Molecular mechanism of inhibition of steroid dehydrogenases by licorice-derived steroid analogs in modulation of steroid receptor function.
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Carbenoxolone↗
Biomedical subjects
F Labrie
Publications and source records attributed to F Labrie.
Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution.
BACKGROUND: The principal human estrogen, 17 beta-estradiol, is a potent stimulator of certain endocrine-dependent forms of breast cancer. Because human estrogenic 17 beta-hydroxysteroid dehydrogenase (type I 17 beta-HSD) catalyzes the last step in the biosynthesis of 17 beta-estradiol from the less potent estrogen, estrone, it is an attractive target for the design of inhibitors of estrogen production and tumor growth. This human enzyme shares less than 15% sequence identity with a bacterial 3 alpha,20 beta-HSD, for which the three-dimensional structure is known. The amino acid sequence of 17 beta-HSD also differs from that of bacterial 3 alpha,20 beta-HSD by two insertions (of 11 and 14 residues) and 52 additional residues at the C terminus. RESULTS: The 2.20 A resolution structure of type I 17 beta-HSD, the first mammalian steroidogenic enzyme studied by X-ray crystallographic techniques, reveals a fold characteristic of the short-chain dehydrogenases. The active site contains a Tyr-X-X-X-Lys sequence (where X is any amino acid) and a serine residue, features that are conserved in short-chain steroid dehydrogenases. The structure also contains three alpha-helices and a helix-turn-helix motif, not observed in short-chain dehydrogenase structures reported previously. No cofactor density could be located. CONCLUSIONS: The helices present in 17 beta-HSD that were not in the two previous short-chain dehydrogenase structures are located at one end of the substrate-binding cleft away from the catalytic triad. These helices restrict access to the active site and appear to influence substrate specificity. Modeling the position of estradiol in the active site suggests that a histidine side chain may play a critical role in substrate recognition. One or more of these helices may also be involved in the reported association of the enzyme with membranes. A model for steroid and cofactor binding as well as for the estrone to estradiol transition state is proposed. The structure of the active site provides a rational basis for designing more specific inhibitors of this breast cancer associated enzyme.
Amino Acid Sequence↗
Occurrence of cytochrome P450c17 mRNA and dehydroepiandrosterone biosynthesis in the rat gastrointestinal tract.
We have investigated 17 alpha-hydroxylase and C17,20-lyase activities and the presence of cytochrome P450c17 mRNA in the esophagus, stomach, duodenum, and colon of adult rats of both sexes. All tissues converted [4-14C]pregnenolone mainly to dehydroepiandrosterone (DHEA) through the 5-ene-3 beta-hydroxysteroid route as opposed to the 4-ene-3-ketosteroid pathway in a control testicular incubate. Synthesis of dehydroepiandrosterone was particularly high in the duodenum and was found to be lower in the stomach, colon and esophagus, in decreasing order. 20 alpha-Hydroxypregnenolone and progesterone were also formed primarily by the esophagus and colon, respectively. P450c17 mRNA was demonstrated by ribonuclease protection assay in the stomach and duodenum, but not in esophagus and colon. However, a 335 bp-long cDNA fragment, whose sequence corresponded to that of rat P450c17 cDNA, was amplified by reverse transcription (RT) and polymerase chain reaction (PCR) from the poly(A)+ RNAs of all four tissues. This result was further confirmed by Southern blotting using a 794-bp testicular probe. The complete sequence of P450c17 cDNA in the stomach and duodenum was identical to that reported for rat testis P450c17 cDNA. No amplification and no positive signal in Southern blotting were observed with the total RNAs from adult male adrenal and spleen, which were taken as negative controls since they had been previously found unable to form androgens from pregnenolone. Although the levels of transcription in gonads, duodenum and stomach were found to be equivalent, as indicated by the RNase protection assay and semiquantitative RT-PCR assay, P450c17 enzyme activity was much higher in the testis, pointing at a possible dissimilarity in the respective rates of mRNA translation. Thus, P450c17 is differentially expressed in the rat gastrointestinal tract, where it leads to the synthesis of the sex steroid precursor DHEA, especially in the duodenum and stomach.
