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Potencial do inibidor de farnesil-transferase Lonafarnib na terapêutica da infeção pelo vírus da hepatite delta
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Resumo(s)
O vírus da Hepatite Delta (HDV) é o vírus hepatotrópico responsável pela forma mais severa de hepatite viral. O HDV co-infeta ou super-infeta hepatócitos previamente infetados pelo vírus da hepatite B (HBV), aumentando o risco de hepatite fulminante, desenvolvimento de cirrose e carcinoma hepatocelular (CHC). A associação existente entre estes dois vírus explica-se pelo facto do invólucro externo do vírus da hepatite delta ser constituído por proteínas de superfície do HBV (HBsAgs). Considera-se assim que o HDV é um vírus satélite do HBV.
O genoma do HDV é constituído por uma única molécula de RNA circular de cadeia simples, codificando uma única proteína, o antigénio delta (HDAg). Através de um mecanismo de editing do RNA, obtém-se duas formas do HDAg, a pequena (S-HDAg) e a grande (L-HDAg). Ambas as proteínas virais desempenham diferentes funções no ciclo do HDV, o S-HDAg estimula a replicação do RNA viral, enquanto o L-HDAg é responsável pela inibição da replicação e pela interação com os HBsAgs para formar partículas virais.
Nos dias de hoje, não existe uma terapia específica para tratamento de doentes com infeção HDV e vários estudos têm sido efetuados no sentido de serem desenvolvidas novas terapias que visam eliminar esta infeção. A necessidade de encontrar novas opções terapêuticas levou ao desenvolvimento de fármacos dirigidos a moléculas específicas com relevância para o ciclo do HDV. O L-HDAg sofre um mecanismo de prenilação, importante para a interação com os HBsAgs e que é catalisado por enzimas celulares do grupo das farnesil-transferases. Assim, a utilização de inibidores desta enzima celular poderá constituir uma abordagem terapêutica da infeção provocada por HDV.
Neste trabalho desenvolvemos um estudo em que se avaliou o potencial terapêutico do inibidor de farnesil-transferases Lonafarnib em células Huh7D12, avaliando o impacto em termos de viabilidade celular e correlacionando a capacidade do fármaco intervir nos níveis de expressão do genoma viral do HDV. Avaliamos ainda a ação deste inibidor sobre a expressão do oncogene STAT3, oncogene este que está intimamente associado ao desenvolvimento de CHC.
A análise dos efeitos em termos de viabilidade celular demonstrou que o Lonafarnib apresenta uma citotoxicidade muito inferior aos restantes inibidores de farnesil-transferases previamente estudados no nosso laboratório. Verificou-se que o Lonafarnib interfere no ciclo de vida do HDV, provocando um aumento da expressão do RNA genómico do HDV nas células de hepatocarcinoma humano e influenciando a estabilidade do L-HDAg quando expresso isoladamente. Observou-se ainda, quer por análise de RT-PCR quer por Western Blot, que tratamentos com Lonafarnib conduzem a um aumento da expressão de STAT3 e CCND1.
Em conclusão, os resultados obtidos neste trabalho mostram que o Lonafarnib constitui uma nova abordagem terapêutica com grande potencial no tratamento da infeção por HDV, mas que são necessários estudos moleculares adicionais para analisar a existência de enventuais efeitos secundários negativos, tais como o aumento da expressão do RNA genómico do HDV e do oncogene STAT3.
