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.     

Proliferative effect of androst-4-ene-3,17-dione and its metabolites in the androgen-sensitive LNCaP cell line

As a therapeutic approach for the treatment of androgen-sensitive diseases, it would be tempting to lower the level of the potent androgens testosterone (T) and dihydrotestosterone (DHT) by using inhibitors of type 3 and type 5 17beta-hydroxysteroid dehydrogenases (17beta-HSDs). However, the efficiency of such a strategy will be optimal only if androst-4-ene-3,17-dione (Delta4-dione), the precursor of T, does not possess per se agonist activity on the androgen receptor (AR). To determine if the proliferative effect previously observed on AR(+) cells for Delta4-dione originates from its direct (per se) action on AR or from its transformation into a metabolite, we started a series of experimentations using the human prostate cancer LNCaP cell line, which expresses a highly sensitive AR. By real-time RT-PCR analysis, we detected type 1 5alpha-reductase (5alpha-R), a small amount of type 5 17beta-HSD, but not type 2 5alpha-R nor type 3 17beta-HSD. We then studied the transformation of labeled Delta4-dione in LNCaP cells after 1-7 days and the most important metabolite detected was 5alpha-androstane-3,17-dione (A-dione), which is the product of 5alpha-R activity. We measured only low levels of androsterone (ADT) and epi-ADT. This result was next confirmed by using an inhibitor of 5alpha-R that completely inhibited the transformation of Delta4-dione into A-dione, and consequently into ADT and epi-ADT. The proliferative effect of Delta4-dione (carefully purified) on LNCaP (AR(+)) cells was next determined in presence or absence of the 5alpha-R inhibitor. Although the cells proliferate in the presence of Delta4-dione only, no cell proliferation was observed with a combination of Delta4-dione and 5alpha-R inhibitor, suggesting that Delta4-dione is not androgenic per se. We next determined that A-dione and epi-ADT stimulated cell growth with the same pattern and potency as Delta4-dione, whereas ADT had a 3.5-fold lower proliferative activity. In conclusion, Delta4-dione is not in itself an agonist steroid on LNCaP (AR(+)) cells, and its proliferative activity appears to be mediated by its transformation into A-dione and/or into epi-ADT.     

Steroid metabolism and profile of steroidogenic gene expression in Episkin: high similarity with human epidermis

The skin is a well-recognized site of steroid formation and metabolism. Episkin is a cultured human epidermis. In this report, we investigate whether Episkin possesses a steroidogenic machinery able to metabolize adrenal steroid precursors into active steroids. Episkin was incubated with [14C]-dehydroepiandrosterone (DHEA) and 4-androstenedione (4-dione) and their metabolites were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS). The results show that the major product of DHEA metabolism in Episkin is DHEA sulfate (DHEAS) (88% of the metabolites) while the other metabolites are 7alpha-OH-DHEA (8.2%), 4-dione (1.3%), 5-androstenediol (1.3%), dihydrotestosterone (DHT) (1.4%) and androsterone (ADT) (2.3%). When 4-dione is used as substrate, much higher levels of C19-steroids are produced with ADT representing 77% of the metabolites. These data indicate that 5alpha-reductase, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and 3alpha-hydroxysteroid dehdyrogenase (3alpha-HSD) activities are present at moderate levels in Episkin, while 3beta-HSD activity is low and represents a rate-limiting step in the conversion of DHEA into C19-steroids. Using realtime PCR, we have measured the level of mRNAs encoding the steroidogenic enzymes in Episkin. A good agreement is found between the mRNAs expression in Episkin and the metabolic profile. High expression levels of steroid sulfotransferase SULT2B1B and type 3 3alpha-HSD (AKR1C2) correspond to the high levels of DHEA sulfate (DHEAS) and ADT formed from DHEA and 4-dione, respectively. 3beta-HSD is almost undetectable while the other enzymes such as type 1 5alpha-reductase, types 2, 4, 5, 7, 8, and 10 17beta-HSD and 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) (AKR1C1) are highly expressed. Except for UGT-glucuronosyl transferase, similar mRNA expression profiles between Episkin and human epidermis are observed.     

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.     

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 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, 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 5 microM. 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.     