Aldehyde-Lyases↗
Synthesis and in vitro evaluation of 4-substituted N-(1,1-dimethylethyl)-3-oxo-4-androstene-17 beta-carboxamides as 5 alpha-reductase inhibitors and antiandrogens.
4-Substituted N-(1,1-dimethylethyl)-3-oxo-4-androstene-17 beta-carboxamides with the hydroxy (OH) 3d, mercapto (SH) 3e, chloro (Cl) 3f, and bromo (Br) 3g substituents at the 4-position were prepared in a two-step sequence with overall yields of 21%, 27%, 41%, and 37%, respectively. Compounds 3d-g showed weak inhibitory activity on human type I 5 alpha-reductase (IC50 > or = 700 nM) while they had intermediate inhibitory activity on human type II 5 alpha-reductase at IC50S of 172, 437, 192, and 387 nM, respectively. In androgen-sensitive Shionogi cells, the inhibition of dihydrotestosterone (DHT) stimulatory action on the proliferation of the androgen-sensitive cancer cells by all four compounds was high at IC50S of 170-279 nM compared with 117 nM for hydroxyflutamide. The present data show compounds having both moderate inhibition of human type II 5 alpha-reductase activity and relatively potent antiandrogenic action, two beneficial characteristics in the therapy of androgenic-sensitive diseases.
5-alpha Reductase Inhibitors↗
Synthesis and in vitro activity of 17 beta-(N-alkyl/arylformamido)- and 17 beta-[(N-alkyl/aryl)alkyl/arylamido]-4-methyl-4-aza-3-oxo-5 alpha-androstan-3-ones as inhibitors of human 5 alpha-reductases and antagonists of the androgen receptor.
A number of 17 beta-(N-alkyl/arylformamido)- and 17 beta-[(N-alkyl/aryl)alkyl/arylamido]-3-oxo-4-aza-5 alpha-steroids were prepared from 17 beta-hydroxy-4-azasteroids and evaluated as inhibitors of human 5 alpha-reductase and antagonists of the androgen receptor. Jones' oxidation of 17 beta-hydroxy compounds gave the 17-keto-4-azasteroids, which were treated with amines and NaBH(OAc)3/NaBH3CN to give 17 beta-(N-alkyl/arylamino)-4-azasteroids 10-27. Alternatively, the above-indicated compounds were prepared from amines and 17-keto-4-azasteroids to form imines, which were then reduced with NaBH4. Formylation of amines 10-27 gave 17 beta-(N-alkylformamides) 28-41; however, acylation afforded 17 beta-[(N-alkyl/aryl)alkyl/arylamides] 42-53. In comparison to N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA; IC50 = 4.15 nM), 17 beta-(N-alkylformamido)-4-azasteroids were potent inhibitors of human type I 5 alpha-reductase, IC50 values of compounds 29, 30, 36, and 37 being measured as 3.05, 0.91, 2.19, and 2.35 nM, respectively. The structure-activity relationships suggest that the type I enzyme has preference for N-substituted straight alkyl side chains of four to five carbon atoms. On the other hand, formamides 32 (N-heptyl) and 33 (N-octyl), in addition to inhibiting the type I enzyme (IC50s = 9.57 and 16.9 nM, respectively), showed also strong inhibitory activity (IC50s = 14.0 and 18.4 nM, respectively) for human type II 5 alpha-reductase, in comparison to N-(1',1'-dimethylethyl)-3-oxo-4-aza-5 alpha-androst-1-ene-17 beta-carboxamide (MK-906; IC50 = 4.53 nM). Other compounds in this series showed moderate activities (IC50 > 100 nM) on the type II enzyme. 17 beta-[(N-Alkyl/aryl)alkyl/arylamides] 45, 46, 48, and 51 exhibited highly potent inhibitory activity for human type I 5 alpha-reductase with IC50s of 1.77, 2.42, 2.93, and 5.44 nM, respectively, while moderate to no effect was observed on the type II enzyme (100 < IC50s < 1000 nM), except for compound 48 (IC50 = 3.75 nM). In another substitution pattern, N-aryl/alkylamides were studied; an electron-donating group increased the potency of compound 51, whereas an electron-withdrawing group decreased the potency of compounds 52 and 53 compared to parent compound 50. In addition to their 5 alpha-reductase activities, 17 beta-(N-alkylformamides) were also studied for their inhibitory activities on dihydrotestosterone (DHT)-stimulated proliferation of androgen-sensitive Shionogi mouse mammary carcinoma cells (clone SEM-107).(ABSTRACT TRUNCATED AT 400 WORDS)
Androgen Receptor Antagonists↗
Identification and characterization of the G15D mutation found in a male patient with 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency: alteration of the putative NAD-binding domain of type II 3 beta-HSD.
