Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Inhibitors of human and rat testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD) as potential agents for prostatic cancer

In a screening programme for inhibitors of human testis 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3), as potential agents for the treatment of hormone-dependent prostatic cancer, we have used crude human testis microsomal 17beta-hydroxysteroid dehydrogenase as a convenient source of the enzyme. Crude human enzyme was shown to have a similar substrate profile to recombinant Type 3 17beta-HSD from the same source as determined by the low Km/Vmax ratio for the reduction of androstenedione compared to the oxidation of testosterone, and a low level of activity in reduction of oestrone. Screening of a wide range of compounds of different structural types as potential inhibitors of the microsomal enzyme in the reduction step revealed that certain p-benzoquinones and flavones/isoflavones were potent inhibitors of the enzyme, diphenyl-p-benzoquinone (2.7 microM), phenyl-p-benzoquinone (5.7 microM), 7-hydroxyflavone (9.0 microM), baicalein (9.3 microM) and biochanin A (10.8 microM). Some structure-activity relationships within the flavone/isoflavone series are discussed. Studies with rat testis microsomal 17beta-HSD showed that it differed from the human enzyme mainly in its greater ability to accept oestrone as substrate and the pH-optimum for oxidation of testosterone. It was found to be much less sensitive to inhibition by the compounds studied so negating it use as a more readily available tissue for the screening of potential inhibitors.     

New inhibitors of fungal 17beta-hydroxysteroid dehydrogenase based on the [1,5]-benzodiazepine scaffold

The synthesis and activity of a new series of non-steroidal inhibitors of 17beta-hydroxysteroid dehydrogenase that are based on a 1,5-benzodiazepine scaffold are presented. Their inhibitory potential was screened against 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. Some of these compounds are potent inhibitors of 17beta-HSDcl activity, with IC50 values in the low micromolar range and represent promising lead compounds that should be further developed and investigated as inhibitors of human 17beta-HSD isoforms, which are the enzymes associated with the development of many hormone-dependent and neuronal diseases.     

Aromatase inhibition by bioavailable methylated flavones

Previous studies have shown chrysin, 7-hydroxyflavone and 7,4'-dihydroxyflavone to be the most potent flavonoid inhibitors of aromatase. However, very poor oral bioavailability is a major limitation for the successful use of dietary flavonoids as chemopreventive agents. We have recently shown that methylated flavones, including 5,7-dimethoxyflavone, 7-methoxyflavone and 7,4'-dimethoxyflavone, are much more resistant to metabolism than their unmethylated analogs and have much higher intestinal absorption. In this study, we examined these fully methylated flavones as potential aromatase inhibitors for the prevention and/or treatment of hormone-dependent cancers. Whereas 5,7-dimethoxyflavone had poor effect compared to its unmethylated analog chrysin, 7-methoxyflavone and 7,4'-dimethoxyflavone were almost equipotent to their unmethylated analogs with IC(50) values of 2-9 microM. Thus, some fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents.     

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.     

