Human Steroid 5α-reductase Research Articles

Paraben preservatives exhibit inhibition on human and rat steroid 5α-reductase 1: A comprehensive 3D-QSAR and computational analysis

Parabens are preservatives used in personal care products, cosmetics, and pharmaceuticals. Steroid 5α-reductase 1 (SRD5A1) catalyzes the conversion of testosterone to dihydrotestosterone and is present in the brain, contributing to neurosteroid production. This study aimed to assess the effects of nine paraben preservatives on SRD5A1 in human SF126 glioblastoma cell and rat brain microsomes, particularly focusing on dihydrotestosterone production in SF126 cells. The results showed that methyl, ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl paraben inhibited human SRD5A1, with nonylparaben having the strongest effect (7.59 μM). Additionally, kinetic analysis indicated that parabens acted as mixed/noncompetitive inhibitors, leading to a significant decrease in dihydrotestosterone production in SF126 cells. While rat SRD5A1 exhibited lower sensitivity to parabens, docking analysis revealed that parabens bind to the NADPH-binding site of both human and rat SRD5A1. In conclusion, these results highlight the inhibitory effects of paraben preservatives on SRD5A1 and elucidate their binding mechanisms, underscoring their role in hormone production.

Modulatory properties of Thespesia garckeana (F. Hoffm.) Exell & Hillc. Aqueous fruit extract on female rats reproductive hormones and tissues histology and potential inhibitory activity of its constituents against SRD5α2

Ethnopharmacological relevanceThespesia garckeana (F. Hoffm.) Exell & Hillc. is called Gorontula (Kola of Tula) in Nigeria, Morojwa in Bostwana, and Thespesia garckeana in South Africa and is widely distributed across Africa. Its parts reportedly possess multiple medicinal properties and are employed for treating various diseases. In Tula, Gombe State, Nigeria, the ripe fruit decoction is taken as remedy for female infertility as documented by Ochokwu and co in the Journal of Biology, Agriculture and Healthcare in 2015. Aim of the studyThis research examined the effects of aqueous fruit extract of T. garckeana (F. Hoffm.) Exell & Hillc. (AFETG) on selected reproductive tissues and hormones in female rats and also evaluated the inhibitory potentials of its phytoconstituents against human 5-alpha reductase 2 (SRD5α2) using in silico approach. MethodsTwenty-five (25) sexually matured female rats were randomized into 5 groups (i.e. A - E). Oestrous in the rats was synchronized (subcutaneous oestradiol conjugate [10 μg/100 g BW]). Group A received distilled water (control). Group B received standard drug, clomiphene citrate (0.85 mg/kg BW), while groups C, D, and E received AFETG at 50, 100, and 200 mg/kg BW respectively. The animals were treated for five (5) days and sacrificed 24 h after. Their blood was collected and prepared for analysis of serum prolactin, oestradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) while ovaries, uteruses, and oviducts were studied for histological and histomorphometric changes. For the in silico study, the target protein, human steroid 5α-reductase 2 (SRD5α2) was prepared and its receptor grid was generated using Optimized Potential for Liquid Simulations-2005. The ligand 2D structures were prepared using LigPrep 2.4 software and docked using Glide. The binding energy of the ligands to the protein receptor was predicted using Molecular Mechanics/Generalized Born Surface Area (MM-GBSA) analysis. ResultsAFETG significantly increased serum levels of E2 but did not alter serum levels of prolactin, LH and FSH when compared with distilled water and clomiphene citrate. AFETG also significantly increased ovarian tertiary follicular diameter, oviductal epithelial height and serosa thickness as well as uterine endometrial epithelial height, endometrial thickness, and myometrial thickness when compared with control. Ovarian secondary follicular diameter and oviductal submucosa thickness and muscular thickness were significantly decreased by AFETG when compared with control. Two compounds in T. garckeana (F. Hoffm.) Exell & Hillc.; D-Melezitose (−12.55 kcal/mol XP GScore) and 1, 3, 4-trihydroxy-5-oxo cyclohexane-1-carboxylic acid (−9.136 kcal/mol XP GScore) exhibited higher binding affinities for SRD5α2 than the reference ligand, epristeride (−8.096 kcal/mol XP GScore).In conclusion, the ability of AFETG to increase serum E2 level, thickness of uterine endometrium and ovarian tertiary follicles size can be explored for the treatment of female infertility caused by thinning of the uterine endometrium and reduced follicular size. Two compounds in AFETG (i.e. D-Melezitose and 1, 3, 4-trihydroxy-5-oxo cyclohexane-1-carboxylic acid are potential inhibitors of SRD5α2, thus aiding the biosynthesis of E2. Available evidence therefore corroborate the traditional use of T. garckeana (F. Hoffm.) Exell & Hillc fruit as a female fertility enhancer in Northern Nigeria.

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.

