Hydrogen bonding in steroidogenesis: studies on new heterocyclic analogs of estrone that inhibit human estradiol 17 beta-dehydrogenase

New heterocyclic analogs of estrone are reported that inhibit estradiol 17 beta-dehydrogenase (E2-17 beta DH) from human placenta. The inhibitors are efficiently synthesized in two steps from estrone (or its 3-O-methyl ether), giving fully characterized analogs with pyrazole or isoxazole fused to the 16,17-position on the D ring. Dixon plots of enzyme kinetic data show the heterocyclic steroids are competitive inhibitors of E2-17 beta DH. Correlating molecular structures of the inhibitors with their Ki-values yields a pattern suggesting intermolecular hydrogen bonding stabilizes the [(pyrazole)inhibitor-E2-17 beta DH] complexes. A free energy difference of 2.74 Kcal/mol calculated from Ki-value differences between hydrogen bonded (4.08 microM) and non-bonded (425 microM) [inhibitor-E2-17 beta DH] complexes is in the range for intermolecular hydrogen bonding. We conclude that specific intermolecular hydrogen bonds stabilize [hydroxysteroid-enzyme] complexes, thereby making important contributions to the affinity between hydroxysteroids and steroid-specific enzymes of steroidogenesis.     

Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter

A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3'-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 microM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.     

STEROID SULFATASE IN BRAIN: COMPARISON OF SULFOHYDROLASE ACTIVITIES FOR VARIOUS STEROID SULFATES IN NORMAL AND PATHOLOGICAL BRAINS, INCLUDING THE VARIOUS FORMS OF METACHROMATIC LEUKODYSTROPHY

Abstract– The enzymatic hydrolysis by brain homogenate of the sulfate esters of estrone, pregnenolone, dehydroepiandrosterone, testosterone, cholesterol and p-nitrophenol was studied. With homogenate of young rat brain, the pH optima of estrone sulfatase ⁴ 4 The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout.
and arysulfatase C (p-nitrophenyl sulfate as substrate) were 8.2 and all other steroid sulfatases had pH optima at 6.6. Apparent K_ms for these steroid sulfates were widely different. The highest K_m value was 32.2 μm for estrone sulfate and the lowest was 0.66 μm for testosterone sulfate; the K_m for p-nitrophenyl sulfate was 30 fold higher than for estrone sulfate. Specific activity was also highest with estrone sulfatase and lowest with testosterone sulfatase; specific activity with aryl sulfatase C was over 3 fold higher than with estrone sulfatase. Estrone sulfatase activity was inhibited noncompetitively by sulfate esters of dehydroepiandrosterone, pregnenolone, and cholesterol; on the other hand, other steroid sulfatases were inhibited by these latter three sulfates competitively. Developmental changes of these sulfohydrolase activities in rat brain were almost identical with the exception of testosterone sulfatase activity; the latter sulfatase had a peak activity at 30 days old, while all other sulfatase had a peak at 20 days old. Thermal stability of all these activities was identical. Testosterone sulfatase activity in neurological mouse mutants, jimpy, msd, and quaking mice, was less than one half of littermate controls, while other steroid sulfatase levels in these mutants' brain were normal. All sulfatase activities were diminished in the brain of a metachromatic leukodystrophy patient with multiple sulfatase deficiency. The brains of classical metachromatic leukodystrophy patients contained normal levels of all steroid sulfatases and arylsulfatase C, with the single exception of testosterone sulfatase which level was less than 50% of control.     

Regulation of human placental progesterone synthesis in vitro by naturally occurring steroids

A regulatory model of human placental progesterone synthesis is based on studies with isolated placental enzymes. Steroids causing a dose-dependent inhibition are listed in the standing order of their inhibitory potency (I50 (microM)/Ki value (microM)/type of inhibition: c = competitive and nc = non competitive). Cholesterol side chain cleavage enzyme (mitochondria): Mainly regulated by hydroxylated cholesterol derivates. No inhibition was observed by cholesterylesters and by other naturally occurring steroids tested. 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (mitochondria): 6 beta-hydroxyprogesterone (nc), dehydroepiandrosterone (0.32/0.82/c), 20 alpha-dihydroprogesterone (0.38/-/nc), progesterone (0.46/-), estrone (0.56/0.1/c), estradiol (0.1/0.8/c), 17 alpha-hydroxyprogesterone (2.1/-/nc), 17 alpha-hydroxypregnenolone (0.4/-/c), dehydroepiandrosterone sulfate (2.5/-/c), cortisone (5.0/-), cortisol (100/-). 20 alpha-hydroxysteroid dehydrogenase (cytoplasmic): estrone (0.26/0.7/c), estradiol (0.28/0.9/c), pregnenolone (4.4/9.2/c), 5 alpha-pregnan-3 beta-ol-20-one (4.6/-/nc), estriol (5.1/11.5/c); dehydroepiandrosterone (7.2/14.0/c), 5 alpha-dihydrotestosterone (26.0/-/nc), progesterone (33.0/48.0/c), dehydroepiandrosterone sulfate (50.0/23.0/nc), and testosterone (59.0/63.0/c). An autoregulatory mechanism of placental progesterone synthesis is postulated which is in good agreement with data published by others proving that placental progesterone synthesis is independent of the endocrine organs of the mother and the fetus.     

