A three-dimensional model of CYP19 aromatase for structure-based drug design

Aromatase (CYP450_arom, CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme–substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.     

Aromatase--key enzyme of estrogen biosynthesis

Estrogens control a large range pivotal life functions as reproductive development and fertility, bone growth and sexual behavior. Aromatase is a key enzyme of estrogen biosynthesis. The property, structure and reaction mechanism of aromatase as well as detailed structure of human aromatase cytochrome P450 gene (CYP19) was discussed in this article. It was pointed that unique human CYP19 gene expression results from presence of many tissue specific promoters and alternative splicing. The molecular mechanism of control aromatase cytochrome P450 gene expression in various species ovaries, testes and human adipose tissue and placenta was discussed in details. Because of a very important role of estrogen in breast cancer a molecular base of aberrant expression CYP19 gene in breast tumor and adipose tissue proximal to breast tumor and potential possibility of pharmacological silencing of this gene expression was discussed in the article.     

The ferrous-oxy complex of human aromatase

In this communication, we document the self-assembly of heterologously expressed truncated human aromatase (CYP19) into nanometer scale phospholipids bilayers (Nanodiscs). The resulting P450 CYP19 preparation is stable and can tightly associate with the substrate androstenedione to form a nearly complete high-spin ferric protein. Ferrous CYP19 in Nanodiscs was mixed anaerobically in a rapid-scan stopped-flow with atmospheric dioxygen and the formation of the ferrous-oxy complex observed. First order decay of the oxy-complex to release superoxide and regenerate the ferric enzyme was monitored kinetically. Surprisingly, the ferrous-oxy complex of aromatase is more stable than that of hepatic CYP3A4, opening the path to precisely determine the biochemical and biophysical properties of the reaction cycle intermediates in this important human drug target.     

Three-dimensional model of the human aromatase enzyme and density functional parameterization of the iron-containing protoporphyrin IX for a molecular dynamics study of heme-cysteinato cytochromes

Mammalian cytochromes P450 (CYP) are enzymes of great biological and pharmaco-toxicological relevance. Due to their membrane-bound nature, the structural characterization of these proteins is extremely difficult, and therefore computational techniques, such as comparative modeling, may help obtaining reliable structures of members of this family. An important feature of CYP is the presence of an iron-containing porphyrin group at the enzyme active site. This calls for quantum chemical calculations to derive charges and parameters suitable for classical force field-based investigations of this proteins family. In this report, we first carried out density functional theory (DFT) computations to derive suitable charges for the Fe2+-containing heme group of P450 enzymes. Then, by means of the homology modeling technique, and taking advantage of the recently published crystal structure of the human CYP2C9, we built a new model of the human aromatase (CYP19) enzyme. Furthermore, to study the thermal stability of the new model as well as to test the suitability of the new DFT-based heme parameters, molecular dynamics (MD) simulations were carried out on both CYP2C9 and CYP19. Finally, the last few ns of aromatase MD trajectories were investigated following the essential dynamics protocol that allowed the detection of some correlated motions among some protein domains.     

Aromatase expression in a human osteoblastic cell line increases in response to prostaglandin E(2) in a dexamethasone-dependent fashion

Recent progress supports the importance of local estrogen secretion in human bone tissue to increase and maintain bone-mineral density. In a previous report, we found that forskolin (FSK) synergistically induces aromatase (CYP19: a rate-limiting enzyme for estrogen synthesis) expression in dexamethasone (Dex) dependent manner in a human osteoblastic cell line, SV-HFO [Watanabe M, Ohno S, Nakajin S. Forskolin and dexamethasone synergistically induce aromatase (CYP19) expression in the human osteoblastic cell line SV-HFO. Eur J Endocrinol 2005;152:619-24]. In this report, we investigated whether prostaglandin (PG) E(2) induces estrogen production, in other words, if PGE(2) exerts the same effect as FSK because PGE(2) is the major prostanoid in the bone and is one of the key molecules in the osteoblast. We found PGE(2) up-regulates aromatase activity synergistically, but this up-regulation depends on Dex. CYP19 gene expression was also increased synergistically by Dex and PGE(2). Promoter I.4 was activated synergistically by PGE(2) and Dex. PGE(2) receptor, EP(1), EP(2) and EP(4) were involved in the up-regulation of aromatase activity in response to PGE(2) in a Dex-dependent manner. The cAMP-PKA pathway and Ca(2+) signaling pathway were involved in the up-regulation of aromatase activity in response to PGE(2). Furthermore, glucocorticoid response element on promoter I.4 sequence was an essential minimum requirement for its activity and synergism of PGE(2) and Dex. These findings are the first report on osteoblastic cell line which uses predominantly promoter I.4 to drive aromatase expression. These findings also suggest that endogenous PGE(2) produced in bone mainly may synergistically support local estrogen production in osteoblastic cells in the presence of glucocorticoid.     

