Endocrine Abstracts

Introduction: Recently, 11-oxygenated androgens (11OA), particularly 11-ketotestosterone (11KT), have emerged as contributors to the bioactive androgen pool. However, unlike classic androgens testosterone (T) and dihydrotestosterone (DHT), which bind to steroid hormone-binding proteins (sex hormone-binding globulin (SHBG), albumin), little is known about how these proteins interact with 11KT. Observational data from saliva (reflecting free hormone) and serum (reflecting total hormone) suggest that steroid hormone- binding proteins may influence 11KT bioavailability. We aimed to examine 11-ketotestosterone’s (11KT) interaction with steroid hormone receptors and the effect of SHBG on its androgenic activity.

Methods: We used in vitro luciferase reporter assays to assess 11KT’s ability to activate glucocorticoid (GR), mineralocorticoid (MR), progesterone (PR), and oestrogen receptors (ER) in HEK293T cells transiently coexpressing a specific receptor and a steroid receptor–inducible luciferase construct (Sri_Luc). Increasing concentrations of 11KT were added to the cell culture medium. To assess SHBG’s impact on 11KT’s androgenic activity, the cell culture medium of HEK293T cells stably expressing the androgen receptor (AR) and response element (ARE)-driven luciferase reporter was supplemented with a fixed concentration of 11KT (EC50=10-7 M) and varying SHBG concentrations (6–56 nM). The SHBG concentration range was designed to mimic both sub-physiological and physiological levels, reflecting typical values observed in healthy adult men (24–55 nM). Additionally, computational docking (AutoDock Vina) was used to model 11KT’s fit in the SHBG DHT-binding pocket. The docking parameters include the coordinates of the SHBG residue that anchors DHT in its binding pocket.

Results: In addition to its strong androgenic activity, 11KT was found to activate Erα and ERβ, but only at concentrations more than 1,000-fold higher than oestradiol. However, 11KT did not activate GR, MR, or PRα. Interestingly, SHBG co-treatment did not reduce 11KT-induced luciferase activity, as luminescence remained stable even at higher SHBG concentrations. In contrast, T and DHT showed dose-dependent decreases in luminescence with SHBG addition. Exploratory in silico modelling suggests that 11KT may bind SHBG with relatively high affinity.

Conclusion: 11KT is an AR agonist that can also activate ERα and ERβ, though only at supraphysiological concentrations. Our in vitro findings suggest that 11KT maintains its androgenic activity in the presence of SHBG. Albumin, which also binds classic androgens, may play a more prominent role in 11KT binding. Further experimental validation is needed to confirm these findings and investigate the in silico prediction of 11KT’s relatively high SHBG binding affinity.

Keywords: Steroid hormone-binding proteins, 11-oxygenated androgens, free hormone hypothesis, androgen bioactivity, steroid hormone receptors