We report the detection of a homozygous G to A mutation converting codon Gly15 into Asp15 in the type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) gene in a male pseudo-hermaphrodite born from consanguineous parents and suffering from severe salt-losing 3 beta-HSD deficiency. To investigate further the potential involvement of residue 15 in the beta alpha beta dinucleotide-binding fold, we have studied the effect of substituting Gly15 for Ala15. We assessed the effect of the G15D and G15A missense mutations on enzymatic activity by analyzing mutant enzymes generated by site-directed mutagenesis of type II 3 beta-HSD cDNA after their transient expression in COS-1 cells. In intact transfected cells, after a 2-h incubation, the percentage of conversion of [3H]pregnenolone (PREG) into [3H]progesterone (PROG) was 35% and 50% for the G15A and native type II 3 beta-HSD enzymes, respectively, whereas no detectable activity was observed in cells expressing the G15D protein. This finding is in agreement with the severity of the disease in the homozygote G15D index case. On the other hand, in homogenates from cells transfected with the normal pCMV-type II 3 beta-HSD plasmid or with the mutated pCMV-G15D or pCMV-G15A plasmid, the Km values for PREG were 0.72 microM, 3.2 microM, and 3.4 microM, respectively, when incubated for 1 h in the presence of excess (1 mM) NAD+. Moreover, the expressed G15D and G15A proteins had decreased affinities for NAD+ with Km values of 113 microM and 148 microM, respectively, compared with 22 microM for normal type II 3 beta-HSD.(ABSTRACT TRUNCATED AT 250 WORDS)
3-Hydroxysteroid Dehydrogenases↗
A collaborative survey of 80 mutations in the BRCA1 breast and ovarian cancer susceptibility gene. Implications for presymptomatic testing and screening.
OBJECTIVES: To report the initial experience of an international group of investigators in identifying mutations in the BRCA1 breast and ovarian cancer susceptibility gene, to assess the spectrum of such mutations in samples from patients with different family histories of cancer, and to determine the frequency of recurrent mutations. DESIGN: Nine laboratories in North America and the United Kingdom tested for BRCA1 mutations in DNA samples obtained from a total of 372 unrelated patients with breast or ovarian cancer largely chosen from high-risk families. Three of these laboratories also analyzed a total of 714 additional samples from breast or ovarian cancer cases, including 557 unselected for family history, for two specific mutations that had been found to recur in familial samples. PARTICIPANTS: A total of 1086 women with either breast or ovarian cancer. MAIN OUTCOME MEASURE: The detection of sequence variation in patients' DNA samples that is not found in sets of control samples. RESULTS: BRCA1 mutations have now been identified in a total of 80 patient samples. Thirty-eight distinct mutations were found among 63 mutations identified through a complete screen of the BRCA1 gene. Three specific mutations appeared relatively common, occurring eight, seven, and five times, respectively. When specific tests for the two most common mutations were performed in larger sets of samples, they were found in 17 additional patients. Mutations predicted to result in a truncated protein accounted for 86% of the mutations detected by complete screening. CONCLUSIONS: The high frequency of protein-terminating mutations and the observation of many recurrent mutations found in a diverse set of samples could lead to a relatively simple diagnostic test for BRCA1 mutations. More data must be accumulated to address specifically the sensitivity and specificity of such a diagnostic testing procedure and to better estimate the age-specific risk for breast and ovarian cancer associated with such mutations.
