Caracterização das FKBPS de (FK506 binding protein) de Trypanosoma cruzi

Orientador: Ricardo Lehtonen Rodrigues de SouzaCoorientadora: Daniela Parada PavoniMonografia (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Ciências Biológica

Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors

The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors

In-cell analysis of FK506 binding protein 51-glucocorticoid receptor-heat shock protein 90 interaction at single residue resolution

The large immunophilins FKBP51 and FKBP52 play key roles in the Hsp90-mediated maturation of steroid hormone receptors, which is crucial for stress-related disorders and correct sexual embryonic development, respectively. A prominent regulatory target is the glucocorticoid receptor (GR), whose activation is repressed by FKBP51 and facilitated by FKBP52. Despite their vital roles, the molecular modes of action of FKBP51 and FKBP52 are poorly understood since the transient key states of FKBP-mediated GR-regulation have remained experimentally elusive. This work presents a systematic incorporation of a photoreactive amino acid inside human cells that allows capture of the transient FKBP51-GR-Hsp90 interactions. A photoreactive, unnatural amino acid is site specifically incorporated by amber suppression and acts as a proximity sensor to map the interaction at single residue resolution. This is first established for FKBP51, where the FKBP51-Hsp90 interaction interface is explored well beyond the known FKBP51-TPR-domain mediated interaction including both the FK1- and FK2-domain of FKBP51. Additionally, the Hsp90 cochaperone p23 is identified as a direct interaction partner of FKBP51 suggesting that a multi-protein complex is needed for a functional Hsp90 machinery. Creating a suitable, high-throughput analysis for mapping protein interaction via ELISA allows the elucidation of the GR-FKBP51 interaction interface in great detail. The identified crosslinking sites all depend on a functional Hsp90 chaperone cycle, are disrupted by GR activation, and cluster in characteristic patterns, defining the relative orientation and contact surfaces within the FKBP51/p23-apoGR complexes. Moreover, the developed ELISA enables quantification of in-cell activation of the GR by its synthetic agonist dexamethasone. GR-->FKBP52 crosslinks were found to be 5-fold more sensitive to GR activation compared to GR-->FKBP51 crosslinks. The ELISA data show that GR activation is facilitated in the context of a FKBP52-Hsp90-GR complex compared to GR in a FKBP51-Hsp90-GR complex. This in accordance with the well-documented GR-facilitating effect of FKBP52 and the GR-repressing effect of FKBP51. Taken together, this work presents the architecture and functional annotation of FKBP51-, and p23-containing Hsp90-apoGR pre-activation complexes, trapped by large scale in-cell photocrosslinking