Steroid sulfatase inhibitors: promising new tools for breast cancer therapy?

Inhibition of aromatase is currently well-established as the major treatment option of hormone-dependent breast cancer in postmenopausal women. However, despite the effects of aromatase inhibitors in both early and metastatic breast cancer, endocrine resistance may cause relapses of the disease and progression of metastasis. Thus, driven by the success of manipulating the steroidogenic enzyme aromatase, several alternative enzymes involved in steroid synthesis and metabolism have recently been investigated as possible drug targets. One of the most promising targets is the steroid sulfatase (STS) which converts steroid sulfates like estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) to estrone (E1) and dehydroepiandrosterone (DHEA), respectively. Estrone and DHEA may thereafter be used for the synthesis of more potent estrogens and androgens that may eventually fuel hormone-sensitive breast cancer cells. The present review summarizes the biology behind steroid sulfatase and its inhibition, the currently available information derived from basic and early clinical trials in breast cancer patients, as well as ongoing research. Article from the Special Issue on Targeted Inhibitors.     

Characterization, in the guinea pig, of a hepatic cytosol protein binding dehydroepiandrosterone sulfate and estrone sulfate

A protein which binds dehydroepiandrosterone sulfate and estrone sulfate was detected in the cytosolic fraction of female Guinea-pig liver. It is characterized by a molecular mass of 14,400 Da, its affinity for DHEA sulfate (KD = 8.8 microM) and estrone sulfate (KD = 8.5 microM), and its lack of affinity for free steroids such as dehydroepiandrosterone or estrone. It is eluted by gel filtration on Sephadex G-50 simultaneously with the inhibitor of microsomal DHEA sulfatase recently described by some of us. This protein could be implicated in the intracellular transport or in the metabolism of sulfated steroids.     

Steroid sulfate sulfatase in human benign prostatic hyperplasia: characterization and quantification of the enzyme in epithelium and stroma

Characteristics and activities of estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHAS) sulfatases were studied in epithelium and stroma of benign hyperplastic tissues from human prostates. Tissues were obtained by suprapubic prostatectomy, and epithelium and stroma were separated mechanically by standard techniques. The assay procedure comprised homogenization in Tris-buffer, incubation of the homogenate with [3H]E1S or [3H]DHAS, separation of free steroids from nonhydrolyzed steroid sulfates by extraction with ether, and their final quantification by LSC. The main results were: (1) The pH-optimum of the sulfatase was found at pH 7.0. (2) The highest specific sulfatase activity was found in the epithelium and was associated with its nuclear fraction. (3) Michaelis-Menten constants Km (microM) were 8.7 +/- 1.4 (7) and 4.3 +/- 0.8 (5), maximum velocity rates Vmax (nmol/h x mgDNA) were 47.4 +/- 8.8 (7) and 8.4 +/- 1.5 (5) for E1S and DHAS, respectively (means +/- SEM (n]. (4) The enzymatic cleavage of E1-sulfate was competitively inhibited by DHA-sulfate and vice versa with inhibition constants Ki (microM) of 4.0 +/- 0.5 (2) for E1S and 2.7 +/- 0.4 (2) for DHAS. On the basis of these findings, possible roles of steroid sulfate-sulfatases in forming precursors of active androgens and estrogens from the high amounts of E1S and DHAS in blood are discussed.     

Differential metabolism of dehydroepiandrosterone sulfate and estrogen conjugates by normal or malignant AXC/SSh rat prostate cells and effects of these steroid conjugates on cancer cell proliferation in vitro

Normal AXC/SSh rat ventral prostate and clonally derived AXC/SSh rat prostate cancer cells were evaluated for ability to metabolize estrone sulfate (E1S), estrone glucuronide (E1G), or dehydroepiandrosterone sulfate (DHEAS). Both normal and malignant prostate cells converted E1S to estrone. Neither normal nor malignant prostate cells had significant ability to metabolize DHEAS to DHEA, indicating differential specificity of prostate sulfatases(s) for estrogen and androgen sulfates. Both normal and neoplastic prostate cells possess beta-glucuronidase which hydrolyzed E1G to estrone. To assess potential physiologic consequences of these enzymatic activities, we determined the effect of steroid conjugates on in vitro proliferation of selected clonal lines of AXC/SSh rat prostate cancer cells. DHEAS, 10(-6) to 10(-9) M in decade intervals, did not affect in vitro proliferation of AXC/SSh prostate cancer cells; however, 10(-5) M DHEAS decreased in vitro proliferation of these cells. Neither E1S nor E1G, 10(-5) to 10(-9) M in decade intervals, affected in vitro proliferation of AXC/SSh prostate cancer cells. These findings suggest that low residual levels of steroid conjugates, which are not removed by charcoal stripping of serum, do not affect demonstrated in vitro androgen modulation of AXC/SSh rat prostate cancer cell proliferation (Cancer Res. 46, 3775-3781, 1986).     

