RESUMO
Quinolone-3-carboxylic acid represents a highly privileged chemotype in medicinal chemistry and has been extensively explored as antibiotics and antivirals targeting human immunodeficiency virus (HIV) integrase (IN). Herein we describe the synthesis and anti-hepatitis C virus (HCV) profile of a series of C-6 aryl substituted 4-quinlone-3-carboxylic acid analogues. Significant inhibition was observed with a few analogues at low micromolar range against HCV replicon in cell culture and a reduction in replicon RNA was confirmed through an RT-qPCR assay. Interestingly, evaluation of analogues as inhibitors of NS5B in a biochemical assay yielded only modest inhibitory activities, suggesting that a different mechanism of action could operate in cell culture.
Assuntos
4-Quinolonas/farmacologia , Antivirais/farmacologia , Ácidos Carboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , 4-Quinolonas/síntese química , 4-Quinolonas/química , Antivirais/síntese química , Antivirais/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hepacivirus/genética , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Reação em Cadeia da Polimerase em Tempo Real , Replicon/genética , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: Adeno-associated virus type 2 (AAV) has the ability to target integration of its DNA into a specific locus of the human genome. Site-specific AAV integration is mediated by viral Rep proteins, although the role of cellular factors involved in this process is largely unknown. Recent studies provide evidence showing that cellular DNA repair proteins are involved in targeted integration of AAV, although their specific roles are not well defined. METHODS: In the present study, we investigated the interaction between Rep and proteins of the back-up nonhomologous end-joining pathway (B-NHEJ). We then analyzed the effect of one of these proteins, poly(ADP-ribose) polymerase 1 (PARP1) on AAV integration. RESULTS: We show that AAV Rep interacts with B-NHEJ members DNA ligase III and PARP1 but does not associate with the scaffolding factor XRCC1. Moreover, PARP1 and Rep bind directly and not via DNA-protein interactions. We also found that Rep increases the enzymatic activity of PARP1 potentially through the endonuclease activity of Rep. Finally, we demonstrate that both chemical inhibition of PARP1 and PARP1 depletion using small hairpin RNA enhance integration of the AAV genome in HeLa cells. CONCLUSIONS: The findings of the present study indicate that manipulation of PARP1 activity could be used as a tool for developing new, effective AAV-based therapies for the treatment of genetic diseases and cancer.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Dependovirus/genética , Genoma Viral/genética , Poli(ADP-Ribose) Polimerases/fisiologia , Proteínas Virais/fisiologia , Integração Viral/fisiologia , Southern Blotting , Western Blotting , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos/genética , Células HeLa , Humanos , Imunoprecipitação , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/metabolismo , Reação em Cadeia da Polimerase , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Epstein-Barr virus (EBV) causes a range of life-threatening B-lymphocyte malignancies but, despite the use of various strategies, treatment remains problematic. METHODS: In the present study, we developed a non-integrating lentiviral vector (NILV) that mediates specific killing of EBV nuclear antigen 1 (EBNA1)-expressing cells with minimal toxicity to EBNA1-negative cells. The EBV family of repeats (FR) was cloned intok the NILV genome upstream of various transgenes. RESULTS: The presence of the FR in the NILV genome induced transcriptional up-regulation and prolonged the expression of a transgene specifically in EBNA1-positive B cells. Transgene expression from an FR-containing NILV was also prolonged in EBV-transformed cells compared to an FR-negative NILV. We found that the delivery of an FR-containing NILV encoding herpes simplex virus 1 thymidine kinase (TK) lead to the killing of more than 99% of EBNA1-positive B cells with minimal toxicity to EBNA1-negative cells in the presence of gancyclovir. EBNA1-positive cells were not killed by an FR-negative vector containing the TK gene. An FR-TK-containing NILV also specifically killed EBNA1-containing cells in a mixed population of EBNA1-positive and EBNA1-negative cells, thus confirming that NILV-FR-TK-mediated killing is specific for EBNA1-expressing cells. CONCLUSIONS: Transgene expression from our NILVs is both EBNA1-specific and dependent upon the presence of the FR. The results obtained in the present study indicate that NILVs have potential use in the treatment of EBV-associated B cell malignancies.
Assuntos
Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Genes Transgênicos Suicidas , Vetores Genéticos/genética , HIV-1/genética , Linfoma de Células B/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ganciclovir/farmacologia , Regulação da Expressão Gênica , Ordem dos Genes , Técnicas de Transferência de Genes , Terapia Genética , Células HEK293 , Humanos , Linfoma de Células B/virologia , Timidina Quinase/genética , Transcrição Gênica , Transdução Genética , Regulação para Cima/genéticaRESUMO
Adeno-associated viral (AAV) vectors preferentially integrate into the genome of cells that are defective in DNA repair, such as occurs with DNA-PKcs deficiency or poly(ADP-ribose) polymerase-1 down-regulation. As the tumor suppressor protein p53 regulates the transcription of many genes involved in DNA repair, we sought to determine whether functional p53 affects the efficiency of AAV integration. p53 is mutated in more than 50% of cancers, and site-specific integration of AAV into the AAVS1 site of human chromosome 19 has frequently been observed in transformed cancer cell lines, but rarely seen in primary cells or in vivo. We therefore hypothesized that p53-negative cells would be more permissive to AAV integration than p53-positive cells. The integration of a rep- and green fluorescent protein-encoding recombinant AAV vector was quantified in p53-expressing and p53-deficient HCT116 colon cancer cells. Our results show that there is a higher efficiency of AAV integration in p53-negative cells compared with p53-positive cells, indicating that p53 does indeed inhibit AAV integration. Further experiments suggest that this p53-mediated block to AAV integration is likely to be through binding of p53 to the AAV Rep protein and the consequent inhibition of Rep activity during AAV integration.