Chemical synthesis and in vitro biological evaluation of a phosphorylated bisubstrate inhibitor of type 3 17β-hydroxysteroid dehydrogenase

Abstract

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD) catalyzes the last step in the biosynthesis of the potent androgen testosterone (T) by selectively reducing the C17 ketone of 4-androstene-3,17-dione (Δ⁴-dione), with NADPH as cofactor. This enzyme is thus an interesting therapeutic target for androgen-sensitive diseases. Using an efficient convergent chemical approach we synthesized a phosphorylated version of the best Δ⁴-dione/adenosine hybrid inhibitor of type 3 17β-HSD previously reported. An appropriately protected C2′ phosphorylated adenosine was first prepared and linked by esterification to the steroid Δ⁴-dione bearing an alkyl spacer. After three deprotection steps, the phosphorylated bisubstrate inhibitor was obtained. The inhibitory potency of this compound was evaluated on homogenated HEK-293 cells overexpressing type 3 17β-HSD and compared to the best non-phosphorylated bisubstrate inhibitor. Unexpectedly, the phosphorylated derivative was slightly less potent than the non-phosphorylated bisubstrate inhibitor of type 3 17β-HSD. Two hypotheses are discussed to explain this result: 1) the phosphorylated adenosine moiety does not interact optimally with the cofactor-binding site and 2) the bisubstrate inhibitors, phosphorylated or not, interact only with the substrate-binding site of type 3 17β-HSD.

Keywords::

• 17β-Hydroxysteroid dehydrogenase
• bisubstrate
• inhibitor
• steroid
• adenosine
• phosphate
• 17β-HSD type 3

Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR) (financial support and M.B., scholarship). We are grateful to Yannick Laplante and Dr Van Luu-The for providing HEK-293 cells overexpressing type 3 17β-HSD. We also thank Ms Sylvie Méthot for careful reading of the manuscript.