Effects of aromatase inhibitors on in vitro steroidogenesis by Atlantic salmon (Salmo salar) gonadal and brain tissue

In order to assess the efficacy of selected aromatase inhibitors on Atlantic salmon (Salmo salar) ovarian and brain tissue, in vitro systems were developed for measuring 17beta-estradiol (E(2)) production by these tissues. Isolated vitellogenic follicles, or homogenised whole brains were incubated at 10 degrees C in complete Cortlands solution for 18 or 42 h respectively, and E(2) levels in the medium were determined by RIA. The addition of testosterone to the medium increased E(2) production in all preparations. E(2) production by whole brain homogenate was reduced by co-incubation with the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD), 4-androstene-4-ol-3,17-dione (OHA), aminoglutethimide, fadrozole or miconazole. Fadrozole, ATD, and OHA reduced E(2) production by vitellogenic follicles at a medium concentration of 0.1 microg mL(-1), whereas miconazole was only effective at 10 microg mL(-1). This study demonstrates a simple and rapid screening method for assessing the efficacy of aromatase inhibitors on fish tissues, and that the aromatase inhibitors ATD, OHA and fadrozole are potent inhibitors of both brain and gonadal aromatase in vitro, in Atlantic salmon.     

Dissociation of 19-hydroxy- 19-oxo-, and aromatizing-activities in human placental microsomes through the use of suicide substrates to aromatase

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.     

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.     

Metabolism of 19-methyl-substituted steroids by human placental aromatase

The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS)     

The constitutive 7-ethoxycoumarin 0-deethylase of human placental microsomes: relationship to the intermediary steps in steroid aromatization

All oxidative functions of aromatase, i.e., estrogen production, 19-oxygenated androgen production and 7-ethoxycoumarin deethylation, were inhibited in parallel in placental microsomes from non-smokers by the mechanism-based, time-dependent inactivators (suicide substrates) 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione. In contrast, the aromatase suicide substrate androst-4-ene-3,6,17-trione had little or no effect on the conversion of androst-4-ene-3,17-dione to 19-hydroxyandrost-4-ene-3,17-dione or on the conversion of the latter to 3,17-dioxoandrost-4-en-19-al while severely limiting the capacity for estrogen production from androst-4-ene-3,17-dione and 19-hydroxyandrost-4-ene-3,17-dione in such microsomal preparations. Androst-4-ene-3,6,17-trione, therefore, appears to uncouple the 19-hydroxylation of androgens from estrogen synthesis. This agent also produced only a minimal inhibition of 7-ethoxycoumarin deethylation, indicating that this major constitutive transformation of a xenobiotic chemical is associated with the steroid 19-hydroxylating function of the aromatase system.     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.     

Selection of 19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958): a potent aromatase inhibitor in vivo.

19-Mercaptoandrost-4-ene-3,17-dione (ORG 30365) has been reported to be both a competitive and irreversible inhibitor of aromatase. In comparison to the known aromatase inhibitors 4-hydroxy-androst-4-ene-3,17-dione (4OH-AD) and 1-methyl-1,4-androstadiene-3,17-dione (SH 489), ORG 30365 was found to be, respectively, about 16 and 8 times more active in vitro using human placental microsomes. Although the activity profile of ORG 30365 is very attractive, this compound was not selected for further development because it has limited pharmaceutical stability, which is probably due to its free--SH group and therefore a number of more stable dithio-derivatives of ORG 30365 have been synthesized. These derivatives are considered to be converted to ORG 30365 before they become active. The in vivo aromatase inhibiting activity of these derivatives was determined in hypophysectomized rats treated with the estrogen precursor dehydroepiandrosterone sulphate (DHEAS) using inhibition of cornification of vaginal epithelium as parameter. The 19-(ethyldithio)-derivative (ORG 30958) appeared to be the most active inhibitor in this series being twice as active as ORG 30365 and about 8 times as active as inhibitors like 4OH-AD and SH 489. Besides inhibition of cornification of vaginal epithelium ORG 30958 decreased ovarian aromatase and plasma E2 levels in DHEAS-treated hypophysectomized rats. Plasma estradiol levels were also lowered by ORG 30958 in dogs which were treated with pregnant mare serum gonadotrophin in order to induce pro-estrus. ORG 30958 displayed much less than 1/400th of the androgenic activity of testosterone propionate in immature castrated rats and appeared to be devoid of estrogenic and anti-estrogenic activity in ovariectomized mature rats. A twice daily dose of 1.5 mg ORG 30958/kg postponed ovulation in mature female rats. In conclusion: ORG 30958 is a potent aromatase inhibitor in vivo. It probably becomes active after cleavage of the -S-S- bond yielding ORG 30365 a potent irreversible aromatase inhibitor. ORG 30958 does not display other hormonal activities making it an attractive candidate for the treatment of estrogen-dependent diseases.     

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.     

