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J. Biol. Chem., Vol. 282, Issue 11, 8368-8379, March 16, 2007

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Structure-based Inhibitor Design for an Enzyme That Binds Different Steroids

A POTENT INHIBITOR FOR HUMAN TYPE 5 17-HYDROXYSTEROID DEHYDROGENASE^*

From the Canadian Institutes of Health Research Group in Molecular Endocrinology, Laval University Medical Center, Centre Hospitalier de Universités de Québec and Laval University, Quebec G1V 4G2, Canada

Human type 5 17-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 1.30Å_. 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 K_i 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.

Received for publication, July 17, 2006 , and in revised form, November 29, 2006.

The atomic coordinates and structure factors (code 1ZQ5 and 2FGB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Canadian Institute of Health Research and EndoRecherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Figs. S1 and S2, and additional references.

¹ Both authors contributed equally to this work.

² Present address: Structural Genomics Consortium, 100 College St., Banting Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada.

³ To whom correspondence should be addressed: CHUL Research Center, 2705 Blvd. Laurier, Ste-Foy, Quebec G1V 4G2, Canada. Tel.: 418-654-2296; Fax: 418-654-2761; E-mail: sxlin{at}crchul.ulaval.ca.

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