Thyroid hormone receptor: computational studies, surface plasmon resonance and cell based assays

Author(s)

Napoleão Fonseca Valadares

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2008-12-08

Examining board members

Richard Charles Garratt; Adriano Defini Andricopulo; Mauricio da Silva Baptista; Luis Francisco de Assis Rocha Neves; Jose Daniel Figueroa Villar

• Advisor: Richard Charles Garratt

Abstract

The thyroid hormone receptors (TRs) are transcriptional factors involved in cell differentiation, development, metabolism and physiological function of most tissues. Many lines of evidence show that several pharmacological actions of TRs might be beneficial in medical therapy, specially those mediated by TR that target important medical conditions like obesity, hypercholesterolemia and diabetes. Additionally, the findings that TR is the predominant isoform in the heart and mediates most of the TRs deleterious cardiovascular effects, stimulated the research for selective TR ligands which could address important medical needs with an acceptable safety profile. In this PhD thesis, studies of the quantitative structure-activity relationships (QSAR) of a dataset of compounds with reported biologic activity for both TR and TR were performed, and statistically significant Hologram QSAR models with good predictive ability for untested compounds were created. In parallel, a careful virtual screening procedure was executed, leading to the selection of 7 compounds which were purchased for the evaluation of their biological activities. Cell transfection and reporter gene assays were developed, validated and used to evaluate the biological activities of these compounds. Finally, a surface plasmon resonance (SPR) assay was developed and used to assess the agonistic activity of these compounds. The SPR technique was also employed in a careful study of the interaction between the ligand binding domain of TR and peptides derived from its coregulators, which included the determination of the kinetic and thermodynamic parameters for this interaction. The results suggest that flexibility plays an important role in the interaction between the receptor and its coregulators, and point out important aspects of experimental design that should be addressed when using TR LBD and its agonists. Furthermore, the methodology described here may be useful for the identification of new TR ligands. (AU)