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A new antiviral strategy for pandemic and seasonal influenza
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O vírus da gripe continua a representar um importante problema de saúde pública a nível mundial. De acordo com a Organização Mundial de Saúde (WHO), acredita-se que este vírus seja responsável por mil milhões de casos e até 650 000 mortes por ano. A sua capacidade de evoluir rapidamente, associada ao aparecimento de estirpes resistentes aos antivirais, denota o risco de (re)aparecimento de vírus pandémicos e sublinha a necessidade urgente de novas opções antivirais. Recentemente, surgiu o interesse em torno de pequenas moléculas capazes de facilitar o transporte de aniões através de bicamadas lipídicas - anionóforos - e o seu potencial anticancerígeno, antiparasitário e antimicrobiano já foi descrito. No entanto, não existia evidência da sua atividade antiviral.
O trabalho desenvolvido e apresentado nesta dissertação focou-se na investigação do potencial antiviral de uma nova biblioteca destas pequenas moléculas contra o vírus da gripe A (IAV) subtipo H1N1.
Foram estudados cinco anionóforos com o objetivo de selecionar o(s) candidato(s) mais promissor(es) para futura caraterização detalhada, otimização, formulação e validação clínica. A citotoxicidade causada por estas moléculas foi avaliada numa linha celular suscetível animal (MDCK CCL-34™) e em células epiteliais pulmonares humanas (Calu-3 HTB-55™), por citometria de fluxo. Subsequentemente, a atividade antiviral, baseada em duas abordagens diferentes de administração dos compostos - profilática e terapêutica - foi avaliada por ensaios em placa. Estes resultados foram ainda confirmados pela reação em cadeia da polimerase com transcrição reversa quantitativa (RT-qPCR) através da amplificação do gene da matriz (M). Para complementar os testes citotóxicos efetuados, foram realizados ensaios de hemólise para avaliar a capacidade do candidato mais promissor para causar hemólise de glóbulos vermelhos humanos (hRBCs).
Este estudo revelou que uma destas novas moléculas tem propriedades antivirais contra o IAV, com a abordagem terapêutica a apresentar os melhores resultados: o composto AS-46 provocou uma redução estatisticamente significativa do título viral e uma percentagem de redução da atividade viral de 79.7% ± 11.2%. Foi igualmente observada uma redução estatisticamente significativa (p = 0.0096) do número de cópias do gene M obtidas por RT-qPCR, corroborando os resultados obtidos pelos ensaios em placa. Além disso, na concentração testada, o AS-46 não causou hemólise dos hRBCs nem apresentou efeitos citotóxicos em nenhuma das linhas celulares testadas.
No geral, este trabalho indica que o composto anionóforo AS-46 pode inibir eficazmente o IAV in vitro. A nossa hipótese é que a sua ação está relacionada com a desacidificação dos endossomas da célula hospedeira, o que impede a fusão do envelope viral com a membrana endossomal, durante os passos de internalização do ciclo de replicação. Esta nova estratégia antiviral poderá contribuir para a preparação de uma resposta global e eficiente em futuros surtos pandémicos.