Comparative biochemistry of mammalian arylsulfatase C and steroid sulfatase

1. Hepatic arylsulfatase C (ASC) and steroid sulfatase (SS) from six of eleven mammals (rat, dog, baboon, cow, goat, and sheep) coeluted from DEAE-Sephacel as a single anionic species. A minor cationic peak of ASC and SS activity was also recovered from solubilized microsomes derived from the domestic cat. Characterization of the cationic activities indicated they were most likely contributed by a protein structurally related to the anionic isozyme. Properties of ASC and SS activities occurring in these seven species were most consistent with the presence of both activities in the same enzyme. 2. Guinea-pig liver SS activity was partitioned between an alkylsulfatase (hydrolyzing dehydroepiandrosterone sulfate (DHEAS)) and an arylsulfatase (hydrolyzing both estrone sulfate (E1S) and 4-methylumbelliferyl sulfate (4MUS) at a common active site). These enzymes were physically separable by ion-exchange chromatography and possessed distinct immunological and chemical properties. 3. Porcine, squirrel, and human livers possessed a major isozyme of ASC that lacked both E1S- and DHEAS-sulfatase activities. The human hepatic ASC was separable from SS by electrophoresis and was partially resolved from SS by DEAE-Sephacel chromatography. The ASC isozyme lacking SS activity was heat-labile in all three species.     

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.     

Solubilization and partial purification of steroid sulfatase from rat liver: characterization of estrone sulfatase.

Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.     

Steroid sulfatase activities in normal and cirrhotic livers and plasma levels of estrone sulfate,estrone and estradiol-17β in men

Estrone and dehydroepiandrosterone (DHEA) sulfatases were studied in livers of normal and cirrhotic men. Their Km were 3.2μM and 1.2μM respectively. The musomal sulfatases were solubilized by Miranol H2M and ultrasound. After gel filtration, the soluble material gave a single peak of activity for both substrates with a molecular weight of approximately 330,000. In terms of pmol of product.min^?1 per mg of fresh tissue, the mean (±SD) values of estrone and DHEA sulfatase activities were lower in cirrhotic livers [(n=7) (4.09±2.90 and 0.38±0.20)] than in normal livers [(n=13)(8.29±4.00 and 0.69±0.20)]. The differences were statistically significant: p<0.03 for estrone sulfatase and p<0.01 for DHEA sulfatase. In cirrhotic men, the mean level of plasma estrone is increased whereas that of estrone sulfate is decreased. The variations may be related to the decrease of serum albumin in cirrhotic subjects.     

Estrogen sulfotransferase in human placenta

Human placental estrogen sulfotransferase (ESFT) was partially purified from the term placental cytosol by (NH4)2SO4 precipitation and agarose gel chromatography. Additional purifications caused a rapid loss of the enzyme activity. The activity was abolished by isoelectrofocusing but partially retained by chromatofocusing. The value of pI of human placental ESFT is 5.8 and the same value was obtained for bovine adrenal ESFT. The enzyme protein was able to bind to the affinity resin, estradiol-17-hemisuccinyl-1,2-diaminododecane sepharose 4B, but difficult to be extracted by estradiol (E2). The extract of the affinity resin showed one major protein band at 68,000 dalton on SDS-polyacrylamide gel electrophoresis. Kinetic studies using partially purified ESFT revealed that E2 is the best substrate for this enzyme. The relative rate of sulfurylation of E2, estrone, estriol and dehydroepiandrosterone at 4 microM (Km for E2) is 1, 0.3, 0.08 and 0.08, respectively.     

Purification and properties of steroid sulfatase from human placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.     