Improved synthesis of EM-1745, preparation of its C17-ketone analogue and comparison of their inhibitory potency on 17β-hydroxysteroid dehydrogenase type 1

Endocrine therapies are widely used for the treatment of estrogen-sensitive diseases. 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is involved in the last step of the biosynthesis of potent estrogen estradiol (E₂). This enzyme catalyzes the reduction of the C17-ketosteroid estrone (E₁) into the C17β-hydroxy steroid E₂ using the cofactor NAD(P)H. The X-ray analysis of E₂/adenosine bisubstrate inhibitor EM-1745 proven that this compound interacts with both the substrate- and the cofactor-binding sites. However, E₁ is a better substrate of 17β-HSD1 than E₂. Thus, in order to improve the inhibitory potency of EM-1745, the C17-ketone analogue was prepared. During this work, a new and more efficient method for synthesizing EM-1745 was developed using an esterification and a cross-metathesis as key steps. Contrary to what was expected, the C17-ketone analogue of EM-1745 is a less potent inhibitor (IC₅₀ = 12 nM) than the C17-alcohol (IC₅₀ = 4 nM) in homogenated HEK-293 cells overexpressing 17β-HSD1. Our results contribute to the knowledge of an unexpected observation: the C17-ketone steroidal inhibitors of 17β-HSD1 are less potent than their corresponding C17-alcohol derivatives.     

Inhibition of type 2 17beta-hydroxysteroid dehydrogenase by estradiol derivatives bearing a lactone on the D-ring: structure-activity relationships

The peripheral conversion of steroid precursors into biologically active forms can be a major source of steroid synthesis, and these steroids support the growth of hormone-dependent diseases. The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) enzyme family is involved in the biosynthesis of active steroids and its inhibition constitutes an interesting approach for treating estrogen- and androgen-dependent cancers. We previously found that a compound formed by the introduction of a spiro-gamma-lactone at position 17 of estradiol (E2) produces a significant inhibition of type 2 17beta-HSD. To optimize the inhibitory potency of such compounds, we synthesized a series of estradiol derivatives bearing a lactone on the D-ring and tested their ability to inhibit the type 2 17beta-HSD transformation of 4-androstenedione into testosterone. The results of our structure-activity relationship study determined the importance of the 17beta-orientation of the oxygen atom. Indeed, the 17beta-O-isomer of spiro-gamma-lactone-E2 is a much more potent inhibitor than the 17alpha-O-analog (respectively 85 and 9% of inhibition at 1 microM). The carbonyl function is essential since the percentage of inhibition shifts from 85 to 30%, 15, or 3%, when the carbonyl group is transformed into a hydroxyl, a methoxy or a methylene (cycloether) group, respectively. Our results lead us to realize the importance of the spirolactone versus the C17beta-O/C16beta lactone (respectively 32 and 2% of inhibition at 0.1 microM, for the same size of lactone ring). The optimal size for the spirolactone was also established to be six members. All the types of substituents (methyl, dimethyl, allyl, propyl, and methoxycarbonyl) that we added on the spiro-delta-lactone moiety decreased the inhibitory activity, suggesting steric restrictions for the space that can be occupied in proximity of the spiro-delta-lactone functionality. 17-(Spiro-delta-lactone)-E2, compound 6, was thus the most potent inhibitor of type 2 17beta-HSD with a K(i) value of 29 +/- 5 nM. This compound reversibly inhibits type 2 17beta-HSD in a non-competitive manner.     

Estradiol and estrone C-16 derivatives as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: blocking of ER+ breast cancer cell proliferation induced by estrone

Estrogens play an important role in the development of breast cancer. Inhibiting 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1)--the enzyme responsible for the last step in the biosynthesis of the most potent estrogen, estradiol (E2)--would thus allow hindering the growth of estrogen-sensitive tumors. Based on a previous study identifying 16beta-benzyl-E2 (1) as a lead compound for developing inhibitors of the transformation of estrone (E1) into E2, we modified the benzyl group of 1 to improve its inhibitory activity. Three strategies were also devised to produce compounds with less residual estrogenic activity: (1) replacing the hydroxy group by a hydrogen at position 3 (C3); (2) adding a methoxy at C2; and (3) adding an alkylamide chain known to be antiestrogenic at C7. In order to test the inhibitory potency of the new compounds, we used the human breast cancer cell line T-47D, which exerts a strong endogenous 17beta-HSD1 activity. In this intact cell model, 16beta-m-carbamoylbenzyl-E2 (4m) emerged as a potent inhibitor of 17beta-HSD1 with an IC50 value of 44 nM for the transformation of [14C]-E1 (60 nM) into [14C]-E2 (24-h incubation). In another assay aimed at assessing the unwanted estrogenic activity, a 10-day treatment with 4m at a concentration of 0.5 microM induced some proliferation (38%) of T-47D estrogen-sensitive (ER+) breast cancer cells. Interestingly, when 4m (0.5 microM) was given with E1 (0.1 nM) in a 10-day treatment, it blocked 62% of the T-47D cell proliferation induced by E1 after its reduction to E2 by 17beta-HSD1. Thus, in addition to generating useful structure-activity relationships for the development of 17beta-HSD1 inhibitors, our study demonstrates that using such inhibitors is a valuable strategy for reducing the level of E2 and consequently its proliferative effect in T-47D ER+ breast cancer cells.     

