Molecular Interactions Between Sex Hormone-Binding Globulin and Nonsteroidal Ligands That Enhance Androgen Activity

Sex hormone-binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two non-steroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (-)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast a, synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71-1.80 Å resolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other non-steroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ~20 fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how non-steroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids.Peer reviewe

Characterization and comparison of recombinant full-length ursine and human sex hormone-binding globulin

Sex hormone‐binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell‐based expression system for recombinant full‐length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10‐fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~ 58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in the presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [(3)H]DHT were determined to 0.21 ± 0.04 nm for human and 1.32 ± 0.10 nm for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology

Hepatic and adipose phenotype in Alström syndrome

BACKGROUND AND AIMS: Alström syndrome (AS) is a recessive monogenic syndrome characterized by obesity, extreme insulin resistance and multi-organ fibrosis. Despite phenotypically being high risk of non-alcoholic fatty liver disease (NAFLD), there is a lack of data on the extent of fibrosis in the liver and its close links to adipose in patients with AS. Our aim was to characterize the hepatic and adipose phenotype in patients with AS. METHODS: Observational cohort study with comprehensive assessment of metabolic liver phenotype including liver elastography (Fibroscan® ), serum Enhanced Liver Fibrosis (ELF) Panel and liver histology. In addition, abdominal adipose histology and gene expression was assessed. We recruited 30 patients from the UK national AS clinic. A subset of six patients underwent adipose biopsies which was compared with control tissue from nine healthy participants. RESULTS: Patients were overweight/obese (BMI 29.3 (25.95-34.05) kg/m2 ). A total of 80% (24/30) were diabetic; 74% (20/27) had liver ultrasound scanning suggestive of NAFLD. As judged by the ELF panel, 96% (24/25) were categorized as having fibrosis and 10/21 (48%) had liver elastography consistent with advanced liver fibrosis/cirrhosis. In 7/8 selected cases, there was evidence of advanced NAFLD on liver histology. Adipose tissue histology showed marked fibrosis as well as disordered pro-inflammatory and fibrotic gene expression profiles. CONCLUSIONS: NAFLD and adipose dysfunction are common in patients with AS. The severity of liver disease in our cohort supports the need for screening of liver fibrosis in AS.Alström UKThis is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.1111/liv.1316

Sex hormone-binding globulin (SHBG) : interaction with non-steroidal ligands and the enhancement of sex steroid action

Sex hormone-binding globulin (SHBG) determines the equilibrium between “free” and “protein-bound” androgens and estrogens in blood and regulates their access to target tissues. I present in this thesis how a steroid derivative, and two non-steroidal compounds bind to human SHBG by crystallographic studies and radiolabeled competitive binding capacity assays. I then show how these compounds influence protein pulldown assays, and androgen action in cell culture experiments. Danazol, a steroid derivative was structurally determined to be bound very similarly as steroids, by SHBG. While danazol was shown to increase the activity of testosterone through displacement from SHBG, the number of other steroid receptors danazol interacts with, limits danazols potential as a SHBG inhibitor. One of the non-steroidal compounds, (-)3,4-Divanillyltetrahydrofuran (DVT), binds SHBG with relatively low affinity and is present in stinging nettle root extracts used as a nutraceutical. By contrast, a synthetic non-steroidal compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), binds SHBG with an affinity like that of testosterone and estradiol. Crystal structures of the N-terminal LG4 domain of SHBG in complex with DVT or IPI revealed their unique orientations in the SHBG ligand-binding pocket and reveal opportunities for the design of other non-steroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ~20 fold in the presence of zinc, while DVT binding was almost completely lost. Estradiol dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound. Both DVT and IPI increase the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. IPI was then used as a model for the computational design and chemical synthesis of IPI-derived compounds, which were predicted to bind SHBG more effectively. Three of the five IPI-derived compounds synthesized bound SHBG with lower affinity then IPI, the remaining two did not bind. At higher concentrations, these compounds maintained the ability to increase testosterone activity through displacement of testosterone from SHBG. This work established SHBG as a druggable target for increasing sex steroid activity and laid the ground work for further development of novel non-steroidal SHBG ligands for use as pharmaceuticals.Medicine, Faculty ofGraduat