17Alpha-alkan (or alkyn) amide derivatives of estradiol as inhibitors of steroid-sulfatase activity

To develop inhibitors of steroid sulfatase without residual estrogenic activity, we have designed a series of estradiol (E2) derivatives bearing an alkan (or alkyn) amide side chain at position 17alpha. A hydrophobic alkyl group was selected from our previous study where 17alpha-octyl-E2 was found to inhibit strongly the steroid-sulfatase activity. Furthermore, it is known that an alkylamide side chain blocks the estrogen-receptor activation. Starting from ethynylestradiol, the chemical synthesis of target compounds was short and efficient with overall yields of 22-42% (3 or 4 steps). Among these compounds, N-octyl,N-methyl-3-(3',17'beta-dihydroxy-1',3',5'(10')-estratrien- 17'alpha-yl)-propanamide (15) was the most potent inhibitor, with an IC50 value of 0.08 microM for the transformation of estrone sulfate (E1S) to estrone (E1) by homogenated JEG-3 cells. N-butyl, N-hexyl, and N,N-dioctyl propanamide derivatives of E2 (IC50 values of 6.4, 2.8, and >20 microM, respectively) were less potent inhibitors than N-octyl analog 15. Furthermore, the unsaturated propynamide analog of 15 gave lower inhibition (four times) than the saturated compound. Compound 15 is also about 100-fold more effective in interacting with the enzyme than substrate E1S itself. The ability of target compounds to bind the estrogen receptor, to stimulate the proliferation of estrogen-sensitive ZR-75-1 cells, or to inhibit the E2-stimulation of ZR-75-1 cells was also evaluated. Although a mixed estrogenic/anti-estrogenic activity was obtained for tested compounds at 1 microM, no estrogenic activity was observed at 0.03 microM for 15. In conclusion, a promising inhibitor of steroid-sulfatase activity was obtained by introducing a hydrophobic octyl group in a 17alpha-propanamide side chain of E2, but further structure-activity relationships (SAR) studies are necessary to minimize the residual estrogenic activity.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

Estradiol formation by human osteoblasts via multiple pathways: relation with osteoblast function.

The importance of estrogens in bone metabolism is illustrated by the accelerated bone loss and increase in osteoporotic fractures associated with postmenopausal estrogen deficiency. In this study, the expression and activity of the enzymes involved in estrogen metabolism in human osteoblastic cells were investigated in relation to differentiation of these cells. PCR reactions using mRNA from an in vitro differentiating human cell line (SV-HFO) were performed to assess mRNA expression of the enzymes aromatase, different subtypes of 17beta-hydroxysteroid dehydrogenase (17beta-HSD), and steroid sulfatase. Aromatase, sulfatase, and 17beta-HSD type 2 and 4 were found to be expressed throughout differentiation. Expression of 17beta-HSD type 3, however, was relatively weak, except for early time points in differentiation. Type 1 17beta-HSD expression was not detected. Aromatase activity decreased during differentiation, as was demonstrated by the conversion of androstenedione (A) and testosterone (T) into estrone (E(1)) and estradiol (E(2)), respectively. The 17beta-HSD isozymes catalysing a reductive reaction convert androstenedione and estrone into testosterone and estradiol, respectively. Their activity declined with differentiation. Analysis of 17beta-HSD activity indicated both oxidative (E(2) to E(1); T to A) and reductive (E(1) to E(2); A to T) metabolism at all stages of osteoblast differentiation. Both activities declined as cells moved toward a differentiating mineralizing phenotype. However, the oxidative reaction was increasingly in favor of the reductive reaction at all times during differentiation. Sulfatase activity, as demonstrated by the conversion of estrone-sulfate into estrone, was constant during differentiation. In conclusion, we have demonstrated that all enzymes necessary for estrogen metabolism are expressed and biologically active in differentiating human osteoblasts. The activity of aromatase and 17beta-HSD was found to be dependent on the stage of cell differentiation. In addition, human osteoblasts effectively convert estradiol into estrone. The efficacy of osteoblasts to synthesize estradiol may determine the ultimate change in rate of bone turnover after menopause, as well as the development of osteoporosis. Moreover, the enzymes involved in the metabolism of estradiol may form a target for intervention.     

Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.     

Dehydroepiandrosterone sulfate (DHEAS) suppresses P2X purinoceptor-coupled responses in PC12 cells.

Some steroids rapidly alter neuronal excitability through interaction with neurotransmitter-gated ion channels in addition to their well-known genomic effects via intracellular steroid receptors. Such effects were found in GABA receptor, nicotinic receptors, yet not investigated in P2X purinoceptors. In this study, the effects of dehydroepiandrosterone sulfate on the P2 purinoceptor was investigated. Results show that dehydroepiandrosterone sulfate acutely inhibits P2X purinoceptor functions in PC12 cells. Dehydroepiandrosterone sulfate suppressed ATP-induced cytosolic free calcium concentration ([Ca(2+)](i)) rise, cytosolic free sodium concentration ([Na(+)](i)) rise, and dopamine secretion in the presence of external calcium, but had no effect on ATP-induced [Ca(2+)](i) rise in the absence of external calcium or on UTP-induced [Ca(2+)](i) rise in the absence or presence of external calcium. Our data show that dehydroepiandrosterone sulfate exerted its effect on P2X, but not on the P2Y purinoceptors found in PC12 cells. Estradiol and estrone have similar effects on P2X purinoceptor, but dehydroepiandrosterone and progesterone do not.