Development of a new class of aromatase inhibitors: design, synthesis and inhibitory activity of 3-phenylchroman-4-one (isoflavanone) derivatives

Aromatase (CYP19) catalyzes the aromatization reaction of androgen substrates to estrogens, the last and rate-limiting step in estrogen biosynthesis. Inhibition of aromatase is a new and promising approach to treat hormone-dependent breast cancer. We present here the design and development of isoflavanone derivatives as potential aromatase inhibitors. Structural modifications were performed on the A and B rings of isoflavanones via microwave-assisted, gold-catalyzed annulation reactions of hydroxyaldehydes and alkynes. The in vitro aromatase inhibition of these compounds was determined by fluorescence-based assays utilizing recombinant human aromatase (baculovirus/insect cell-expressed). The compounds 3-(4-phenoxyphenyl)chroman-4-one (1h), 6-methoxy-3-phenylchroman-4-one (2a) and 3-(pyridin-3-yl)chroman-4-one (3b) exhibited potent inhibitory effects against aromatase with IC(50) values of 2.4 μM, 0.26 μM and 5.8 μM, respectively. Docking simulations were employed to investigate crucial enzyme/inhibitor interactions such as hydrophobic interactions, hydrogen bonding and heme iron coordination. This report provides useful information on aromatase inhibition and serves as a starting point for the development of new flavonoid aromatase inhibitors.     

Articular cartilage chondrocytes express aromatase and use enzymes involved in estrogen metabolism

Introduction

Sex hormones, especially estrogens, have been implicated in articular cartilage metabolism and the pathogenesis of postmenopausal osteoarthritis. The conversion by aromatase (CYP19A1) of androstenedione into estrone (E1) and of testosterone into 17β-estradiol (E2) plays a key role in the endogenous synthesis of estrogens in tissue.

Methods

We analyzed the expression of aromatase (CYP19A1) in immortalized C-28/I2 and T/C-28a2 chondrocytes, as well as in cultured primary human articular chondrocytes and human articular cartilage tissue, by means of RT-PCR, Western blotting and immunohistochemistry. By means of quantitative RT-PCR and enzyme-linked immunosorbent assay, we also determined whether the aromatase inhibitor letrozole influences estrogen metabolism of cultured chondrocytes in immortalized C-28/I2 chondrocytes.

Results

Aromatase mRNA was detected in both immortalized chondrocyte cell lines, in cultured primary human chondrocytes, and in human articular cartilage tissue. By means of Western blot analysis, aromatase was detected at the protein level in articular cartilage taken from various patients of both sexes and different ages. Cultured primary human articular chondrocytes, C-28/I2 and T/C-28a2, and human articular cartilage tissue reacted with antibodies for aromatase. Incubation of C-28/I2 chondrocytes with 10⁻¹¹ M to 10⁻⁷ M letrozole as an aromatase inhibitor revealed significantly increased amounts of the mRNAs of the enzyme cytochrome P4501A1 (CYP1A1), which is involved in the catagen estrogen metabolism, and of the estrogen receptors ER-α and ER-β. Concomitantly, synthesis of estrone (E1) was significantly downregulated after incubation with letrozole.

Conclusions

We demonstrate that human articular cartilage expresses aromatase at the mRNA and protein levels. Blocking of estrone synthesis by the aromatase inhibitor letrozole is counteracted by an increase in ER-α and ER-β. In addition, CYP1A1, an enzyme involved in catabolic estrogen metabolism, is upregulated. This suggests that articular chondrocytes use ERs functionally. The role of endogenous synthesized estrogens in articular cartilage health remains to be elucidated.     

