Biochemical and biological evaluation of novel potent coumarin inhibitor of 17β-HSD type 1.

Human 17β-hydroxysteroid dehydrogenase (17β-HSD) type 1 is an enzyme that acts at the pre-receptor level. It catalyzes the NADPH-dependent reduction of the weak estrogen estrone into the most potent estrogen 17β-estradiol, which exerts proliferative effects via estrogen receptors. Overexpression of 17β-HSD type 1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis. 17β-HSD type 1 thus represents an attractive target for development of new drugs. Recently, we discovered that substituted coumarin derivatives potently and selectively inhibit 17β-HSD type 1. In the present study, we have performed additional biochemical and biological evaluation of the most promising coumarin derivative. First, we used an efficient method for isolation and purification of the active, soluble recombinant human 17β-HSD type 1 from Escherichia coli. This 17β-HSD type 1 showed a specific activity of 0.64±0.08 μmol min(-1) mg(-1) for estrone reduction in the presence of NADPH at pH 6.5, and a K(m) of 62 nM for estrone. Next, we evaluated the best of the coumarin-derivative inhibitors, showing its reversible and competitive inhibition of 17β-HSD type 1 reductive activity with a K(i) of 53 nM. We confirmed that this coumarin inhibitor acts not only in a cell-free assay, but also decreases endogenous 17β-HSD type 1 activity in human T-47D breast cancer cells. This inhibitor also reduced estrone dependent growth of T-47D cells after 48 h of incubation.