytotoxic effects were observed. These results demonstrated the potential and feasibility of this novel minicircle vector based RNAi system for antiviral therapy against EV 71 and CA 16 infection.


Assuntos
Enterovirus Humano A/fisiologia , Enterovirus/fisiologia , Regulação Viral da Expressão Gênica , Vetores Genéticos , Replicação Viral , Animais , Produtos Biológicos/administração & dosagem , Linhagem Celular , Modelos Animais de Doenças , Enterovirus/genética , Enterovirus Humano A/genética , Produtos do Gene pol/antagonistas & inibidores , Inativação Gênica , Doença de Mão, Pé e Boca/terapia , Humanos , Camundongos , Peptídeo Hidrolases/metabolismo , RNA Interferente Pequeno/genética , Resultado do Tratamento
15.
J Hepatol ; 55(6): 1235-40, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21703180

RESUMO

BACKGROUND & AIMS: Therapy of chronic hepatitis B with HBV-polymerase inhibitors, in particular tenofovir or adefovir, may affect renal function. To assess renal function more accurately in the normal range, we used the recently validated, Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula to calculate the estimated glomerular filtration rate (eGFR). METHODS: Patient subgroups included: patients with HBV-monoinfection treated with lamivudine (n=36), adefovir (n=32), entecavir (n=32), or tenofovir (n=37). HBsAg-positive untreated patients (n=60) served as control. For comparison HIV-monoinfected patients treated with tenofovir (n=120) or zidovudine (n=52) based antiretroviral therapy and antiretroviral naive patients (n=109) were assessed. CKD-EPI equation was used to calculate eGFR. In a more sensitive approach, we modeled the individual change in eGFR over time with linear mixed effects models (LME). RESULTS: Yearly predicted median changes in individual eGFR according to the LME model were: HBV untreated -2.05 ml/min, HBV lamivudine -0.92 ml/min, HBV adefovir -1.02 ml/min, HBV entecavir -1.00 ml/min, and HBV tenofovir -0.92 ml/min (p<0.01 for HBV untreated vs. HBV treated). In HIV-monoinfected patients: HIV untreated -0.62 ml/min, HIV treated with tenofovir -2.64 ml/min, HIV treated with zidovudine -1.0 ml/min (p=0.017 for tenofovir vs. no treatment, p<0.001 for tenofovir vs. zidovudine). CONCLUSIONS: Therapy of HBV infection irrespective of medication seems to result in a milder decrease of renal function. In contrast tenofovir as part of HIV combination therapy seems to impair renal function in this Caucasian population.


Assuntos
Produtos do Gene pol/antagonistas & inibidores , Taxa de Filtração Glomerular/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/fisiopatologia , Inibidores da Transcriptase Reversa/uso terapêutico , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/uso terapêutico , Adolescente , Adulto , Idoso , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/uso terapêutico , Antivirais/efeitos adversos , Antivirais/uso terapêutico , Estudos de Casos e Controles , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/fisiopatologia , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Organofosfonatos/efeitos adversos , Organofosfonatos/uso terapêutico , Inibidores da Transcriptase Reversa/efeitos adversos , Tenofovir , Adulto Jovem
16.
Minerva Med ; 100(6): 447-58, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20010480

RESUMO

Treatment of chronic hepatitis B has shown a rapid development in the last years leading to a shift of treatment strategies from interferon to hepatitis B virus (HBV)-polymerase inhibitors. In particular, treatment with HBV-polymerase inhibitors has changed the indication on how to treat a patient and when to stop therapy. Long-term treatment with HBV-polymerase inhibitors may often be required, even if it raises the possibility of resistance and subsequent treatment failure. This review provides a strategy on how to manage HBV therapy with the currently available treatment options.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Hepatite B Crônica/tratamento farmacológico , Quimioterapia Combinada , Produtos do Gene pol/antagonistas & inibidores , Infecções por HIV/tratamento farmacológico , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Humanos , Interferons/uso terapêutico , Lamivudina/uso terapêutico , Replicação Viral
17.
Expert Rev Anti Infect Ther ; 7(9): 1053-62, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19883325

RESUMO

Hepatitis B virus is a major cause of chronic liver disease worldwide and is associated with an increased risk of hepatocellular carcinoma, progressive hepatic fibrosis and end-stage liver disease. Suppression of HBV replication is recognized as the primary on-treatment goal of antiviral therapy, as reduction of serum HBV DNA to low or undetectable levels is highly likely to have a positive impact on long-term clinical outcomes in HBV-associated chronic liver disease. Entecavir is an oral nucleoside analogue that effectively inhibits HBV polymerase, resulting in rapid viral suppression. Long-term data on patients receiving entecavir for chronic hepatitis B have demonstrated high potency, a low incidence of antiviral drug resistance and good tolerability, making entecavir an ideal first-line agent for the treatment of chronic hepatitis B.