Two homologous fungal carbonyl reductases with different substrate specificities

Two homologous fungal short-chain dehydrogenase/reductase (SDR) proteins have been cloned from the fungus Curvularia lunata (teleomorph: Cochliobolus lunatus) and expressed in Escherichia coli: trihydroxynaphthalene reductase (3HNR), an enzyme of the melanin biosynthetic pathway that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, and 17beta-hydroxysteroid dehydrogenase (17beta-HSDcl), which acts on androgens and estrogens, although its physiological substrate remains to be defined. In the present study, we have compared the structures, specificities to substrates and inhibitors, temperature and pH optima of 3HNR and 17beta-HSDcl. Sequence analysis and homology-built models revealed that these enzymes are highly similar. Both of these enzymes are NADP(H)-preferring reductases and act on steroids at position 17; however, 17beta-HSDcl presented considerably higher initial rates than 3HNR. In vitro, 17beta-HSDcl preferably catalyzed the reduction of 4-estrene-3,17-dione, while the best steroid substrate for 3HNR was 5alpha-androstane-3,17-dione. On the other hand, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), an artificial substrate of 3HNR, was oxidized rapidly by 3HNR, while it was not a substrate for 17beta-HSDcl. Additionally, our data show that tricyclazole, a specific inhibitor of 3HNR, is 100-fold less effective for 17beta-HSDcl inhibition, while flavonoids can inhibit both 3HNR and 17beta-HSDcl. We have also examined the effects of temperature and pH on the oxidation of DDBO by 3HNR and the oxidation of 4-estrene-17beta-ol-3-one by 17beta-HSDcl. The apparent optimal temperature for 3HNR activity was between 25 and 30 degrees C, while it was between 40 and 45 degrees C for 17beta-HSDcl activity. The pH optimum of 3HNR activity was between 8 and 9, and for 17beta-HSDcl, between 7 and 8. Our data show that in spite of high homology and similar backbone structure, differences between 3HNR and 17beta-HSDcl were not only in substrate specificities, but also in temperature and pH optima.     

Cinnamic acid esters as potent inhibitors of fungal 17beta-hydroxysteroid dehydrogenase--a model enzyme of the short-chain dehydrogenase/reductase superfamily

We present the synthesis of a new family of nonsteroidal inhibitors of 17beta-hydroxysteroid dehydrogenase, designed from flavones and chalcones. Their inhibitory potential was screened on 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a model enzyme of the short-chain dehydrogenase/reductase superfamily. In a series of cinnamates and related coumarin-3-carboxylates, a number of compounds proved to be potent inhibitors of both the oxidative and reductive reactions catalyzed by 17beta-HSDcl, with IC(50) values in the low micromolar range.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-hydroxyphenyl ketones as potent and specific inhibitors of the type 3 of 17beta-hydroxysteroid dehydrogenase (17beta-HSD3)

We report the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as potential inhibitors of the enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In particular, we evaluated compounds against the catalysis of the conversion of androstenedione (AD) to testosterone (T) [17beta-HSD type 3 (17beta-HSD3)], furthermore, in an effort to determine the specificity of our compounds, we evaluated the ability of the compounds to inhibit the catalysis of the conversion of estrone (E1) to estradiol (E2) [17beta-HSD type 1 (17beta-HSD1)] as well as the conversion of dehydroepiandrosterone (DHEA) to AD [by 3beta-hydroxysteroid dehydrogenase (3beta-HSD)]. The results of our study suggest that the synthesised compounds are, in general, able to inhibit 17beta-HSD3 whilst being weak inhibitors of 17beta-HSD1. Against 3beta-HSD, we discovered that all of the synthesised compounds were weak inhibitors (all were found to possess less than 50% inhibition at [I]=500 microM). More specifically, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (15) was the most potent against 17beta-HSD3 (IC(50)=2.9 microM) whilst possessing poor inhibitory activity against 17beta-HSD1 ( approximately 36% inhibitory activity against this reaction at [I]=100 microM) and less than 10% inhibition for the conversion of DHEA to AD. We have therefore provided good lead compounds in the design and synthesis of novel non-steroidal inhibitors of 17beta-HSD3.     