Inhibitors of type 1 17beta-hydroxysteroid dehydrogenase with reduced estrogenic activity: modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6beta-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17beta-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6beta-configuration of the side chain led to a much better inhibition than the 6alpha-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17beta-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17beta-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 17beta-HSD without estrogen-like effects.     

A photoaffinity inhibitor of aromatase

Several 7-substituted 4-androstene-3, 17-diones are potent inhibitors of the biosynthesis of estrogens, with the most effective being 7-(4'-amino)phenylthio-4-androstene-3, 17-dione. An azide derivative of this 7-thioether compound has been prepared as a potential photoaffinity inhibitor. The enzyme kinetics of the azide analog were examined under both dark conditions and UV irradiation. In the dark, the azide was a very potent competitive inhibitor, with an apparent K_i of 1.3 nM. Under UV-irradiation, a time-dependent loss of aromatase activity was also observed. These studies indicate that the 7-substituent enhances the affinity of the steroidal analogs for the enzyme site.     

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.     

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.     

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.     

Inhibitory effect of synthetic progestins, 4-MA and cyanoketone on human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene-isomerase activity

Human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene isomerase (3 beta-HSD) purified from human placenta transforms C-21 (pregnenolone and 17 alpha-hydroxy pregnenolone) as well as C-19 (dehydroepiandrosterone and androst-5-ene-3 beta, 17 beta-diol) steroids into the corresponding 3-keto-4-ene-steroids and is thus involved in the biosynthesis of all classes of hormonal steroids. Trilostane, epostane and cyanoketone are potent inhibitors of 3 beta-HSD with Ki values of approximately 50 nM. 4-MA, a well known 5 alpha-reductase inhibitor, is also a potent inhibitor of 3 beta-HSD with a Ki value of 56 nM. Synthetic progestin compounds such as promegestone and RU2323 show relatively strong inhibitory effects with Ki values of 110 and 190 nM, respectively. Cyproterone acetate, a progestin used in the treatment of hirsutism, acne and prostate cancer as well as norgestrel and norethindrone that are widely used as oral contraceptives also inhibit 3 beta-HSD activity at Ki values of 1.5, 1.7 and 2.5 microM, respectively.     

Structure-activity relationships in the in vitro modulation of rat hepatic microsomal androst-4-ene-3,17-dione hydroxylase activities by derivatives of 5 alpha- and 5 beta-androstane

The relationships between structure and inhibitory potency toward microsomal cytochrome P-450 (P-450)-mediated androst-4-ene-3,17-dione hydroxylase activities were investigated in rat liver with a series of 5 alpha- and 5 beta-androstane derivatives. 5 beta-Reduced steroids (containing a cis-A/B ring junction) were more potent inhibitors than the 5 alpha-reduced epimers (containing a trans-A/B ring junction) except in the case of the 17 beta-hydroxy-substituted derivatives. The most effective inhibitor was 5 beta-androstane-3 beta-ol which exhibited I50 values of 7 and 27 microM against androstenedione 16 alpha- and 6 beta-hydroxylase activities, which are catalysed by P-450 IIC11 and IIIA2, respectively. In general, these two pathways of steroid hydroxylation were more susceptible to inhibition than the 7 alpha- and 16 beta-hydroxylase pathways. The 7 alpha-hydroxylase enzyme (P-450 IIA1) was only inhibited by 5 beta-reduced steroids that contained an oxygenated function at C17. All of the test compounds elicited type I spectral binding interactions with P-450 in oxidised microsomes. The most effective steroid inhibitors generally exhibited the greatest capacity to interact with P-450. Additional studies with one of the more potent compounds, 5 beta-androstane-3 beta-ol-17-one, revealed that the inhibition kinetics were competitive and that preincubation of the inhibitor with NADPH-supplemented microsomes prior to substrate (androstenedione) addition decreased the extent of inhibition observed. These findings are consistent with the assertion that the inhibition of hepatic steroid hydroxylases by 5 beta-androstanes involves an effective competitive interaction with the steroid substrate at the P-450 active site. Since the relative overproduction of 5 beta-reduced metabolites of certain androgens has been reported in clinical conditions, such as androgen insensitivity, it now appears important to investigate the hepatic drug oxidation capacity of patients with hormonal abnormalities.     