Adult↗
Mapping of the HSD17B2 gene encoding type II 17 beta-hydroxysteroid dehydrogenase close to D16S422 on chromosome 16q24.1-q24.2.
The enzymes of the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) gene family are responsible for a key step in the formation and degradation of androgens and estrogens: catalyzing the interconversion of 17-ketosteroids and their active 17 beta-hydroxysteroid counterparts. The structure of human type II 17 beta-HSD cDNA was recently reported. This enzyme catalyzes the interconversion of delta 4-androstenedione and testosterone, androstanedione and dihydrotestosterone, and estrone and 17 beta-estradiol, whereas type I 17 beta-HSD catalyzes exclusively the interconversion of estrogens. To locate the HSD17B2 gene, the novel dinucleotide CA repeat sequence found 571 bp downstream from the end of exon 1 was genotyped into eight CEPH reference families by PCR. Two-point linkage analysis was performed between the latter polymorphism and the 2066 microsatellite markers of Généthon. The maximal pairwise lod score (Zmax = 33.3) with a maximal recombination fraction (theta max) of 0.008 was obtained with the marker D16S422 located on 16q24.1-q24.2. To define further the localization of the HSD17B2 gene, we constructed a high-resolution genetic map of the region flanking the polymorphic HSD17B2 gene including eight Généthon markers. The order of the HSD17B2 gene and markers is qter-D16S516-D16S504-D16S507-D16S505-D16S511+ ++-[HSD17B2-D16S422]-D16S520- D16S413-tel.
17-Hydroxysteroid Dehydrogenases↗
Cloning and expression of human tyrosine aminotransferase cDNA.
Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.
Adult↗
Rapid restriction fragment analysis for screening four point mutations of the low-density lipoprotein receptor gene in French Canadians.
Familial hypercholesterolemia (FH) has an estimated frequency of 1:154 among French Canadians in Northeastern Quebec, compared with 1:500 in most other populations. FH is caused by numerous mutations of the low-density lipoprotein (LDL) receptor gene, but only six well-characterized mutations are known to cause FH in French Canadians. High prevalence of the phenotype, along with a limited number of mutations in this population, provides a unique opportunity to study genotype-phenotype variation. Since the current methods for detection of point mutations in this population use complicated approaches, we report polymerase chain reaction (PCR)-based restriction fragment analysis to detect all four point mutations. This approach provides a rapid diagnosis and is suitable to screen large number of samples for studies in genetic epidemiology; it should be useful in identifying FH in other populations bearing the same mutations.
Base Sequence↗
Molecular mass and carbohydrate structure of prostate specific antigen: studies for establishment of an international PSA standard.
Despite the widely accepted use of prostate specific antigen (PSA) as a marker of prostate cancer, this molecule has not yet been completely characterized. Past studies have well established, however, using both amino acid and cDNA sequencing techniques, that PSA contains 237 amino acids, with a molecular mass of 26,079 Da for the peptide moiety of the molecule. The present study reports analysis of this protein by ion spray mass spectrometry (ISMS) and analysis of its carbohydrate moiety by NMR spectroscopy. The predominant PSA molecular species detected by ISMS was at relative molecular mass (M(r)) of 28,430, indicating that PSA contains a carbohydrate residue of M(r) 2,351, for a total percentage of carbohydrate of 8.3%. Analysis of PSA by SDS-PAGE, however, showed a M(r) of 32,000 to 33,000, suggesting an overestimation of the molecular weight by the latter technique. The complete primary structure of the PSA carbohydrate chain was determined by NMR spectroscopy in combination with carbohydrate composition analysis. The experimentally determined carbohydrate content of PSA confirms that only one N-glycosylation site is occupied in the protein. The proposed carbohydrate structure is a diantennary N-linked oligosaccharide of the N-acetyllactosamine type, with a sialic acid group at the end of each of the two branches. In addition, our data indicate that approximately 70% of the PSA molecules contain a fucose group in the core chitobiose moiety. The calculated molecular weight of this carbohydrate structure (M(r) 2,351.8) is in excellent agreement with the predicted molecular weight of the carbohydrate group, based on the M(r) 28,430 for PSA measured by ion spray mass spectrometry and M(r) 26,079 calculated from the consensus sequence for the peptide portion of the molecule. ISMS of PSA is thus proposed as a convenient and reliable method of quality control, an indispensible step towards international standardization of this very important tumor marker for detection and monitoring of prostatic diseases, especially prostate cancer.
