Human 5α-reductase Research Articles

The Genotype-Phenotype Correlation in Human 5α-Reductase Type 2 Deficiency: Classified and Analyzed from a SRD5A2 Structural Perspective.

The phenotype of the 5α-reductase type 2 deficiency (5αRD2) by the SRD5A2 gene mutation varies, and although there have been many attempts, the genotype-phenotype correlation still has not yet been adequately evaluated. Recently, the crystal structure of the 5α-reductase type 2 isozyme (SRD5A2) has been determined. Therefore, the present study retrospectively evaluated the genotype-phenotype correlation from a structural perspective in 19 Korean patients with 5αRD2. Additionally, variants were classified according to structural categories, and phenotypic severity was compared with previously published data. The p.R227Q variant, which belongs to the NADPH-binding residue mutation category, exhibited a more masculine phenotype (higher external masculinization score) than other variants. Furthermore, compound heterozygous mutations with p.R227Q mitigated phenotypic severity. Similarly, other mutations in this category showed mild to moderate phenotypes. Conversely, the variants categorized as structure-destabilizing and small to bulky residue mutations showed moderate to severe phenotypes, and those categorized as catalytic site and helix-breaking mutations exhibited severe phenotypes. Therefore, the SRD5A2 structural approach suggested that a genotype-phenotype correlation does exist in 5αRD2. Furthermore, the categorization of SRD5A2 gene variants according to the SRD5A2 structure facilitates the prediction of the severity of 5αRD2 and the management and genetic counseling of patients affected by it.

Bacterial expression, purification, and reconstitution of human steroid 5α-reductases in phospholipid liposomes and nanodiscs.

The two human steroid 5α-reductase (5αR) enzymes catalyze the conversion 3-keto-Δ4-steroids to their 5α-reduced congeners. In the genital skin and prostate, the type 2 isoenzyme converts testosterone (T) to the more potent androgen 5α-dihydrotestosterone (DHT), and intracellular DHT is essential for the morphogenesis of the undifferentiated external genitalia to the male phenotype. Both isoenzymes also metabolize other 19- and 21-carbon 3-keto-Δ4-steroids, both endogenous compounds and some steroid-based drugs. Rigorous biochemical studies have been limited due to the extremely hydrophobic nature of these proteins. We have described the heterologous expression of these enzymes in bacteria, their purification with affinity chromatography, and the reconstitution of activity in liposomes. This article details these procedures, as well as reconstitution in phospholipid nanodiscs and enzyme assay.

In silico model and design of novel 5α-reductase inhibitors for treatment of benign prostatic hyperplasia

Purpose: To carry out in silico design of 5α-reductase inhibitors and study their potential for use in the treatment of benign prostate hyperplasia (BPH).
 Methods: In silico molecular docking simulation-based virtual screening of NCI Diversity Set-II containing 1880 diverse ligands was performed against human 5α-reductase for identification of potential lead molecules. The pharmacological properties and toxicity of the lead compounds were determined using software, Marvin Sketch and OSIRIS online programs, respectively.
 Results: Three compounds: ZINC13099050, ZINC01569237 and ZINC17995347_2 showed potent inhibition of 5α-reductase enzyme protein and good pharmacokinetic properties without any serious toxic effects.
 Conclusion: The selected lead molecules are promising inhibitors of 5α-reductase. They are recommended for further structure-based development of drugs for the treatment of BPH.

Structural basis for the catalysis and inhibition of human steroid 5α‐reductase 2

Human steroid 5α-reductases (SRD5As) are membrane-embedded NADPH-dependent steroid and lipid reductases with diverse physiological and pathological roles. Among them, SRD5A2 is a critical enzyme in steroid metabolism that catalyzes testosterone to dihydrotestosterone. Mutations on its gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride as SRD5A2 inhibitors are widely used anti-androgen drugs for benign prostate hyperplasia and androgenic alopecia, which have recently been indicated in the treatment of COVID-19. The molecular mechanisms underlying enzyme catalysis and inhibition had remained elusive for SRD5A2 and other eukaryotic integral membrane steroid reductases due to a lack of structural information. We recently solved a crystal structure of human SRD5A2 at 2.8 Å with finasteride using the lipid mesophase (or LCP) crystallization method. The structure reveals a unique structural topology of 7-transmembrane helices and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride, which binds in a largely enclosed binding cavity inside the membrane. To our knowledge, similar NADPH adducts have not been reported previously. Structural analysis together with computational and mutagenesis studies reveals molecular mechanisms for the 5α-reduction of testosterone and the semi-irreversible finasteride inhibition involving residues E57 and Y91 of SRD5A2. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region of SRD5A2 including three cytosolic loops regulating the NADPH/NADP+ exchange and their entry into the transmembrane region of SRD5A2. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. In summary, our results offer critical and unprecedented structural insights into the function of integral membrane steroid reductases and will facilitate drug development.

