Development of fluorescent probes for determination of binding sites in albumin: study of the relationship between molecular structure, association constant and specificity

Author(s)

Luiza de Carvalho Bertozo

• Document type: Doctoral Thesis
• Press: Bauru. 2021-07-16.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências. Bauru
• Defense date: 2021-06-08
• Advisor: Valdecir Farias Ximenes

Abstract

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. (AU)

• FAPESP's process: 16/22014-1 - Development of Fluorescent Probes for the Determination of Binding Sites on Albumin: Studies of the Relationship of the Molecular Structure, Association Constant and Specificity
• Grantee: Luiza de Carvalho Bertozo
• Support Opportunities: Scholarships in Brazil - Doctorate