Desenvolvimento de sondas fluorescentes para determinação de sítios de ligação em albumina: estudo da relação entre estrutura molecular, constante de associação e especificidade
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Data
2021-06-08
Autores
Bertozo, Luiza de Carvalho [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A albumina sérica humana, HSA, é a principal proteína do plasma sanguíneo e responsável pelo transporte de compostos endógenos e exógenos. As propriedades farmacocinéticas de um fármaco podem ser alteradas devido a sua interação com a albumina. Por isso, quando um novo fármaco está em desenvolvimento, a sua interação com a albumina é extensivamente estudada, sendo, por exemplo, determinado sua constante de associação e sítios de ligação na albumina, sítio I ou sítio II. A determinação do local preferencial de ligação de um novo composto pode ser realizada por estudos de competição envolvendo sondas fluorescentes. Neste contexto, se destaca os aminoácidos dansilados, que são sondas fluorescentes que possuem especificidade pelo sítio I ou pelo sítio II da HSA. O levantamento crítico da literatura mostra que os aminoácidos dansilados de sítio I são menos aplicados como sondas do que os de sítio II, isso porque, eles parecem apresentar menor especificidade. Neste trabalho estudamos a interação entre oito aminoácidos dansilados e a HSA buscando compreender a diferença de especificidade encontrada nos dois sítios de ligação da proteína. Além disso, utilizamos sondas de diferentes hidrofobicidade para verificar a influência desta propriedade na interação de compostos com a albumina. Os resultados de rendimento quântico, tempo de vida, anisotropia de fluorescência e dicroísmo circular mostraram a maior afinidade entre os aminoácidos dansilados de sítio II e a HSA. Os aminoácidos dansilados de sítio II também apresentaram maiores valores de constante de associação. Os estudos de calorimetria de titulação isotérmica, ITC, confirmaram a menor afinidade dos dansilados de sítio I com a HSA. A aplicação da dansil-glutamina (DGlu) como sonda do sítio I da HSA evidenciou que a carga positiva encontrada nas estruturas dos demais dansilados de sítio I parece prejudicar suas ligações com a proteína. Os estudos envolvendo ligantes com diferentes hidrofobicidade mostraram que o ligante mais hidrofóbico apresenta maior afinidade com a albumina. Neste caso, a interação é entropicamente dirigida e por isso o critério envolvendo a temperatura na distinção do tipo de quenching não foi o mais adequado. Os estudos de docking e dinâmica molecular confirmaram os achados experimentais. Em conclusão, a menor afinidade dos aminoácidos dansilado de sítio I em HSA está relacionada a maior energia que esses compostos devem ter para acessar esta cavidade da proteína. Isso ocorre devido a uma repulsão eletrostática entre aminoácidos Lys195, Lys199, Arg218 e Arg222 na entrada do sítio I e a carga positiva dos aminoácidos dansilados do sítio I.
Human serum albumin, HSA, is the main protein in blood plasma and it is responsible for the transport of endogenous and exogenous compounds. The pharmacokinetic properties of a drug can be altered due to its interaction with albumin. Therefore, when a new drug is under development, its interaction with albumin is extensively studied, for example, in determining its association constant and binding sites on albumin, site I or site II. The determination of the preferred binding site for a new compound can be carried out by competition studies involving fluorescent probes. In this context, we highlight the dansyl amino acids which are fluorescent probes that have specificity for site I or site II of HSA. The critical survey of the literature shows that the dansyl amino acids of site I are less applied as probes than those of site II, because, they seem to have less specificity. In this work, we study the interaction between the eight dansyl amino acids and HSA in order to understand the difference in specificity found between the two protein binding sites. In addition, we use probes of different hydrophobicity to verify the influence of this property on the interaction of compounds with albumin. The results of quantum yield, lifetime, fluorescence anisotropy and circular dichroism showed the greatest affinity between the dansyl amino acids of site II and HSA. The dansyl amino acids from site II also showed higher values of association constant. The isothermal titration calorimetry, ITC, studies confirmed the lower affinity of the site I dansyl amino acids with HSA. The application of dansyl-glutamine (DGlu) as a probe of the site I of HSA showed that the positive charge found in the structures of the other dansyl amino acids of site I seems to impair its binding with the protein. Studies involving ligands with different hydrophobicity have shown that the more hydrophobic ligand has greater affinity with albumin. In this case, the interaction is entropically-driven and, therefore, the criterion involving temperature in distinguishing the type of quenching was not the most appropriate. Docking and molecular dynamics studies confirmed the experimental findings. In conclusion, the lower affinity of the dansyl amino acids of site I of HSA is related to the greater energy that these compounds must have to access this protein cavity. This is due to an electrostatic repulsion between amino acids Lys195, Lys199, Arg218 and Arg222 at the entrance to site I and the positive charge of dansylated amino acids at site I.
Human serum albumin, HSA, is the main protein in blood plasma and it is responsible for the transport of endogenous and exogenous compounds. The pharmacokinetic properties of a drug can be altered due to its interaction with albumin. Therefore, when a new drug is under development, its interaction with albumin is extensively studied, for example, in determining its association constant and binding sites on albumin, site I or site II. The determination of the preferred binding site for a new compound can be carried out by competition studies involving fluorescent probes. In this context, we highlight the dansyl amino acids which are fluorescent probes that have specificity for site I or site II of HSA. The critical survey of the literature shows that the dansyl amino acids of site I are less applied as probes than those of site II, because, they seem to have less specificity. In this work, we study the interaction between the eight dansyl amino acids and HSA in order to understand the difference in specificity found between the two protein binding sites. In addition, we use probes of different hydrophobicity to verify the influence of this property on the interaction of compounds with albumin. The results of quantum yield, lifetime, fluorescence anisotropy and circular dichroism showed the greatest affinity between the dansyl amino acids of site II and HSA. The dansyl amino acids from site II also showed higher values of association constant. The isothermal titration calorimetry, ITC, studies confirmed the lower affinity of the site I dansyl amino acids with HSA. The application of dansyl-glutamine (DGlu) as a probe of the site I of HSA showed that the positive charge found in the structures of the other dansyl amino acids of site I seems to impair its binding with the protein. Studies involving ligands with different hydrophobicity have shown that the more hydrophobic ligand has greater affinity with albumin. In this case, the interaction is entropically-driven and, therefore, the criterion involving temperature in distinguishing the type of quenching was not the most appropriate. Docking and molecular dynamics studies confirmed the experimental findings. In conclusion, the lower affinity of the dansyl amino acids of site I of HSA is related to the greater energy that these compounds must have to access this protein cavity. This is due to an electrostatic repulsion between amino acids Lys195, Lys199, Arg218 and Arg222 at the entrance to site I and the positive charge of dansylated amino acids at site I.
Descrição
Palavras-chave
Aminoácidos dansilados, Fluorescência molecular, Dicroísmo circular induzido, Calorimetria de titulação isotérmica, Albumina, Dansyl amino acids, Molecular fluorescence, Induced circular dichroism, Isothermal titration calorimetry, Albumin