Abstract Influenza virus continues to pose a major global public health issue. According to the World Health Organization (WHO), this virus is believed to be responsible for one billion cases and up to 650,000 deaths annually. Its ability to rapidly evolve coupled with the emergence of antiviral-resistant strains denotes the risk of the (re)emergence of pandemic viruses and underscores the urgent need for new antiviral options. Recent interest has risen around small molecules capable of facilitating the transport of anions through lipid bilayers - anionophores - and their anticancer, antiparasitic, and antimicrobial potential has already been described. However, there was no evidence of their antiviral activity. The work developed and presented in this dissertation focused on investigating the antiviral potential of a new library of these small molecules targeting the influenza A virus (IAV) subtype H1N1. Five anionophores were studied aiming to select the most promising candidate(s) for future detailed characterization, optimization, formulation, and clinical validation. Cytotoxicity caused by these molecules was assessed in an animal susceptible cell line (MDCK CCL-34™) and in human lung epithelial cells (Calu-3 HTB-55™), by flow cytometry. Subsequently, the antiviral activity, based on two different approaches to administering the compounds - prophylactic and therapeutic - was evaluated by plaque assays. These findings were further confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) by amplification of the matrix (M) gene. To further complement the cytotoxic tests performed, hemolysis assays were conducted to assess the ability of the most promising candidate to cause hemolysis of human red blood cells (hRBCs). This study revealed that one of these new molecules has antiviral properties against IAV, with the therapeutic approach showing the best results: the compound AS-46 caused a statistically significant reduction in viral titer and a percentage of viral activity reduction of 79.7% ± 11.2%. A statistically significant reduction (p = 0.0096) in the number of copies of the M gene obtained by RT-qPCR was also observed, corroborating the results obtained by the plaque assays. Furthermore, at the tested concentration, AS-46 did not cause hemolysis of hRBCs nor did it show cytotoxic effects in either of the cell lines tested. Overall, this work indicates that the anionophore compound AS-46 can effectively inhibit IAV in vitro. We hypothesized that its action relates to the deacidification of the host cell’s endosomes, which prevents the fusion of the viral envelope with the endosomal membrane, during the internalization steps of the replication cycle. This novel antiviral strategy could contribute to preparing a global and efficient response in future pandemic outbreaks.
Abstract Influenza virus continues to pose a major global public health issue. According to the World Health Organization (WHO), this virus is believed to be responsible for one billion cases and up to 650,000 deaths annually. Its ability to rapidly evolve coupled with the emergence of antiviral-resistant strains denotes the risk of the (re)emergence of pandemic viruses and underscores the urgent need for new antiviral options. Recent interest has risen around small molecules capable of facilitating the transport of anions through lipid bilayers - anionophores - and their anticancer, antiparasitic, and antimicrobial potential has already been described. However, there was no evidence of their antiviral activity. The work developed and presented in this dissertation focused on investigating the antiviral potential of a new library of these small molecules targeting the influenza A virus (IAV) subtype H1N1. Five anionophores were studied aiming to select the most promising candidate(s) for future detailed characterization, optimization, formulation, and clinical validation. Cytotoxicity caused by these molecules was assessed in an animal susceptible cell line (MDCK CCL-34™) and in human lung epithelial cells (Calu-3 HTB-55™), by flow cytometry. Subsequently, the antiviral activity, based on two different approaches to administering the compounds - prophylactic and therapeutic - was evaluated by plaque assays. These findings were further confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) by amplification of the matrix (M) gene. To further complement the cytotoxic tests performed, hemolysis assays were conducted to assess the ability of the most promising candidate to cause hemolysis of human red blood cells (hRBCs). This study revealed that one of these new molecules has antiviral properties against IAV, with the therapeutic approach showing the best results: the compound AS-46 caused a statistically significant reduction in viral titer and a percentage of viral activity reduction of 79.7% ± 11.2%. A statistically significant reduction (p = 0.0096) in the number of copies of the M gene obtained by RT-qPCR was also observed, corroborating the results obtained by the plaque assays. Furthermore, at the tested concentration, AS-46 did not cause hemolysis of hRBCs nor did it show cytotoxic effects in either of the cell lines tested. Overall, this work indicates that the anionophore compound AS-46 can effectively inhibit IAV in vitro. We hypothesized that its action relates to the deacidification of the host cell’s endosomes, which prevents the fusion of the viral envelope with the endosomal membrane, during the internalization steps of the replication cycle. This novel antiviral strategy could contribute to preparing a global and efficient response in future pandemic outbreaks.
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Palavras-chave
Biologia molecular Medicina tropical Vírus da gripe A H1N1 Pandemia Antiviral Anionóforos