Endocrine Abstracts

Sex Hormone binding globulin (SHBG) is a plasma steroid binding protein that is the major determinant androgen and estrogen access to target tissues. Several ligands of SHBG have been predicted by computational methods or discovered through basic research, they include pharmaceutical, natural plant extracts and anthropogenic endocrine disrupting compounds. In this study we characterized 3,4-Divanillyltetrahydrofuran (DVT), a non-steroidal ligand of SHBG, and Danazol, a synthetic steroid-like molecule, using X-ray crystallography, steroid binding capacity assays, and cell reporter assays. DVT is present in organic extracts of Urtica dioica root that are used in natural health supplements to enhance the anabolic activities of testosterone by virtue of its ability to displace testosterone from SHBG. Danazol treatment has been used pharmaceutically to treat endometriosis, but more recently has been reported to lead to telomere elongation; an effect that is known to estrogen-dependent and may be related to the ability of Danazol to displace estradiol from the SHBG binding site. The binding affinities of DVT and Danazol for SHBG, are 1.47% and 2.81%, respectively, relative to that for testosterone. The crystal structure of E176K SHBG LG4 Domain with DVT or Danazol in the binding site was solved to 1.7 Æ and 1.75 Æ resolution, respectively. Danazol resides in the SHBG steroid-binding site in essentially the same orientation as Dihydrotestosterone (DHT) and like DHT its binding affinity was unaffected by Zn²⁺. By contrast, the binding pose of DVT in the SHBG steroid-binding site was similar to those of estrogens, resulting in a similar positioning of key residues in and around the binding pocket, and as observed from estrogens the SHBG binding affinity for DVT is substantially reduced in the presence of Zn²⁺. These results indicate that the SHBG binding site can accommodate ligands with structures that diverge significantly from the natural steroid structure. To examine the physiological relevance of DVT binding to SHBG, we used an in vitro luciferase reporter assays of androgen receptor activity in kidney cell lines. This showed that DVT is capable of displacing DHT from SHBG in media, resulting in dose dependant increase in androgen receptor mediated luciferase reporter activity, thus confirming that non-steroidal ligands of SHBG can displace natural steroid from their SHBG binding site and may be of utility in promoting the biological activities of endogenous sex steroids.