Assuntos
Antivirais/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Produtos do Gene pol/antagonistas & inibidores , Guanina/análogos & derivados , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Antivirais/química , Antivirais/farmacologia , Farmacorresistência Viral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Guanina/administração & dosagem , Guanina/química , Guanina/farmacologia , Vírus da Hepatite B/enzimologia , Humanos , Assistência de Longa Duração , Ensaios Clínicos Controlados Aleatórios como Assunto , Resultado do Tratamento , Carga Viral/efeitos dos fármacos
18.
Antiviral Res ; 81(2): 147-55, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19028525

RESUMO

Telbivudine, a nucleoside analog inhibitor of the viral polymerase of hepatitis B virus (HBV), has been approved for the treatment of chronic HBV infection, along with the nucleoside inhibitors lamivudine and entecavir, and the nucleotide inhibitors adefovir and tenofovir. The resistance profiles of these agents were investigated via drug treatment of HepG2 cells stably transfected with wild-type or mutant HBV genomes bearing known resistance mutations. Telbivudine was not active against HBV strains bearing lamivudine mutations L180M/M204V/I but remained active against the M204V single mutant in vitro, potentially explaining the difference in resistance profiles between telbivudine and lamivudine. Against HBV genomes with known telbivudine-resistance mutations, M204I and L80I/M204I, telbivudine, lamivudine and entecavir lost 353- to >1000-fold activity whereas adefovir and tenofovir exhibited no more than 3-5-fold change. Conversely, against HBV cell lines expressing adefovir resistance mutations N236T and A181V, or the A194T mutant associated with resistance to tenofovir, telbivudine remained active as shown by respective fold-changes of 0.5 (N236T) and 1.0 (A181V and A194T). These in vitro results indicate that nucleoside and nucleotide drugs have different cross-resistance profiles. The addition of telbivudine to ongoing adefovir therapy could provide effective antiviral therapy to patients who develop adefovir resistance.


Assuntos
Adenina/análogos & derivados , Antivirais/farmacologia , Farmacorresistência Viral , Vírus da Hepatite B/efeitos dos fármacos , Nucleosídeos/farmacologia , Organofosfonatos/farmacologia , Pirimidinonas/farmacologia , Adenina/farmacologia , Substituição de Aminoácidos/genética , Linhagem Celular Tumoral , Produtos do Gene pol/antagonistas & inibidores , Produtos do Gene pol/genética , Humanos , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Telbivudina , Tenofovir , Timidina/análogos & derivados
19.
Enferm Infecc Microbiol Clin ; 26 Suppl 7: 49-55, 2008 May.
Artigo em Espanhol | MEDLINE | ID: mdl-19100231

RESUMO

The development hepatitis B virus (HBV) polymerase inhibitors has revolutionised the treatment of chronic HBV infection. However, the emergence of resistance mutations can compromise their clinical efficacy and it is mandatory to know the mechanisms of these resistances, its clinical implications, strategies for prevention and how to deal with the rescue. Since HBV has a high degree of replication and a high error rate, during their life cycle it will produce a large number of punctual mutations in individuals with active replication. Due to the large size of the HBV genome, all the possible changes may occur daily and should be screened before starting any antiviral therapy. Therefore, in individuals infected with HBV there is a mixture of similar viruses that evolves over time (quasispecies), some of which are carriers of resistance mutations to antivirals, which explains why they can be selected quickly after exposure to drug. Of the five drugs approved in Europe for the treatment of hepatitis B, three of them (lamivudine, adefovir and entecavir) are likely to be affected directly by these mutations, as well as other active drugs, such as telbivudine, tenofovir and the emtricitabine. The characterization of the resistance mutations is helpful for the prevention and the optimization of antiviral therapies.


Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral , Produtos do Gene pol/antagonistas & inibidores , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B Crônica/tratamento farmacológico , Inibidores da Transcriptase Reversa/uso terapêutico , Adenina/administração & dosagem , Adenina/análogos & derivados , Adenina/farmacologia , Adenina/uso terapêutico , Antivirais/administração & dosagem , Antivirais/farmacologia , Farmacorresistência Viral Múltipla/genética , Farmacorresistência Viral/genética , Quimioterapia Combinada , Produtos do Gene pol/química , Produtos do Gene pol/genética , Genótipo , Guanina/administração & dosagem , Guanina/análogos & derivados , Guanina/farmacologia , Guanina/uso terapêutico , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Humanos , Lamivudina/administração & dosagem , Lamivudina/farmacologia , Lamivudina/uso terapêutico , Modelos Moleculares , Organofosfonatos/administração & dosagem , Organofosfonatos/farmacologia , Organofosfonatos/uso terapêutico , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/farmacologia , Tenofovir , Resultado do Tratamento , Replicação Viral/efeitos dos fármacos
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