No evidence for the in vivo activity of aromatase-inhibiting flavonoids

Measurements of the aromatase-inhibiting and antioxidative capacities of flavonoids in vitro showed that slight changes in flavonoid structure may result in marked changes in biological activity. Several flavonoids such as 7-hydroxyflavone and chrysin (5,7-dihydroxyflavone) were shown to inhibit the formation of 3H-17beta-estradiol from 3H-androstenedione (IC(50)<1.0 microM) in human choriocarcinoma JEG-3 cells and in human embryonic kidney cells HEK 293 transfected with human aromatase gene (Arom+HEK 293). Flavone and quercetin (3,3',4',5,7-pentahydroxyflavone) showed no inhibition (IC(50)>100 microM). None of the requirements for optimal antioxidative capacity (2,3-double bond with 4'-hydroxy group, 3-hydroxyl group, 5,7-dihydroxy structure and the orthodihydroxy structure in the B-ring) is relevant for the maximum inhibition of aromatase by flavonoids. After oral administration to immature rats at a dose of 50 mg/kg body weight, which considerably exceeds amounts found in daily human diets, neither aromatase-inhibiting nonestrogenic flavonoids, such as chrysin, nor estrogenic flavonoids, such as naringenin and apigenin, induced uterine growth or reduced estrogen- or androgen-induced uterine growth. The inability of flavonoids to inhibit aromatase and, consequently, uterine growth in short-term tests may be due to their relatively poor absorption and/or bioavailability.     

Chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities

Chalcones were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. In the present study, we have demonstrated for the first time that chalcones are potent inhibitors of aromatase and 17beta-hydroxysteroid dehydrogenase activities: these enzymes being considered as important targets in the metabolic pathways of human mammary hormone-dependent cells. Our results showed that naringenin chalcone and 4-hydroxychalcone were the most effective aromatase and 17beta-hydroxysteroid dehydrogenase inhibitors with IC50 values of 2.6 and 16 microM respectively. In addition, inhibitory effects of some flavones and flavanones were compared to those of the corresponding chalcones. A structure-activity relationship was established and regions or/and substituents essential for these inhibitory activities were determined.     

Triphenyltin and Tributyltin inhibit pig testicular 17beta-hydroxysteroid dehydrogenase activity and suppress testicular testosterone biosynthesis

We previously reported that tributyltin chloride (TBT) and triphenyltin chloride (TPT) powerfully suppressed human chorionic gonadotropin- and 8-bromo-cAMP-stimulated testosterone production in pig Leydig cells at concentrations that were not cytotoxic [Nakajima Y, Sato Q, Ohno S, Nakajin S. Organotin compounds suppress testosterone production in Leydig cells from neonatal pig testes. J Health Sci 2003;49:514-9]. This study investigated the effects of these organotin compounds on the activity of enzymes involved in testosterone biosynthesis in pig testis. At relatively low concentrations of TPT, 17beta-hydroxysteroid dehydrogenase (17beta-HSD; IC(50)=2.6microM) and cytochrome P450 17alpha-hydroxylase/C(17-20) lyase (IC(50)=117microM) activities were inhibited, whereas cholesterol side-chain cleavage cytochrome P450 and 3beta-HSD/Delta(4)-Delta(5) isomerase activities were less sensitive. Overall, TPT was more effective than TBT. TPT also inhibited both ferredoxin reductase and P450 reductase activities at concentrations over 30microM; however, TBT had no effect, even at 100microM. The IC(50) values of TPT were estimated to be 25.7 and 22.8microM for ferredoxin reductase and P450 reductase, respectively. The inhibitory effect of TPT (30microM) on microsomal 17beta-HSD activity from pig testis was eliminated by pretreatment with the reducing agents dithiothreitol (1mM) and dithioerythritol (1mM). On the other hand, TPT (0.03microM) or TBT (0.1microM) exposure suppressed the testosterone production from androstenedione in pig Leydig cells indicating that these organotins inhibit 17beta-HSD activity in vivo as well as in vitro, and the IC(50) values of TPT and TBT for 17beta-HSD activity were estimated to be 48 and 114nM, respectively. Based on these results, it appears possible that the effects of TBT and TPT are largely due to direct inhibition of 17beta-HSD activity in vivo.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a series of 4-hydroxyphenyl ketones as potential inhibitors of 17beta-hydroxysteroid dehydrogenase type 3 (17beta-HSD3)

We report the preliminary results of the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as inhibitors of the isozyme of the enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD) responsible for the conversion of androstenedione (AD) to testosterone (T), more specifically type 3 (17beta-HSD3). The results of our study suggest that we have synthesised compounds which are, in general, potent inhibitors of 17beta-HSD3, in particular, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (8) was the most potent (IC(50) = 2.86 +/- 0.03 microM). We have therefore provided good lead compounds in the synthesis of novel non-steroidal inhibitors of 17beta-HSD3.     