Amino Acid Sequence↗
Inhibitory effects of medroxyprogesterone acetate (MPA) and the pure antiestrogen EM-219 on estrone (E1)-stimulated growth of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat.
Estrogens are well known to play a predominant role in promoting the growth of DMBA-induced mammary tumors in the rat. Estrone (E1), a steroid having weak estrogenic activity, is one of most important estrogens in post-menopausal women, where it is converted into the potent estrogen estradiol (E2) by 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in many peripheral tissues, including the mammary gland. In this report, we have studied the effect of a new antiestrogen (EM-219) (N-butyl, N-methyl-11-(3', 17'beta-dihydroxy-17'alpha-ethinyl-estra-1'3'5'(10'), 14'-tetraen-7'alpha-yl) undecanamide) on E1-stimulated growth of DMBA-induced mammary tumors and compared its effect with that of medroxyprogesterone acetate (MPA) alone or in combination. After 18 days, ovariectomy (OVX) reduced total tumor area to 29.6 +/- 7.1% of the original size, while E1 (1.0 microgram, twice daily) caused a 139 +/- 21% increase in tumor size in OVX animals. MPA (1.5 mg, twice daily) partially reversed the stimulatory effect of E1 to 66.0 +/- 9.0%, while the antiestrogen EM-219 (40 micrograms, twice daily) decreased tumor size to 70.0 +/- 10%. Combination of these two compounds led to a further inhibition of tumor size to 30.7 +/- 7.4% of the value found in OVX animals treated with E1. Tumor E2 levels decreased from 1688 +/- 155 pmoles/kg tissue in OVX animals receiving E1 to 709 +/- 92, 1347 +/- 98, and 184 +/- 11 pmoles/kg tissue in MPA-, EM-219-, and MPA+EM-219-treated OVX-E1 animals, respectively. Treatment of OVX animals with E1 increased by 69% the reductive activity of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) while MPA abolished completely this effect of E1. In the oxidative direction, treatment with E1, E1 + MPA, or E1 + EM-219 had minimal or no significant effect on the activity of 17 beta-HSD (vs OVX), while the combined treatment with MPA+EM-219 induced a 2-fold increase in 17 beta-HSD activity, thus leading to an increased conversion of E2 into E1. The present data show that combination of the pure antiestrogen EM-219 with MPA exerts a greater reduction in DMBA-induced mammary tumor growth and intratumoral E2 levels stimulated by E1 than either compound used alone. This interactive effect of the antiestrogen and MPA could at least partially be related to the increased inactivation of E2 into E1.(ABSTRACT TRUNCATED AT 400 WORDS)
17-Hydroxysteroid Dehydrogenases↗
Inhibitory effect of a steroidal antiestrogen (EM-170) on estrone-stimulated growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat.