Inhibitory effect of Rhus verniciflua Stokes extract on human aromatase activity; butin is its major bioactive component

Rhus verniciflua Stokes has been used as a traditional herbal medicine in Asia. In this study, the effect of R. verniciflua extract on human aromatase (cytochrome P450 19, CYP19) activity was investigated to elucidate the mechanism for the effect of R. verniciflua extract on androgen hormone levels. Androstenedione was used as a substrate and incubated with R. verniciflua extract in cDNA-expressed CYP19 supersomes in the presence of NADPH, and estrone formation was measured using liquid chromatography–tandem mass spectrometry. R. verniciflua extract was assessed at concentrations of 10–1000 μg/mL. The resulting data showed that R. verniciflua extract inhibited CYP19-mediated estrone formation in a concentration-dependent manner with an IC₅₀ value of 136 μg/mL. Subsequently, polyphenolic compounds from R. verniciflua extract were tested to identify the ingredients responsible for the aromatase inhibitory effects by R. verniciflua extract. As a result, butin showed aromatase inhibitory effect in a concentration-dependent manner with an IC₅₀ value of 9.6 μM, whereas the inhibition by other compounds was negligible. These results suggest that R. verniciflua extract could modulate androgen hormone levels via the inhibition of CYP19 activity and butin is a major ingredient responsible for this activity.     

两种泥鳅芳香化酶基因的克隆与时空表达

鱼类的性别分化易受发育环境的影响。向性成熟的泥鳅和大鳞副泥鳅个体注射绒毛膜促性腺激素,获得卵子和精子进行人工授精。把胚胎分别置于20℃、25℃和30℃条件下,使其发育。经性腺检查发现随着温度的升高两种泥鳅中雄性个体所占的比例明显升高,获得明显的偏雄比率群体。根据已知细胞色素P450芳香化酶CYP19 b基因序列设计嵌套简并引物用巢式PCR扩增并克隆出了两种泥鳅的CYP19 b的DNA片段。MaCYP19 b片段和Pd-CYP19 b片段分别长1337bp和1473bp。在此基础上用各自的特异引物克隆出两种泥鳅CYP19 b的相应cDNA片段。通过基因组DNA和cDNA序列的比较证明两种泥鳅的CYP19 b基因均包含三个内含子和四个外显子,编码的蛋白质序列长145氨基酸残基。以GAPDH基因为对照,分别对两种泥鳅成体组织和不同发育阶段的胚胎的CYP19 b进行了半定量RT-PCR表达分析,结果表明泥鳅CYP19 b基因只在成体泥鳅卵巢、肾以及原肠胚和神经胚中表达。大鳞副泥鳅CYP19 b基因在成体的脑、卵巢和肾以及神经胚和卵黄吸收期表达。这些结果为揭示细胞色素P450芳香化酶基因与环境性别决定机制的关系奠定了基础。       

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E₂ (PGE₂) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE₂ can bind to four receptor subtypes, EP₁–EP₄, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE₂, dexamethasone, and agonists for the EP₁ and EP₂ receptor subtypes. An EP₁ antagonist, SC-19220, inhibited the induction of enzyme activity by PGE₂ or 17-phenyltrinor-PGE₂, an EP₁ agonist. Sulprostone, an EP₃ agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.     

Expression profiles of key candidate genes involved in steroidogenesis during follicular atresia in the pig ovary

More than 99?% of follicles in mammalian ovaries undergo a degenerative process known as atresia, and thus only a limited number of ovarian follicles actually ovulate after full growth and development. The endocrinological regulatory mechanisms involved in follicular development have been studied extensively, but the precise and systematic molecular mechanisms of steroidogenesis enzymes involved in atresia are unclear. In the present study, we examined whether and how the steroidogenesis enzymes are involved in porcine ovary follicular atresia. Expression of steroidogenic acute regulatory protein, CYP11, CYP17, 3β-hydroxysteroid dehydrogenase (3β-HSD), CYP19, as well as related pituitary and ovarian hormone receptors were quantified in ovaries. During porcine follicular atresia, expressions of P450 cholesterol side chain cleavage enzyme, progesterone and androgen receptors increased significantly during the late atretic stage, while the expression of aromatase and follicle-stimulating hormone receptors decreased significantly in the early stage. These data suggested that the regulation of aromatase by follicle-stimulating hormone might induce follicular atresia, and that progesterone and androgen production further promoted follicular atresia. Additionally, a correlation analysis indicated a large and complex interactive network among these genes and the endocrinological microenvironment of the follicles. Significant correlations were observed between expression of steroidogenic enzymes and their receptors, and also between progesterone and 17β-estradiol (E2) levels in follicular fluid. Taken together, these results suggest that CYP19 plays a role during early atresia by regulating the production of E2, whereas CYP11 and 3β-HSD increase atresia progression by increasing progesterone levels.