Extracts of Phyllostachys pubescens Leaves Represses Human Steroid 5-Alpha Reductase Type 2 Promoter Activity in BHP-1 Cells and Ameliorates Testosterone-Induced Benign Prostatic Hyperplasia in Rat Model.

Benign prostatic hyperplasia (BPH) is the most common symptomatic abnormality of the human prostate characterized by uncontrolled proliferation of the prostate gland. In this study, we investigated the effect of bamboo, Phyllostachys pubescens, leaves extract (PPE) on human 5α-reductase type 2 (SRD5A2) gene promoter activity in human prostate cell lines and the protective effect of PPE on a testosterone-induced BPH rat model. PPE repressed human SRD5A2 promoter activity and its mRNA expression. The rats treated with PPE for 4 weeks showed a significantly attenuated prostate weight compared to vehicle control. PPE-treated rats also showed reduced serum dihydrotestosterone, testosterone, prostate-specific antigen, and SRD5A2 levels by testosterone injection. Quantitative real-time polymerase chain reaction showed that PPE treatment significantly decreased mRNA expression of SRD5A2, androgen receptor (AR), proliferating cell nuclear antigen (PCNA), and fibroblast growth factor 2 compared with the vehicle-treated, testosterone-injected rats in the prostate. Furthermore, PPE treatment showed reduced AR, PCNA, and tumor necrosis factor alpha expression in the prostate via immunohistofluorescence staining. In conclusion, oral administration of PPE prevented and inhibited the development and progression of enlarged prostate lesions in testosterone-induced animal models through various anti-proliferative and anti-inflammatory pharmacological effects and induced suppression of SRD5A2 gene expression.

Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases.

The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

Photochemical Rearrangement of a 6-Azasteroid Oxaziridine to a Novel 17β-Carbomethoxy-A-homo-B-seco-6-aza-3, 5-androstanedione

4- and 6-azasteroids have been shown to be potent inhibitors of human 5α-reductase and certain azasteroids have shown significant clinical benefit in the treatment of androgen-related disorders. In an effort to expand the diversity of steroidal heterocycles synthetically accessible for structure-activity relationship exploration, a novel reaction sequence was applied to the preparation of the 6-azasteroid framework. To this end, photolysis of the oxaziridine derived from 17β-carbomethoxy-3β-triisopropylsilyloxy-6-azaandrost-5-ene (1) yielded a novel 7, 5-steroidal ring system that was evaluated for inhibition of human type 1 and 2 5α-reductase.

MON-219 In Silico and in Vitro Studies of Human 5α-Reductase Type II Variants in Search for Activating Variants Explaining Androgen Excess Reveal New Loss-Of-Function Variants