Metabolism of 19-methyl substituted steroids and a proposal for the third aromatase monooxygenation

The article summarizes the results of recent studies on the metabolism of 10-ethylestr-4-ene-3,17-dione, 10-[(1R)-1-hydroxyethyl]-,and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione, in placenta. These compounds are the 19-methyl analogs of androstenedione, 19-hydroxyandrostenedione, and 19-oxoandrostenedione, respectively. No conversion of 10-ethylestr-4-ene-3,17-dione to either estrogens or oxygenated metabolites was detected. Both 10-[(1R)-1-hydroxyethyl]- and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione were oxygenated to 10-(1,1-dihydroxyethyl)estr-4-ene-3,17-dione and isolated following in situ dehydration as 10-acetylestr-4-ene-3,17-dione. Evidence for the involvement of aromatase in these conversions is discussed. No conversion of 10-acetylestr-4-ene-3,17-dione to either estrogens or other oxygenated products was detected. These results lead us to propose a new mechanism for the third aromatase monooxygenation. We propose that the third oxygenation is initiated by 1β-hydrogen abstraction at C₁ of 19,19-dihydroxyandrostenedione, followed by homolytic cleavage of the C₁₀−C₁₉ bond with concurrent formation of a Δ^1(10),4−3-ketosteroid and a C₁₉ carbon radical, and terminated by oxygen rebound at C₁₉.     

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Synthesis and evaluation of analogues of 4-androstene-3,17-dione as aromatase inhibitors

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.     

Catalytic differences between porcine blastocyst and placental aromatase isozymes.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.     

7-substituted 1,4,6-androstatriene-3,17-diones as enzyme-activated irreversible inhibitors of aromatase

7-Phenyl-1,4,6-androstatriene-3,17-dione (4), 7-benzyl-1,4,6-androstatriene-3,17-dione (5) and 7-phenethyl-1,4,6-androstatriene-3,17-dione (6) were synthesized and evaluated in vitro in human placental microsomes as enzyme-activated irreversible inhibitors of aromatase. The compounds were synthesized from appropriate 7-substituted 4,6-androstadiene-3,17-diones by reaction with DDQ under neutral conditions. All the compounds produced a first order inactivation of aromatase in the presence of NADPH but not in the absence of NADPH. Substrate 4-androstene-3,17-dione protected the enzyme from inactivation by the inhibitors. Furthermore, cysteine failed to protect aromatase from inactivation by compounds 5 and 6. In contrast, cysteine partially protected aromatase from inactivation by compound 4. Irreversibility studies illustrated the covalent nature of the inactivation by 4, 5 and 6. The above experimental evidence demonstrated that compounds 5 and 6 are effective enzyme-activated irreversible inhibitors of aromatase.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture.

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, 7-substituted 4,6-androstadiene-3,17-diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase activity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC50 values ranging from 490 nM to 4.5 microM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7 alpha-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-1,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC50 value of 80 nM. Other mechanism-based inhibitors, 7 alpha-(4'-amino)phenylthio-1,4-androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase activity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7 alpha-position.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP 19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys 119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles ofAsp309 and His480 in the aromatization of the steroid A ring.     

Induction of implantation by aromatase inhibitors in ovariectomized mice

The ability of aromatase inhibitors to induce implantation in mice was tested in animals in which implantation was delayed by ovariectomy and progesterone treatment. Implantation was consistently induced by 7 mg 4-hydroxyandrostene-3,17-dione (4-OH-A), 7 X 5 mg 1,4,6-androstatriene-3,17-dione (ATD) or 15 mg 4-acetoxyandrostene-3,17-dione, an activity comparable to that of 1 mg testosterone. In intact mice treated with 2 or 10 mg 4-OH-A or ATD/day from Day 2 of pregnancy (Day 1 = vaginal plug), the number and size of implantation sites were not affected. These results may not be necessarily due to inhibitory effects of the compounds on aromatase.     

Three-dimensional model of cytochrome P450 human aromatase

A three-dimensional (3-D) structure of human aromatase (CYP19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles of Asp309 and His480 in the aromatization of the steroid A ring.     

Synthesis and biochemical evaluation of the novel steroid androsta-4,6,8(9)-triene-3,17-dione

According to a proposed aromatisation mechanism by which estrogens are biosynthesized from androgens, the novel steroid androsta-4,6,8(9)-triene-3,17-dione (FCE 24918) should behave as a suicide substrate for aromatase. The synthesis of this triene steroid has been accomplished starting from androsta-4,7-diene-3,17-dione (4) by the acid-catalysed cleavage of the corresponding 7,8 alpha-epoxide, 5, and it was obtained together with androsta-4,6,8(14)-triene-3,17-dione (FCE 24917) as a side product. The time-dependent inactivation of placental aromatase by the two isomers was studied comparatively and showed that the 4,6,8(9)-triene moiety acts as a latent alkylating group.