Structure-based Inhibitor Design for an Enzyme That Binds Different Steroids: A POTENT INHIBITOR FOR HUMAN TYPE 5 17β-HYDROXYSTEROID DEHYDROGENASE

Wei Qiu,Ming Zhou,Mausumi Mazumdar,Arezki Azzi,Dalila Ghanmi,Van Luu-The,Fernand Labrie,Sheng-Xiang Lin

doi:10.1074/jbc.m606784200

Abstract

Human type 5 17beta-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30 A. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9+/-1.4 nM, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy.

Highlights

According to the American Cancer Society, prostate cancer is the most common malignant tumor, excluding skin cancers, in American men
Overall Structure—The inhibitor EM1404 (3-carboxamido-1,3,5(10)-estratrien-17(R)-spiro-2Ј-(5Ј, 5Ј-dimethyl-6Ј-oxo)tetrahydropyran, C25H30O3N, Fig. 1A) was first obtained from a chemical screen [34] where it was shown to have an IC50 of 3.2 Ϯ 1.5 nM, the best among the inhibitors designed for 17␤-HSD5
The ternary complex structure of 17␤-HSD51⁄7EM14041⁄7NADP was determined by molecular replacement using a searching model obtained from the 17␤-HSD51⁄7 testosterone1⁄7NADP complex [18]

Summary

Introduction

According to the American Cancer Society, prostate cancer is the most common malignant tumor, excluding skin cancers, in American men. Prostatic tissue is able to efficiently transform the inactive adrenal steroid precursor dehydroepiandrosterone, into the active androgen dihydrotestosterone, in a new androgen biosynthetic pathway in which human type 5 17␤-hydroxysteroid dehydrogenase (17␤-HSD5) is involved (4 –7). It has been proven that this enzyme has a relatively high 17␤-HSD activity that transforms ⌬4-androstene-3,17-dione (4-dione, a weak androgen) to testosterone (a potent androgen) This enzyme has been immunocytochemically localized in human prostate tissues, and it has been proposed to contribute to local androgen formation in prostate (4 – 6, 16, 17). 17␤-HSD5 immunoreactivity was detected in 77% of carcinoma cells from prostate cancer tissues and was positively associated with the clinical stage of the disease [17] This provides some evidence that 17␤-HSD5 may possibly be involved in the increase of the local concentration of testosterone. A remarkable decrease in the oxidative 17␤-hydroxysteroid dehydrogenase activity was seen, whereas the reductive activity (including 17␤-HSD5 and 17␤HSD7) seemed to increase [28]

Methods

Results

Conclusion