edoc

Yeast-based strategy for identification of 11β-HSD1 inhibitors

Vanella, Rosario. Yeast-based strategy for identification of 11β-HSD1 inhibitors. 2015, PhD Thesis, University of Basel, Faculty of Science.

[img]
Preview
PDF
4Mb

Official URL: http://edoc.unibas.ch/diss/DissB_11741

Downloads: Statistics Overview

Abstract

Glucocorticoids are hormones with a vital role in regulation of metabolic and defence responses. Their metabolism plays an important role in the pathogenesis of obesity, a major risk factor for metabolic syndrome, a collection of disorders such as insulin resistance, dyslipidemia and hypertension.
At intracellular level the production of active glucocorticoids is regulated by two hydroxysteroid dehydrogenase enzyme isoforms 11β-HSD1 and 11β-HSD2, respectively. 11β-HSD2 is predominantly expressed in mineralocorticoid target tissues and acts as oxidase catalysing conversion of cortisol into cortisone. The isoform 11β-HSD1, expressed in a wide array of tissues and with highest levels in the liver, acts mainly as a reductase converting the cortisone into the biologically active hormone cortisol, in a NADPH dependent manner. Due to its implication in the metabolism of glucocorticoids, 11β-HSD1 has become a primary target for the treatment of metabolic syndrome.
In the last years many strategies have been developed for the screening of molecules with inhibitory effects against this target enzyme. All known approaches present unique features and are suitable for specific screening models. Nevertheless, due to the great therapeutic and economical interest around the treatment of metabolic syndrome, many research groups are constantly focused on the development of new and more successful strategies for identification of hits with improved pharmaceutical properties.
In this study an innovative synthetic biology platform for synthesis and screening of 11β-HSD1 inhibitors has been conceived and investigated. This strategy is based on the production of diverse chemical scaffolds in the yeast Saccharomyces cerevisiae, in which the molecules are directly screened through intracellular functional assays.
Providing all necessary genetic information, the biosynthetic pathway of plant triterpenoids, a big class of natural compounds with many beneficial effects on human health, was reconstructed in the baker’s yeast. Moreover, by applying DNA family shuffling methods, a library of chimeric triterpenoid synthase cDNAs was constructed. The chimeric sequences, potential carriers of new enzymatic functions, were assembled together with the wild-type molecules in expressible yeast artificial chromosomes (eYACs).
Furthermore, the construction of two yeast assays strains functionally co-expressing the murine glucocorticoid receptor (GR) and the human 11β-HSD1 enzyme is presented. In both developed strains, with different mechanisms, the reductase activity of the enzyme 11β-HSD1 on cortisone is connected with an easily detectable fluorescent signal. Cells that produce compounds with inhibitory activity against 11β-HSD1 can be identified through changes in fluorescence and isolated to further investigate the active molecules. The developed yeast-assays were validated using carbenoxolone, a known 11β-HSD1 inhibitor, and may represent useful tools for a first quick and easy screening of large number of membrane permeable putative inhibitory compounds.
Advisors:Hall, Michael N. and Kübler, Eric and Odermatt, Alex
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
Item Type:Thesis
Thesis no:11741
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (111 Seiten)
Language:English
Identification Number:
Last Modified:06 Sep 2016 07:40
Deposited On:06 Sep 2016 07:40

Repository Staff Only: item control page