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