20 beta-hydroxysteroid dehydrogenase of neonatal pig testis: 3 alpha/beta-hydroxysteroid dehydrogenase activities catalyzed by highly purified enzyme

Pig testicular 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) has also 3 alpha- and 3 beta-HSD (3 alpha/beta-HSD) activities. The purified 20 beta-HSD preparation from neonatal pig testes could catalyze the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) in the presence of beta-NADPH to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol at the ratio of 4:3, and the specific 3 alpha/beta-HSD activity of 20 beta-HSD for 5 alpha-DHT was about 10 or 15 times larger than the 20 beta-HSD activities for 17 alpha-hydroxypregn-4-ene-3,20-dione (17 alpha-hydroxyprogesterone) or progesterone, respectively. The result indicates that the testicular 20 beta-HSD has high 3 alpha(axial, 3R)- and 3 beta(equatorial, 3S)-HSD activity. The testicular 20 beta-HSD could catalyze the reversible conversion of various 5 alpha- or 5 beta-dihydrosteroids which have a 3-carbonyl or 3-hydroxyl group with beta-NADP(H) as the preferred cofactor. The enzyme transferred the 4-proS hydrogen of NADPH to the 5 alpha-DHT for both 3 alpha- and 3 beta-hydroxylation and it was the same as the 20 beta-hydroxylation of 17 alpha-hydroxyprogesterone. Although the 3 alpha/beta-HSD activity has been known to be present in 3 alpha,20 beta-HSD of Streptomyces hydrogenans, the enzymological properties for 3 alpha/beta-HSD activity catalyzed by testicular 20 beta-HSD were different from the properties for 3 alpha/beta-HSD activity catalyzed by prokaryotic 3 alpha, 20 beta-HSD with respect to the specificity of the catalytic reaction and the cofactor requirement.     

Expression of liver-specific member of the 3 beta-hydroxysteroid dehydrogenase family, an isoform possessing an almost exclusive 3-ketosteroid reductase activity.

We have recently characterized two types of rat 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) isoenzymes expressed in adrenals and gonads. In addition, we have cloned a third type of cDNA encoding a predicted type III 3 beta-HSD protein specifically expressed in the male rat liver which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells of the cDNAs reveals that the type III 3 beta-HSD protein does not display oxidative activity for the classical substrates of 3 beta-HSD, in contrast to the type I 3 beta-HSD isoenzyme. However, in the presence of NADH, type III isoenzyme, in common with the type I isoform, converts 5 alpha-androstane-3,17-dione (A-dione) and 5 alpha-dihydrotestosterone (DHT) to the corresponding 3 beta-hydroxysteroids. In fact, the type I and the type III isoenzymes have the same affinity for DHT with Km values of 5.05 and 6.16 microM, respectively. When NADPH is used as cofactor, the affinity for DHT of the type III isoform becomes higher than that of the type I isoform with Km values of 0.12 and 1.18 microM, respectively. The type III isoform is thus a 3-ketoreductase using NADPH as preferred cofactor which is responsible for the conversion of 3-keto-saturated steroids such as DHT and A-dione into less active steroids.     

The effects of the licorice derivative, glycyrrhetinic acid, on hepatic 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and 5 alpha- and 5 beta-reductase pathways of metabolism of aldosterone in male rats

Ingestion of licorice or treatment with chemical derivatives of glycyrrhetinic acid (GA), an active principle of licorice, can cause hypertension, sodium retention, and hypokalemia. Although GA has been shown to inhibit 11 beta-hydroxysteroid dehydrogenase, it may not be the only hepatic enzyme affected by this licorice derivative. Therefore, we studied the effects of GA on other major hepatic steroid-metabolizing enzymes from adrenalectomized male rats using aldosterone as the substrate; namely, delta 4-5 alpha- and delta 4-5 beta-reductases and 3 alpha- and 3 beta-hydroxysteroid dehydrogenases (3 alpha- and 3 beta-HSD). From these in vitro studies, we demonstrated that GA does not affect either microsomal 5 alpha-reductase or cytosolic 3 alpha-HSD activity. However, GA is a potent inhibitor of cytosolic 5 beta-reductase; the K(is) and K(ii) were calculated from enzyme kinetic analysis to be 6.79 and 5.41 microM, respectively, using the Cleland equation, indicating that GA is a noncompetitive inhibitor of aldosterone. In addition, GA specifically inhibited microsomal 3 beta-HSD enzyme activity by what appears to be a competitive inhibition mechanism, causing a build-up of the intermediate, 5 alpha-dihydroaldosterone (DHAldo). Thus, this study has indicated that GA has a profound effect on hepatic ring A-reduction of aldosterone. Inhibition of 5 beta-reductase and 3 beta-HSD results in decreased synthesis of both 3 alpha, 5 beta-tetrahydroaldosterone (THAldo) and 3 beta, 5 alpha-THAldo and, hence, accumulation of aldosterone and 5 alpha-DHAldo, both potent mineralocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flavonoid inhibition of overexpressed human 3beta-hydroxysteroid dehydrogenase type II

The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.