Background: Androgens are steroid hormones necessary for human sex development. Testosterone (T) and the more potent dihydrotestosterone (DHT) are maybe the best known androgens, which exert their effect by binding and activating the androgen receptor. Steroid reductases 5α (SRD5As) catalyse the conversion of T to DHT in the classic androgen production pathway, or from 17-hydroxyprogesterone to 17OH-dihydroprogesterone, and androstenedione to androstanedione in alternate pathways leading to DHT. There are two enzymes with differential expression, of which SRD5A2 is expressed in reproductive organs and liver, and catalyses the reaction of T to DHT more efficiently than SRD5A1. Human SRD5A2 loss-of-function mutations are known, and cause severe 46XY undervirilization, while gain-of-function variants have been suggested in androgen excess syndromes such as premature adrenarche, the polycystic ovary syndrome or prostate tumors, but they have not been found so far. Aim: Therefore, we aimed to search for gain-of-function mutations in the human SRD5A2 gene. Methods: For that, we searched databases for candidate variants and performed bioinformatic and functional tests on selected variants. After conservation analysis of SRD5A2, a novel 3D protein model was constructed to locate the exact position of amino acids in the tertiary structure and predict their effect on protein function and substrate interaction. We then collected 116 coding SNPs in SRD5A2 from OMIM, dbSNP, Pubmed, Clinvar, HGMD and Uniprot databases. These SNPs were ranked according to their association with phenotypes, physical location in our 3D model, and molecular dynamics simulation studies. Finally, we selected 9 coding SNPs for in vitro studies. These SNPs were located within or close to highly conserved areas that form the binding cavities for substrates or cofactor NADPH. SRD5A2 variants were expressed in HEK293 cells and activity was assessed by conversion of testosterone (T), progesterone (Prog) and androstenedione (Δ4Α) to their 5α-reduced metabolites. Result: Variants R50A and P173S showed partial activity with substrates T (34% and 28%) and Δ4Α (37% and 22%). With substrate Prog variants P106L, P106A, L167S and R168C in addition showed partial activity (15% to 64%). Functional testing of all other variants showed loss-of-function. As predicted in our in silico analysis, all coding SNPs affected enzyme activity, however none of them showed gain-of-function In conclusion, we provide a novel protein model for studies of SRD5A2. No gain-of-function variants were identified, but we have characterized 7 human SRD5A2 variants, which might be of clinical relevance for their enzyme activity loss. It is possible that individuals carrying these SNPs show a minor phenotype that is not yet identified.

Synthesis and biological evaluation of novel unsaturated carboxysteroids as human 5α-reductase inhibitors: A legitimate approach

In the present study, novel steroidal 17a-substituted 3-cyano-17a-aza-D-homo-3,5-androstadien-17-ones (12–19) and 17a-substituted 17-oxo-17a-aza-D-homo-3,5-androstadien-3-oic acids (20–26) were synthesized from dehydroepiandrosterone acetate (6) along with 17-oxo-19-nor-3,5-androstadien-3-oic acid (30) through a multistep synthesis. Compounds were evaluated for their in vitro 5α-reductase inhibitory activity by measuring the conversion of [3H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity was also determined using rat prostate weighing method. Compounds 21–23 and 25 showed potent inhibition of 5α-reductase II enzyme with IC50 values of 54.1 ± 9.5, 22.1 ± 2.4, 72.8 ± 2.3 and 26.5 ± 4.4 nM respectively as compared to Finasteride (30.3 nM) along with a significant (p < 0.05) reduction in rat prostate weight.

Ligand Based 3D-QSAR Study on a Series of Steroidal Human 5&amp;#945;-Reductase Inhibitors

Azasteroids constitute a class of modified steroids in which one or more carbon atoms in the steroid ring system have been substituted with nitrogen atom, having human 5α-reductase inhibitory activities for the management of benign prostatic hyperplasia (BPH). We have performed ligand based 3D-QSAR study using Self Organizing Molecular Field Analysis (SOMFA) on a series of 4-azasteroids including clinically used drug Finasteride. The SOMFA studies results indicate significant q2 (0.809), r2 (0.834) and F-test (70.060) values showing good predictive ability. The analysis of SOMFA grids provides an insight for generation of optimal molecular architecture for better human 5α-reductase inhibitory activity. Keywords: 4-Azasteroids, BPH, Finasteride, 3D-QSAR, 5α-Reductase, SOMFA

Self-organizing molecular field analysis on pregnane derivatives as human steroidal 5α-reductase inhibitors

Normal growth and development of human prostate is regulated by the androgens which balances cell proliferation and apoptosis. Testosterone (T) and dihydrotestosterone (DHT) are the two key androgens that stimulate most of the androgen action in prostate. Testosterone is converted to DHT by the membrane bound NADPH-dependent 5α-reductase enzyme. As a consequence of the important observation that progesterone and deoxycortisone inhibits the synthesis of DHT by competing with 4-en-3-one function of the testosterone for the 5α-reductase enzyme a number of pregnane derivatives were synthesized and have been reported as inhibitors of human 5α-reductase enzyme. Due to lack of information on the crystal structure of human 5α-reductase, ligand-based 3D-QSAR study has been performed on pregnane derivatives using self-organizing molecular field analysis (SOMFA) for rationalizing the molecular properties and human 5α-reductase inhibitory activities. The statistical results having good cross-validated rcv2 (0.881), non-cross-validated r2 (0.893) and F-test value (175.527), showed satisfied predictive ability rpred2 (0.777). Analysis of SOMFA models through electrostatic and shape grids provide useful information for the design and optimization of steroidal structure as novel human 5α-reductase inhibitors.