Design, synthesis and biological evaluation of bis(hydroxyphenyl) azoles as potent and selective non-steroidal inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) for the treatment of estrogen-dependent diseases

The 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the reduction of the weakly active estrone (E1) into the most potent estrogen, 17beta-estradiol (E2). E2 stimulates the growth of hormone-dependent diseases via activation of the estrogen receptors (ERs). 17beta-HSD1 is often over-expressed in breast cancer cells. Thus, it is an attractive target for the treatment of mammary tumours. The combination of a ligand- and a structure-based drug design approach led to the identification of bis(hydroxyphenyl) azoles as potential inhibitors of 17beta-HSD1. Different azoles and hydroxy substitution patterns were investigated. The compounds were evaluated for activity and selectivity with regard to 17beta-HSD2, ERalpha and ERbeta. The most potent compound is 3-[5-(4-hydroxyphenyl)-1,3-oxazol-2-yl]phenol (18, IC(50)=0.31 microM), showing very good selectivity, high cell permeability and medium CaCo-2 permeability.     

Parallel solid-phase synthesis of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-one derivatives for inhibition of type 3 17beta-hydroxysteroid dehydrogenase.

Type 3 17beta-hydroxysteroid dehydrogenase (17beta-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Delta(4)-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3beta-peptido-3alpha-hydroxy-5alpha-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23-58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17beta-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3beta-(N-heptanoyl-L-phenylalanine-L-leucine-aminomethyl)-3alpha-hydroxy-5alpha-androstan-17-one (42) inhibited the enzyme with an IC(50) value of 227nM, which is twice as potent as the natural substrate Delta(4)-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR(+)) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 microM (less than previously reported type 3 17beta-HSD inhibitors) and, interestingly, no proliferation at 0.1 microM.     

Dose-response effects of phytoestrogens on the activity and expression of 3beta-hydroxysteroid dehydrogenase and aromatase in human granulosa-luteal cells

There is evidence that certain phytoestrogens can inhibit key steroidogenic enzymes although most studies have been carried out on microsomal or purified enzyme preparations, some using cell lines. This study was designed to test the hypothesis that low doses of phytoestrogens, at concentrations that would be attained through the diet, could inhibit 3beta-hydroxysteroid dehydrogenase (HSD) and/or aromatase in primary cultures of human granulosa-luteal (GL) cells and that this effect was due to a decrease in the expression of these proteins. Based on published evidence, eight compounds were selected for investigation and these included the flavones apigenin and quercetin, the isoflavones genistein, biochanin A and daidzein, the lignans, enterodiol and enterolactone, and the mycotoxin zearalenone. Human GL cells were cultured for 48 h in the presence of these phytoestrogens at concentrations ranging from 0.01 to 100 microM and after addition of fresh media the conversion of pregnenolone to progesterone or androstenedione to oestradiol over a 4h period was measured. Biochanin A was the only phytoestrogen that displayed any dose-dependent inhibition of 3beta-HSD, others showing inhibition at doses >/=10 microM. Apigenin and quercetin only inhibited aromatase/17beta-HSD at high doses as did genistein, biochanin A and daidzein. The lignans had weak inhibitory effects on aromatase/17beta-HSD, whilst zearalenone showed potent inhibition at 0.1 microM. Phytoestrogens did not exert any significant effects on protein expression of 3beta-HSD or aromatase as determined by Western blots. It is concluded that steroidogenic enzymes are inhibited by phytoestrogens in primary cultures of human GL cells but these cells are less sensitive to the effects of phytoestrogens than cell-free systems. This may be due to poor lipid solubility or cellular metabolism. We have also shown for the first time that phytoestrogens do not act by inhibiting the cellular concentration of 3beta-HSD and aromatase even though exposure time would have allowed for changes in gene expression.     