Hepatitis Delta virus (HDV) is the hepatotropic virus responsible for the most severe form of viral hepatitis. HDV co-infects or super-infects hepatocytes previously infected with the hepatitis B virus (HBV), increasing the risk of fulminant hepatitis, development of cirrhosis and hepatocellular carcinoma (HCC). The association between these two viruses is explained by the fact that the outer envelope of the hepatitis delta virus is made of HBV surface proteins (HBsAgs). HDV is therefore considered to be an HBV satellite virus. The HDV genome consists of a single molecule of single stranded circular RNA encoding a single protein, the delta antigen (HDAg). Through a RNA editing mechanism, two forms of the HDAg are obtained, the small (S-HDAg) and the large (L-HDAg). Both viral proteins perform different functions in the HDV infectious cycle, the S-HDAg stimulates viral replication, whereas L-HDAg is responsible for replication inhibition and interaction with HBsAgs to form viral particles. Currently, there is no specific therapy for the treatment of patients with HDV infection and several studies have been carried out in order to develop new therapies to overcome this limitation. The need to find new therapeutic options has led to the development of drugs aimed at specific molecules with relevance to the HDV infectious cycle. L-HDAg undergoes a prenylation modification, which is important for its interaction with HBsAgs and which is catalysed by cellular enzymes from the farnesil-transferases group. Thus, the use of inhibitors of these cellular enzymes may constitute a novel therapeutic approach to HDV-induced infection. In this study, we evaluated the therapeutic potential of the inhibitor of farnesil-transferases Lonafarnib in Huh7D12 cells, analyzing the impact in terms of cell viability and correlating the drug's ability to intervene in the levels of expression of the HDV viral genome. We also evaluated the effect of this inhibitor on the expression of the STAT3 oncogene, which is intimately associated with the development of HCC. The analysis of the effects on cell viability showed that Lonafarnib has a much lower cytotoxicity than other farnesil-transferase inhibitors previously studied in our laboratory. Lonafarnib was found to interfere with the life cycle of HDV, causing an increase in the expression of HDV genomic RNA in human hepatocarcinoma cells and influencing the stability of L-HDAg when expressed isolated. We also show, both by RT-PCR and Western Blot, that treatments with Lonafarnib result in an increase of STAT3 and CCND1 expression. In conclusion, the results obtained in this study indicate that Lonafarnib may constitute a promising therapeutical approach for the treatment of HDV infection but additional molecular studies are necessary to assess for possible negative side effects, such as the increased expression of the HDV genomic RNA and the STAT3 oncogene.
Hepatitis Delta virus (HDV) is the hepatotropic virus responsible for the most severe form of viral hepatitis. HDV co-infects or super-infects hepatocytes previously infected with the hepatitis B virus (HBV), increasing the risk of fulminant hepatitis, development of cirrhosis and hepatocellular carcinoma (HCC). The association between these two viruses is explained by the fact that the outer envelope of the hepatitis delta virus is made of HBV surface proteins (HBsAgs). HDV is therefore considered to be an HBV satellite virus. The HDV genome consists of a single molecule of single stranded circular RNA encoding a single protein, the delta antigen (HDAg). Through a RNA editing mechanism, two forms of the HDAg are obtained, the small (S-HDAg) and the large (L-HDAg). Both viral proteins perform different functions in the HDV infectious cycle, the S-HDAg stimulates viral replication, whereas L-HDAg is responsible for replication inhibition and interaction with HBsAgs to form viral particles. Currently, there is no specific therapy for the treatment of patients with HDV infection and several studies have been carried out in order to develop new therapies to overcome this limitation. The need to find new therapeutic options has led to the development of drugs aimed at specific molecules with relevance to the HDV infectious cycle. L-HDAg undergoes a prenylation modification, which is important for its interaction with HBsAgs and which is catalysed by cellular enzymes from the farnesil-transferases group. Thus, the use of inhibitors of these cellular enzymes may constitute a novel therapeutic approach to HDV-induced infection. In this study, we evaluated the therapeutic potential of the inhibitor of farnesil-transferases Lonafarnib in Huh7D12 cells, analyzing the impact in terms of cell viability and correlating the drug's ability to intervene in the levels of expression of the HDV viral genome. We also evaluated the effect of this inhibitor on the expression of the STAT3 oncogene, which is intimately associated with the development of HCC. The analysis of the effects on cell viability showed that Lonafarnib has a much lower cytotoxicity than other farnesil-transferase inhibitors previously studied in our laboratory. Lonafarnib was found to interfere with the life cycle of HDV, causing an increase in the expression of HDV genomic RNA in human hepatocarcinoma cells and influencing the stability of L-HDAg when expressed isolated. We also show, both by RT-PCR and Western Blot, that treatments with Lonafarnib result in an increase of STAT3 and CCND1 expression. In conclusion, the results obtained in this study indicate that Lonafarnib may constitute a promising therapeutical approach for the treatment of HDV infection but additional molecular studies are necessary to assess for possible negative side effects, such as the increased expression of the HDV genomic RNA and the STAT3 oncogene.
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Palavras-chave
Ciências biomédicas Microbiologia médica Biologia molecular Vírus da hepatite delta Prenilação Inibidores de farnesil-transferases Carcinoma hepatocelular STAT3