Synthesis and biological activity of progesterone derivatives as 5α-reductase inhibitors, and their effect on hamster prostate weight

In this study, we report the synthesis and biological evaluation of four 6- and 17-substituted progesterone derivatives (7–10). These compounds were prepared from the commercially available 17α-acetoxyprogesterone. The biological effect of these steroids was demonstrated in in vivo as well as in vitro experiments. In the in vivo experiments, we measured the activity of 6–10 on the weight of the prostate glands of gonadectomized hamsters treated with testosterone (T). For the studies in vitro, we determined the IC50 value by measuring the concentration of steroidal derivative that inhibited 50% of the activity of 5α-reductase present in the human prostate. The results from this work indicated that compounds 6–9 significantly decreased the weight of the prostate as compared to testosterone-treated animals and this reduction of prostate weight was comparable to that produced by finasteride. Steroid 8 was the most effective of the tested compounds. However, compound 10 did not exhibit this capacity. On the other hand, 6–9 exhibited a high inhibitory activity for the human 5α-reductase enzyme with IC50 values of 10, 70, 22, and 19 nM, respectively. However, 10 was not effective for the inhibition of 5α-reductase activity. In conclusion, the compounds that contained the acetate ester moiety in the molecule (6, 7, 8, and 9) inhibited the activity of 5α-reductase and decreased the weight of the prostate. Nevertheless, the double bond in ring B seems to diminish the inhibitory potency (7 and 9), since 6, which does not possess a double bond at C-6, had the highest inhibitory activity (the lowest IC50 value).

3D-QSAR studies on unsaturated 4-azasteroids as human 5α-reductase inhibitors: A self organizing molecular field analysis approach

Azasteroids have been reported as inhibitors of human 5α-reductase enzyme. These were designed by substitution of one carbon atom of steroidal A ring by heteroatom nitrogen. Due to lack of information on the crystal structure of human 5α-reductase, 3D-QSAR study has been performed on a series of unsaturated 4-azasteroids using Self Organizing Molecular Field Analysis (SOMFA) for rationalizing the molecular properties and human 5α-reductase inhibitory activities. The statistical results having good cross-validated r2cv (0.783), non cross-validated r2 (0.806) and F-test value (87.282), showed satisfied predictive ability. Analysis of SOMFA models through electrostatic and shape grids provide useful information for the design and optimization of new steroidal human 5α-reductase inhibitors.

Self organizing molecular field analysis on a series of human 5α-reductase inhibitors: Unsaturated 3-carboxysteroid

Steroidal 5 α-reductase is a NADPH dependent enzyme that catalyzes the irreversible conversion of 4-en-3-oxo-steroid testosterone to the corresponding 5 α-H-3-oxo-steroid dihydrotestosterone thus involved in Benign Prostatic Hyperplasia (BPH). As the crystal structure of target enzyme is not available; we have carried out ligand based designing using Self Organizing Molecular Field Analysis (SOMFA). SOMFA, a novel 3D-QSAR methodology used in present case to study the correlation between molecular properties and human 5α-reductase inhibitory activities of a series of unsaturated 3-carboxysteroid. The statistical results, good cross-validated r 2 cv (0.693) and non cross-validated r 2 (0.732), showed satisfied predictive ability. All analysis of SOMFA model may provide some useful information in the design of human steroidal 5α-reductase inhibitors with better spectrum of activity.

Novel C-6 substituted and unsubstituted pregnane derivatives as 5α-reductase inhibitors and their effect on hamster flank organs diameter size

