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

Abstract

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 1.30 Å . 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.According to the American Cancer Society, prostate cancer is the most common malignant tumor, excluding skin cancers, in American men. It is estimated that ϳ232,090 new cases of prostate cancer will be diagnosed in the US during 2005 with 30,350 deaths (www.cancer.org). The growth and function of the prostate is dependent on androgens, which play important roles in the pathogenesis of prostate cancer (1). Androgen withdrawal triggers the programmed cell apoptosis in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen withdrawal; however, they do retain the cellular machinery necessary to activate the apoptotic cascade when sufficiently damaged by exogenous agents (2).In prostate cancer, the balance between cell proliferation and programmed cell death is lost, thus more cell proliferation results in net cell growth. An important finding in prostate cancer research is that ϳ50% of dihydrotestosterone, the most potent natural androgen, remains in the prostatic tissue of patients who have undergone surgical or chemical castration (3). In fact, prostatic tissue is able to efficiently transform the inactive adrenal steroid precursor dehydroepiandrosterone, into the active androgen dihydrotestosterone, in a new androgen biosynthetic pathwa...

Selective Inhibition of Human Type-5 17β-Hydroxysteroid Dehydrogenase (AKR1C3) by Baccharin, a Component of Brazilian Propolis

“…The largest impairment of the affinity for 1 by Phe311Leu may also result from the inability of AKR1C3 to undergo a proper induced-fit conformational change upon the binding of 1, because Phe311 is one of the residues involved in the induced fit to accommodate several ligands in crystallographic studies of these isoforms. 14,16 Inhibitory Effects of Baccharin (1) on Cellular Metabolism and Proliferation by AKR1C3. Compound 1, artepillin C (2), and drupanin (3) were compared for their effects on cellular metabolism with an isoprenyl aldehyde, farnesal, which is rapidly reduced into farnesol by AKR1C3 in vitro and in MCF7 cells.…”

Androgen synthesis in adrenarche

“…The 17βHSD activity of 17βHSD5 is quite labile in vitro [107], hence its activity in androgen biosynthesis was initially controversial, but it appears to be responsible for low levels of testosterone synthesis in the adrenal [107,108], ovary [109], and adipose tissue [110], and may also convert androstenedione to testosterone in muscle. While there has been considerable interest in the potential role of 17βHSD5 in the intra-prostatic metabolism of androgens [46,111], and its crystallographic structure has been determined [112], its potential role in adrenarche has not been investigated.…”

Structures of complexes of type 5 17β-hydroxysteroid dehydrogenase with structurally diverse inhibitors: insights into the conformational changes upon inhibitor binding

“…At present, there are 30 crystal structures of 17-HSD5 in the Protein Data Bank (Lovering et al, 2004;Komoto et al, 2004Komoto et al, , 2006Qiu et al, 2004Qiu et al, , 2007Chen et al, 2012;Liedtke et al, 2013;Jamieson et al, 2012;Heinrich et al, 2013;Flanagan et al, 2012Flanagan et al, , 2014Jackson et al, 2012). Lovering and coworkers reported the first crystal structure of 17-HSD5 (Lovering et al, 2004).…”