Insight into the mode of action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1

17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the biosynthesis of estradiol, the major bioactive endogenous estrogen in mammals, and constitutes an interesting therapeutic target for estrogen-dependent diseases. A steroidal derivative, 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestra-1,3,5(10)-trien-16-yl]methyl} benzamide (PBRM), has recently been described as a non-estrogenic, irreversible inhibitor of 17β-HSD1. However, the mode of action of this inhibitor and its selectivity profile have not yet been elucidated. We assessed PBRM potency via in vitro kinetic measurements. The mechanism of enzyme inactivation was also investigated using interspecies (human, mouse, pig and monkey) comparisons via both in vitro assays and in silico analysis. Mouse and human plasma protein binding of PBRM was determined, whereas its selectivity of action was studied using a wide range of potential off-targets (e.g. GPCR, hERG, CYPs, etc.). The affinity constant (Ki=368nM) and the enzyme inactivation rate (kinact=0.087min-1) values for PBRM were determined with purified 17β-HSD1. PBRM was found to be covalently linked to the enzyme. A long delay period (i.e. 3-5days) is required to recover 17β-HSD1 activity following a pretreatment of breast and placenta cell lines with PBRM. Mechanistic analyses showed important interspecies differences of 17β-HSD1 inhibition which support the importance of inactivation for PBRM effect. Evidences of the potency and selectivity of action presented herein for this first non-estrogenic and steroidal covalent irreversible inhibitor of 17β-HSD1 warrant its further development as a potential drug candidate for estrogen-dependent disorders.