Recently, compounds having pure antiestrogenic activity have become available. In this study, we examined the activity of the new steroidal antiestrogen EM-170 (N-n-butyl, N-methyl-11-(16' alpha-chloro-3',17' alpha-dihydroxy-estra-1',3',5'-(10')-trien-7' alpha-yl) undecanamide) on the growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma stimulated by treatment with estrone (E1), a steroid known to play an important role as precursor of 17 beta-estradiol (E2), especially in postmenopausal women. Twenty-five days after ovariectomy (OVX), tumor volume in control OVX animals decreased to 51.4 +/- 11% of the initial volume; treatment with E1, administered by Silastic implants, stimulated tumor growth to 179 +/- 21%. Treatment with the antiestrogen EM-170 at a dose of 200 micrograms (twice daily) not only completely reversed the stimulatory effect of E1, but also inhibited tumor growth to 30.5 +/- 9.6%, an effect that is 41% (P < 0.01 vs OVX control) greater than that of ovariectomy alone. At a relatively low dose of 40 micrograms (twice daily), 20 days of treatment with EM-170 reversed by 55% the stimulatory effect of E1 (1.0 micrograms, subcutaneously, twice daily) on tumor growth in OVX animals. On the other hand, the antiestrogen also induced a significant inhibitory effect on 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity in the DMBA-induced mammary tumors, an effect that is in agreement with the marked reduction caused by the same treatment on tumor estradiol (E2) levels in E1-treated OVX animals.(ABSTRACT TRUNCATED AT 250 WORDS)
17-Hydroxysteroid Dehydrogenases↗
Reduced testosterone and adrenal C19 steroid levels in obese men.
It has been reported that a high proportion of abdominal fat is associated with increased plasma androgen concentrations in women. Although less evidence is available, abdominal obesity appears to be associated with low plasma testosterone (T) levels in men. We have therefore examined in 80 men (aged 36.3 +/- 3.2 years, mean +/- SD) the correlations between body fatness, adipose tissue (AT) distribution measured by computed tomography (CT), and circulating levels of the following steroids measured by radioimmunoassay after extraction from serum and chromatography: dehydroepiandrosterone (DHEA), androstenedione (delta 4-DIONE), androst-5-ene-3 beta,17 beta-diol (delta 5-DIOL), T, estrone, and estradiol. Sex hormone-binding globulin (SHBG) levels were also determined. T, adrenal C19 steroids, and SHBG levels were negatively correlated with total body fatness indices and abdominal fat deposition measured by CT (-.23 < or = -.55, .0001 < or = P < or = .05), whereas estrone showed positive correlations with these body fatness and AT distribution indices. Covariance analysis showed that after control for the concentration of the adrenal steroid precursor delta 5-DIOL, there was no residual association between T levels and adiposity variables. Furthermore, multivariate analyses showed that steroid and SHBG levels could explain from 20% (visceral AT area measured by CT) to 40% and 42% (body mass index [BMI], waist circumference, and waist to hip ratio [WHR]) of the variation in adiposity variables (.0001 < or = P < or = .05), with delta 5-DIOL being the best single correlate of body fatness and abdominal fat deposition in men.(ABSTRACT TRUNCATED AT 250 WORDS)
Adipose Tissue↗
The enzyme and inhibitors of 4-ene-3-oxosteroid 5 alpha-oxidoreductase.
Since evidence of 5 alpha-reductase activity in rabbit liver homogenate was discovered in 1954, the presence of this enzyme has been demonstrated in many other organs and tissues of mammalian species. 5 alpha-Reductase selectively transforms a 4-ene-3-oxosteroid (e.g., testosterone) irreversibly to the corresponding 5 alpha-3-oxosteroid (e.g., 5 alpha-dihydrotestosterone) in the presence of NADPH as an essential coenzyme at an optimal pH. However, excessive production of 5 alpha-dihydrotestosterone is the major cause of many androgen-related disorders, such as prostate cancer, benign prostatic hyperplasia, acne, female hirsutism, and male pattern baldness; therefore, inhibition of androgenic action by 5 alpha-reductase inhibitors is a logical treatment. During the past two decades, research has focused on understanding the biological functions and effects of 5 alpha-reductase and its 5 alpha-reduced metabolites: purification of the enzyme, substrates, and metabolites; characterization of their physical, chemical, and biochemical properties; analysis of the amino acid sequence of the enzyme; synthesis of various classes of molecules as potential inhibitors; and examination of the biological activity of the inhibitors in vitro and/or in vivo. This review summarizes the biochemical studies on this enzyme, suggests the mechanisms of action of the enzyme or inhibitors, and discusses the chemistry necessary for the preparation, structure-activity relationships, and in vitro and/or in vivo data obtained from the evaluation of nonsteroidal and steroidal compounds that have been tested as inhibitors of 5 alpha-reductase. In particular, IC50 and Ki values for relevant compounds will be compared according to molecular class. This review could function as a comprehensive working reference of what research has been accomplished so far and what problems remain to be solved in the future for those engaged in this interesting field.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Synthesis and biological activities of thioether derivatives related to the antiestrogens tamoxifen and ICI 164384.
