Estudo da interação da crisina e seu derivado monoacetilado com a proteína NS1 do vírus sincicial respiratório humano (hRSV) por espectroscopia de fluorescência e docking molecular
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Data
2019-03-28
Autores
Quintino, Evelyn Pedroso Toscano
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O Vírus Sincicial Respiratório Humano (hRSV) é um dos principais causadores de infecções respiratórias agudas (IRA), do trato inferior, em crianças menores de cinco anos de idade. O hRSV, membro da família Pneumoviridae, sintetiza a proteína NS1 que promove ao vírus evasão do sistema imunológico do hospedeiro. Diante dos efeitos colateriais, alto custo e baixa eficácia dos tratamentos disponíveis, buscam-se tratamentos alternativos para hRSV com melhor eficácia, baixo custo e menos efeitos colaterais. A crisina, um flavonóide proveniente de produtos naturais, dentre suas atividades farmacológicas, apresenta atividade antiviral em estudos in vitro contra HSV-I e Influenza. Diante disso, o objetivo desse trabalho foi estudar se há interação entre a proteína NS1 do hRSV e os flavonóides crisina e derivado monoacetilado de crisina, utilizando espectroscopia de fluorescência e docking molecular. Para isso, foi realizada a clonagem, expressão e purificação da proteína NS1 e caracterização dos ligantes. Os resultados da supressão de fluorescência da NS1 com ambos os ligantes apontam que o mecanismo de interação é do tipo dinâmico, já que as constantes de Stern-Volmer (Ksv) é diretamente proporcional ao aumento da temperatura. A partir da análise do duplo log determinou-se a ordem da constante de ligação (Kb) da ordem de 105 M-1, entretanto a interação NS1-crisina apresentou maior afinidade do que na interação NS1-derivado monoacetilado de crisina. Os parâmetros termodinâmicos indicaram que a reação de ligação é favorável, entropicamente dirigida e predominantemente hidrofóbica. Esses resultados, juntamente com estudos de Docking molecular, nos sugerem que apesar da diminuição na afinidade da proteína pelo derivado monoacetilado de crisina, a modificação não resultou em maior probabilidade de afinidade do ligante por outro sítio que não o mesmo da NS1-crisina. A acetilação, em sistemas mais complexos, poderá resultar em um aumento na sua absorção intestinal, biodisponibilidade e acesso ao seu alvo por facilitar sua passagem pela membrana citoplasmática.
Human Respiratory Syncytial Virus (hRSV) is a major cause of acute lower respiratory tract infections (ALRIs) in children under 5 years of age. HRSV, a member of the Pneumoviridae family, synthesizes the NS1 protein that promotes virus evasion of the host's immune system. In view of the collateral effects, high cost and low efficacy of available treatments, alternative treatments for hRSV was sought with better efficiency, low cost and fewer side effects. Chrysin, flavonoid from natural products, among its pharmacological activities, has antiviral activity in in vitro studies. Therefore, the aim of this work was to study the interaction between hRSV-NS1 and chrysin flavonoids and monoacetylated chrysin derivative using fluorescence spectroscopy and molecular docking. For this, cloning, expression and purification of the NS1 protein and characterization of the ligands were performed. The results of hRSV-NS1 fluorescence suppression with both ligands indicate that the interaction mechanism is of the collisional type, since the Stern-Volmer (Ksv) constants increase with increasing temperature. From the double log analysis, the order of the binding constant (Kb) of 105 M-1 was determined, however, the NS1-chrysin interaction showed higher affinity in comparation to the NS1-monoacetylated derivative of chrysin interaction. The thermodynamic parameters indicated that the binding reaction is favorable, entropically directed. These results, together with molecular docking studies, suggest us that despite the decrease in protein affinity for the monoacetylated derivative of chrysin, the modification did not result in a greater likelihood of affinity for the linker at a site other than that of NS1-chrysin. Chrysin acetylation, in more complex systems, may result in an increase in its intestinal absorption, bioavailability and access to its target by facilitating its passage through the cytoplasmic membrane.
Human Respiratory Syncytial Virus (hRSV) is a major cause of acute lower respiratory tract infections (ALRIs) in children under 5 years of age. HRSV, a member of the Pneumoviridae family, synthesizes the NS1 protein that promotes virus evasion of the host's immune system. In view of the collateral effects, high cost and low efficacy of available treatments, alternative treatments for hRSV was sought with better efficiency, low cost and fewer side effects. Chrysin, flavonoid from natural products, among its pharmacological activities, has antiviral activity in in vitro studies. Therefore, the aim of this work was to study the interaction between hRSV-NS1 and chrysin flavonoids and monoacetylated chrysin derivative using fluorescence spectroscopy and molecular docking. For this, cloning, expression and purification of the NS1 protein and characterization of the ligands were performed. The results of hRSV-NS1 fluorescence suppression with both ligands indicate that the interaction mechanism is of the collisional type, since the Stern-Volmer (Ksv) constants increase with increasing temperature. From the double log analysis, the order of the binding constant (Kb) of 105 M-1 was determined, however, the NS1-chrysin interaction showed higher affinity in comparation to the NS1-monoacetylated derivative of chrysin interaction. The thermodynamic parameters indicated that the binding reaction is favorable, entropically directed. These results, together with molecular docking studies, suggest us that despite the decrease in protein affinity for the monoacetylated derivative of chrysin, the modification did not result in a greater likelihood of affinity for the linker at a site other than that of NS1-chrysin. Chrysin acetylation, in more complex systems, may result in an increase in its intestinal absorption, bioavailability and access to its target by facilitating its passage through the cytoplasmic membrane.
Descrição
Palavras-chave
hRSV, Proteína NS1, Crisina, Derivado monoacetilado de crisina, Espectroscopia de fluorescência, Docking molecular, hRSV-NS1, Chrysin, Monoacetylated derivative of chrysin, Fluorescence spectroscopy, Molecular docking