Effects of flavonoid phytochemicals on cortisol production and on activities of steroidogenic enzymes in human adrenocortical H295R cells

Inhibitory effects of flavonoid phytochemicals, flavones, flavonols and isoflavones on cortisol production were examined in human adrenal H295R cells stimulated with di-buthylyl cAMP. In addition, the inhibitory effects of these chemicals on the activity of P450scc, 3beta-HSD type II (3beta-HSD II), P450c17, P450c21 and P45011beta, steroidogenic enzymes involved in cortisol biosynthesis, were examined in the same cells. Exposure to 12.5 microM of the flavonoids 6-hydroxyflavone, 4'-hydroxyflavone, apigenin, daidzein, genistein and formononetin significantly decreased cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8 and 11.3%, respectively), and biochanin A significantly decreased cortisol production (by 47.3%) at a concentration of 25 microM without any significant cytotoxic effects or changes in cell number. Daidzin, the 7-glucoside of daidzein, did not alter cortisol production by H295R cells at concentrations over 10 microg/ml (24 microM). Daidzein-induced reduction of cortisol production by H295R cells was not inhibited by the estrogen receptor antagonist ICI 182,780. The flavonoids 6-hydroxyflavone, daidzein, genistein, biochanin A and formononetin strongly and significantly inhibited microsomal 3beta-HSD II activity at concentrations from 1 to 25 microM, and I(50) values were estimated to be 1.3, 2, 1, 0.5 and 2.7 microM, respectively. In addition, these flavonoids significantly inhibited microsomal P450c21 activity at 12.5 and/or 25 microM. In addition, 6-hydroxyflavone inhibited activity of microsomal P450c17 and mitochondrial P45011beta at 12.5 and/or 25 microM. Results of Lineweaver-Burk's plot analysis indicate that daidzein is a competitive inhibitor of the activity of 3beta-HSD II and P450c21. K(m) and V(max) values of 3beta-HSD II for DHEA were estimated to be 6.6 microM and 328pmol/minmg protein, respectively. K(m) and V(max) values of P450c21 for progesterone were estimated to be 2.8 microM and 16pmol/minmg protein, respectively. K(i) values of 3beta-HSD II and P450c21 for daidzein were estimated to be 2.9 and 33.3 microM, respectively.     

Effects of phytoestrogens on aromatase, 3beta and 17beta-hydroxysteroid dehydrogenase activities and human breast cancer cells

Isoflavones and others phytoestrogens have been suggested to be anticarcinogenic. Anti-aromatase, antiestrogenic or antiproliferative actions of these compounds have been postulated and related to the observation that there is a reduced incidence of breast cancer associated with diet. In this study, we explored some mechanisms by which they can exert cancer-preventive effects. Phytoestrogens were tested for estimating anti-aromatase, anti-3beta-hydroxysteroid dehydrogenase delta5/delta4 isomerase (3beta-HSD) and anti-17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities in human placental microsomes. We found that isoflavonoids and compounds which presented the phenolic B ring in the 3 position on the pyran ring preferentially inhibited 3beta-HSD and/or 17beta-HSD activities than aromatase activity. We also evaluated their interactions with the estrogen receptor using a stably transfected human breast cancer cell line (MVLN). On the other hand phytoestrogens were evaluated for their effects on the proliferation in estrogen-dependent (MCF-7) and independent (MDA-MB231) human breast cancer cells. We established a relationship structure-activity and determined regions or/and substituents essential for these different activities. However, at high concentrations it seems that some phytoestrogens exert their protection against breast cancer through other estrogen-independent mechanisms.     

His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.