The present study is addressed to ascertain the inhibitory effect of several progesterone derivatives having a chlorine substituent at C-6 (12a–12d), 15 with a bromine substituent at C-6 and 14a–14d, without any halogen atom at C-6 all having an ester side chain at C-17 (benzoate ester bearing a Cl, F and a Br atom at C-4 position of the phenyl ring) on the 5α-reductase enzyme activity present in human prostate. In addition, it was also of interest to investigate the pharmacological effect on hamster flank organs diameter size.In order to study the structure–activity relationships of steroids 12a–12d, 14a–14d and 15 we determined the concentration of these steroids that inhibited 50% of the activity of human prostate 5α-reductase enzyme (IC50), as well as the in vivo effect of these compounds in the hamster flank organs diameter size. We also ascertained, the capacity of these steroids to bind to the androgen receptors present in the rat prostate cytosol using labeled mibolerone (MIB) for monitoring the binding to the androgen receptor.The results from this study indicated that compounds 12a–12d (having a chlorine substituent at C-6), 14a–14d (lacking a halogen atom at C-6), 13 and 15 (having a bromine atom at C-6) showed an increased antiandrogenic effect (lower value for the diameter of the flank organs) as compared to the flank organs from testosterone-treated hamsters. On the other hand, the series of compounds containing a chlorine substituent at C-6 compounds (12a–12d) showed a higher antiandrogenic activity as compared to the compounds lacking a halogen atom at C-6 (14a, 14b and 14d). Although compounds 13 and 15 decreased the flank organs diameter size, however, this increase was not statistically significant as compared to that of the commercially available product finasteride. The steroidal derivatives 13, 14a–14d (lacking the chlorine substituent at C-6) and 15 (having a bromine atom at C-6) exhibited a higher 5α-reductase inhibitory activity (lower IC50 values) as compared to the series of compounds 12a–12d having the halogen substituent at C-6.Finasteride reduced the diameter size of the flank organs. The effect of this steroid and compounds 12a–12d, 13, 14a–14d and 15 on hamster flank organs can be explained by the fact that these steroids did not bind to the androgens receptor, which indicates that its mechanism of action is an inhibiting for the 5α-reductase activity. This enzyme is present in the hamster flank organs and was inhibited by the novel steroids in the human prostate homogenates.

Changes in human placental 5α-reductase isoenzyme expression with advancing gestation: effects of fetal sex and glucocorticoid exposure

5alpha-reduced steroids, including allopregnanolone, suppress neuronal activity and can have neuroprotective effects in the fetus. 5alpha-reductases in the placenta may contribute precursors to brain allopregnanolone synthesis. Preterm birth and glucocorticoids, administered for fetal lung maturation or for maternal asthma, may influence reductase expression. The aims of the present study were to evaluate placental 5alpha-reductase isoform expression during late gestation and to examine fetal sex differences and the effects of glucocorticoid therapies on the expression of these enzymes. Expression of the two 5alpha-reductase isoenzymes was measured in placental samples, whereas cortisol concentrations were measured in cord blood, from two cohorts. The first cohort consisted of women who delivered preterm and received betamethasone treatment (n=41); the second cohort consisted of women who delivered at term and were either healthy controls (n=30) or asthmatics who had used glucocorticoids (n=24). Placental expression of both isoenzymes increased with advancing gestation and there were marked sex differences in levels of 5alpha-reductase I (P<0.05), but not of 5alpha-reductase II. The expression of both enzymes was positively correlated with cortisol levels (P<0.05), but there was no effect of recent glucocorticoid exposure. These findings suggest that the preterm neonate may have lower developmental exposure to 5alpha-reduced steroids and may lack steroid-mediated neuroprotection depending on fetal sex.

In vivo and in vitro effect of novel 4,16-pregnadiene-6,20-dione derivatives, as 5α-reductase inhibitors

In this study, we report the synthesis and biological evaluation of several new 3-substituted pregna-4,16-diene-6,20-dione derivatives ( 11a– 11d). These compounds were prepared from the commercially available 16-dehydropregnenolone acetate. The biological effect of these steroids was demonstrated in in vivo and in vitro experiments. In the in vivo experiments, we measured the activity of the 11a– 11d on the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with the new steroids. For the studies in vitro, we determined the IC 50 values by measuring the steroid concentration that inhibits 50% of the activity of 5α-reductase present in human prostate. In order to study the mechanism of action of 11a– 11d, we also determined the capacity of these steroids to bind to the androgen receptor (AR) present in the rat prostate cytosol using labeled mibolerone as a tracer. The results from this work indicated that compounds 11a– 11d significantly decreased the weight of the prostate as compared to testosterone treated animals and this reduction of the weight of the prostate was comparable to that produced by the finasteride. On the other hand 11a– 11d exhibited a high inhibitory activity for the human 5α-reductase enzyme with IC 50 values of 1.4 × 10 −8, 1.8 × 10 −9, 1.0 × 10 −8 and 4 × 10 −5 respectively. However the IC 50 value of 11a (1.8 × 10 −9) was the only one lower than that of finasteride (8.5 × 10 −9). Nevertheless this compound did not show a higher potency in vivo as compared to that of compounds 11b– 11d. The competition analysis for the androgen receptor indicated that the IC 50 value of non-labeled mibolerone used in this experiment was 1 nM, whereas steroids 10, 11a– 11d did not inhibit the labeled mibolerone binding to the androgen receptor. On the other hand, steroid 10 did not show any activities in vitro or in vivo, and for this reason these steroidal derivatives ( 11a– 11d) cannot be considered as prodrugs of compound 10. In conclusion, the compounds containing chlorine 11a, bromine 11b, iodine 11c atoms, and 11d (without any substituent in the ester moiety) at C-3 produce a significant decrease of the prostate weight in castrated animals treated with T and inhibits the activity of the 5α-reductase. Apparently the presence of the halogen atoms in compounds 11a– 11c enhances the inhibitory activity for the 5α-reductase enzyme.

