Abstract
BackgroundIntracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. At the peripheral level the concentration of active glucocorticoids is mainly regulated by the 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) enzyme, involved in the conversion of cortisone into the biologically active hormone cortisol. Cortisol interacts with the glucocorticoid receptor and regulates the expression of different classes of genes within the nucleus. Due to its implication in glucocorticoid metabolism, the inhibition of 11β-HSD1 activity has become a dominant strategy for the treatment of metabolic syndrome. Moreover, inhibitors of this target enzyme can be used for development of formulations to counteract skin ageing. Here we present the construction of two yeast cell based assays that can be used for the screening of novel 11β-HSD1 inhibitors.ResultsThe yeast Saccharomyces cerevisiae is used as a host organism for the expression of human 11β-HSD1 as well as a genetically encoded assay system that allows intracellular screening of molecules with 11β-HSD1 inhibitory activity. As proof of concept the correlation between 11β-HSD1 inhibition and fluorescent output signals was successfully tested with increasing concentrations of carbenoxolone and tanshinone IIA, two known 11β-HSD1 inhibitors. The first assay detects a decrease in fluorescence upon 11β-HSD1 inhibition, whereas the second assay relies on stabilization of yEGFP upon inhibition of 11β-HSD1, resulting in a positive read-out and thus minimizing the rate of false positives sometimes associated with read-outs based on loss of signals. Specific inhibition of the ABC transporter Pdr5p improves the sensitivity of the assay strains to cortisone concentrations by up to 60 times.ConclusionsOur yeast assay strains provide a cost-efficient and easy to handle alternative to other currently available assays for the screening of 11β-HSD1 inhibitors. These assays are designed for an initial fast screening of large numbers of compounds and enable the selection of cell permeable molecules with target inhibitory activity, before proceeding to more advanced selection processes. Moreover, they can be employed in yeast synthetic biology platforms to reconstitute heterologous biosynthetic pathways of drug-relevant scaffolds for simultaneous synthesis and screening of 11β-HSD1 inhibitors at intracellular level.
Highlights
Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin
The production of active glucocorticoid hormones at the intracellular level is regulated by the two hydroxysteroid dehydrogenase enzyme isoforms 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and 11β-HSD2, microsomal enzymes belonging to the family of short chain dehydrogenases/reductases
Flow cytometric analysis and fluorescence microscope images of RVY97 cells treated with dimethyl sulfoxide (DMSO) and 250 μM cortisone are shown in Fig. 2b, c
Summary
Intracellular metabolism of glucocorticoid hormones plays an important role in the pathogenesis of metabolic syndrome and regulates, among many physiological processes, collagen metabolism in skin. We present the construction of two yeast cell based assays that can be used for the screening of novel 11β-HSD1 inhibitors Glucocorticoids, such as cortisone and cortisol in human, are stress hormones with a vital role in regulation of metabolic and defence responses. A key step in the molecular elucidation of these biological processes and conditions has been the demonstration that glucocorticoid receptor activation in target tissues is not determined exclusively by circulating glucocorticoids and by intra-cellular interconversion of active and inactive forms of the steroids [7] Identification of this mechanism for local regulation of glucocorticoid action has prompted the concept that tissue-specific glucocorticoid excess, in the face of normal circulating concentrations, may contribute to metabolic syndromes [8] and plays an important role in the skin ageing process, reducing collagen metabolism and promoting skin atrophy [5]. Its role is to protect the non-selective mineralocorticoid receptor from the unspecific binding from cortisol, catalysing its conversion to cortisone [10]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