The catalyzed coupling reaction of activated alcohol and mercaptan was used for the short and efficient synthesis of 14 thioether compounds. Two types of side chains, the methyl butyl alkylamide related to the pure steroidal antiestrogen ICI 164384 and the dimethylamino ethyloxy phenyl related to the clinically used nonsteroidal antiestrogen tamoxifen, were introduced by a thioether link on two types of nuclei (triphenylethane or estradiol). The new thioether derivatives were tested to assess their relative binding affinity for the estrogen receptor and their estrogenic or antiestrogenic activity in the ZR-75-1 (ER+) cell line. The results indicate that of the three types of compounds studied, only the nonsteroidal derivatives with an alkylamide side chain possess antiestrogenic activity. In the steroidal series, displacement of the alkylamide side chain from the 7 to the 6 position produced compounds with chemical characteristics similar to ICI 164384 or EM-139 but without antiestrogenic activity. In the nonsteroidal series of compounds with an aryl side chain, compounds with estrogenic activity were obtained. One compound, a nonsteroidal derivative with a methyl butyl alkylamide side chain 20, possesses a relative binding affinity for the estrogen receptor identical to EM-139 (1.1 and 1.2%, respectively) and a relatively good antiestrogenic activity that is 10-fold lower than EM-139 (IC50 values of 250 and 25 nM, respectively). This nonsteroidal thioether with an alkylamide side chain is free of estrogenic activity.
Animals↗
Molecular basis of human 3 beta-hydroxysteroid dehydrogenase deficiency.
The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3 beta-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3 beta-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3 beta-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3 beta-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3 beta-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The finding of a normal type I 3 beta-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from approximately 1-10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (approximately 1%) similar to that observed for the mutant N100S enzyme detected in a homozygous male patient suffering from a nonsalt-losing form of this disorder.(ABSTRACT TRUNCATED AT 400 WORDS)
3-Hydroxysteroid Dehydrogenases↗
Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition.
Following transfection of types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) cDNAs into transformed embryonal kidney (293) cells, we have characterized the selective directional and inhibitory characteristics of these activities. While homogenates of transfected cells could catalyze interconversion of the substrate and product, in agreement with the general belief on the activity of these enzymes, the same activities measured in intact cells, in order to better reflect the physiological conditions, showed an unidirectional reaction. Types 1 and 3 17 beta-HSD catalyzed the reduction of estrone to estradiol and 4-androstenedione to testosterone, respectively, while type 2 17 beta-HSD catalyzed the oxidative transformation of both testosterone and 17 beta-estradiol to 4-androstenedione and estrone, respectively. In addition, types 1, 2 and 3 17 beta-HSD activities showed different pH optima. While types 1 and 3 showed pH optimum values centered at around 5 and 6, respectively, type 2 17 beta-HSD activity, which preferentially, catalyzes the oxidation reaction, has higher activity at an alkaline pH (8-10). Differences in the optimum incubation temperatures were also observed: type 1 17 beta-HSD shows a relatively high temperature tolerance (55 degrees C). In contrast, type 2 and 3 functioned best at 37 degrees C. Types 1, 2 and 3 17 beta-HSD activities could be also differentiated by their sensitivity toward various specific inhibitors: type 1 was potently inhibited by an estradiol derivative containing a bromo/or iodopropyl group at position 16 alpha. On the other hand a derivative of estrone containing a spiro-gamma-lactone at position 17 showed a potent inhibitory effect on type 2 17 beta-HSD, whereas type 3 was strongly inhibited by 1,4-androstadiene-1,6,17- trione.
17-Hydroxysteroid Dehydrogenases↗