3,3-Diphenylpentane skeleton as a steroid skeleton substitute: Novel inhibitors of human 5α-reductase 1

We designed and synthesized novel type 1 5α-reductase inhibitors by using 3,3-diphenylpentane skeleton as a substitute for the usual steroid skeleton. 4-(3-(4-( N-Methylacetamido)phenyl)pentan-3-yl)phenyl dibenzylcarbamate ( 11k) is a competitive 5α-reductase inhibitor with the IC 50 value of 0.84 μM.

Steroids with a carbamate function at C-17, a novel class of inhibitors for human and hamster steroid 5α-reductase

In order to study the biological activity of the two novel steroidal carbamates derivatives: 8a and 8b, we determined the concentration of both compounds that inhibit the 50% of the activity of human prostate 5α-reductase enzyme, as well as the in vivo effect of these compounds in the weight of hamster prostate and flank organs diameter size. We determined also, the capacity of these steroids to bind to the androgen receptors present in the rat prostate cytosol. Furthermore the activity of these compounds on the mRNA expression of glycerol 3-phosphate acyl transferase (GPAT) in flank organs was analyzed by RT-PCR. This enzyme induces the triglycerides synthesis, which is increased by T in flank organs. The results from this study indicated that steroids 8a and 8b inhibited the human 5α-reductase activity. Compound 8b, which contains a bromine atom in the molecule, decreased the inhibitory effect of the human 5α-reductase activity, whereas steroid 8a, which lacks a halogen atom did not show any decrease in the activity of this enzyme. The competition studies demonstrated that 8a and 8b did not inhibit mibolerone binding to the androgen receptor present in the rat prostate cytosol. However, the in vivo activity of both steroids was similar; steroids 8a and 8b had a tendency to decrease the weight of the hamster prostate although this parameter was not statistically significant. These compounds also significantly reduced the diameter of the pigmented spot of hamster flank organs, which are androgen dependent skin's pilosebaceous structures. Steroids 8a and 8b, decreased the transcription of mRNA encoding for GPAT in intact hamster's flank organs topically treated in a similar way as in gonadectomized non-treated animals. These results suggest that mRNA encoding for GPAT is induced by DHT in this tissue.

Biocatalytic synthesis of 4-pregnen-20,21-diol-3-one, a selective inhibitor of human 5α-reductase type II

Biocatalysis, the conversion of substrates into valuable products by the use of enzymes, has some striking advantages in comparison to standard organic chemistry for drug synthesis. By biocatalysis, substrates that contain several identical reactive groups at different positions can be converted with high regio-selectivity and enantio-selectivity. In this study, an E. coli isolate (E132) was identified which was able to convert the steroid desoxycorticosterone into the product 4-pregnen-20,21-diol-3-one in real terms. The product was purified from the cell culture supernatant by HPLC and its structure was demonstrated by mass spectrometry and NMR spectroscopy. It was tested on inhibition of human 5α-reductases type I and type II. At a concentration of 10 μM, inhibition was 49.0% for type I and 81.8% for type II, whereas there was no inhibition of human aromatase (CYP19) at 20 μM and human 17α-hydroxylase-C17,20-lyase (CYP17) at 2.5 μM detectable. The IC50 value of 4-pregnen-20,21-diol-3-one for human 5α-reductase type II was determined to be 1.56 μM.