Phytochemical, Chemotaxonomic and Bioinformatics Study on Laportea bulbifera (Urticaceae).

Phytochemical investigation of the aerial part of Laportea bulbifera (Siebold & Zucc.) Wedd. (L. bulbifera) showed the isolation of seventeen compounds, including five flavonoids (1-4 and 6), one terpenoid (5), five phenolic acids (7-11), one coumarin (12), two steroids (13-14), and three alkaloids (15-17). Structure elucidations of these compounds were performed on the basis of extensive NMR experiments and compared with the published data in the references. It is remarkable that compounds (3-5) were firstly isolated from the Urticaceae family, compounds (3-8, 11 and 15-17) were firstly obtained from genus Laportea. Furthermore, the result of the chemotaxonomic significance discussion showed that compounds (2-4) may can be served as compound fingerprints to distinguish between species of L. bulbifera and genus Urtica, and what' more, we proposed a bold conjecture that isoflavones can distinguish between species of L. bulbifera and genus Urtica. At the same time, the molecular docking method was used to evaluate the inhibitory effect of these compounds on human steroid 5α-reductase 2 (SRD5α2). The results showed that compounds (1-4 and 6) had better expected effects than the positive drug finasteride can by effectively binding to the active sites of SRD5α2. This study assisted in the future phytochemical and chemotaxonomic research on genus Laportea. Simultaneously, this research provided the theoretical evidence for the application of L. bulbifera in treating benign prostatic hyperplasia (BPH).

Structure of human steroid 5\u03b1-reductase 2 with the anti-androgen drug finasteride

Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme in steroid metabolism and catalyzes the reduction of testosterone to dihydrotestosterone. Mutations in the SRD5A2 gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride, as SRD5A2 inhibitors, are widely used antiandrogen drugs for benign prostate hyperplasia. The molecular mechanisms underlying enzyme catalysis and inhibition for SRD5A2 and other eukaryotic integral membrane steroid reductases remain elusive due to a lack of structural information. Here, we report a crystal structure of human SRD5A2 at 2.8 Å, revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the transmembrane domain. Structural analysis together with computational and mutagenesis studies reveal the molecular mechanisms of the catalyzed reaction and of finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region that regulate NADPH/NADP+ exchange. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and may facilitate drug development.

Mutational analysis of SRD5A2: From gene to functional kinetics in individuals with steroid 5α-reductase 2 deficiency

Human steroid 5α-reductase 2 (SRD5A2) plays a determinative role in the masculinization of external genitalia. To date, approximately 114 different mutations of the SRD5A2 gene have been reported; however, little information is available about their impact on catalytic function or their three-dimensional (3D) structures. We determined the effect of point mutations on the testosterone-depend kinetic constants (Km,app and Vmax,app) and structural characteristics of SRD5A2 from Mexican patients with 46,XY-steroid 5α-reductase 2 deficiency. PCR-SSCP assays identified ten distinct gene variants and sequencing analysis identified missense mutations [p.V3I, p.S14R, p.A52T, p.F118L, p.R145W, p.R171S, p.L226P, p.F229S, p.S245Y, and p.A248V]. Mutations were re-created by site-directed mutagenesis and expressed in HEK293 cells. Functional studies demonstrated that 8 variants led to partial (Km,app = 0.16–2.6 μM; Vmax,app = 224−2640 pmol/mg P/min) or complete losses of activity compared to the wild-type enzyme (Km,app = 0.7 μM; Vmax,app = 4044 pmol/mg P/min). All the mutations were assessed using multiple software tools and the results predicted that all of the mutations were associated with disease or damage. Mapping mutations on the model of a 3D structure of SRD5A2 demonstrated alterations in contact sites with their proximal amino acids. Our data show that mutations affect the catalytic efficiency (Vmax/Km) or result in residual enzymatic activity, which could be due to erroneous interactions between amino acid residues, the substrate testosterone, or NADPH.

Subcellular localization of the five members of the human steroid 5α-reductase family

In humans the steroid 5α-reductase (SRD5A) family comprises five integral membrane enzymes that carry out reduction of a double bond in lipidic substrates: Δ4-3-keto steroids, polyprenol and trans-enoyl CoA. The best-characterized reaction is the conversion of testosterone into the more potent dihydrotestosterone carried out by SRD5A1-2. Some controversy exists on their possible nuclear or endoplasmic reticulum localization.We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

Erratum to: In silico structural prediction of human steroid 5α-reductase type II

Erratum to: In silico structural prediction of human steroid 5α-reductase type II

In silico structural prediction of human steroid 5α-reductase type II

Steroid 5α-reductase type II is a membrane-associated enzyme in an oxidoreductase family. This enzyme, which is found in male sexual organs, plays the important biological actions toward steroid metabolism. Overexpression of 5α-reductase type II has affected the balance between testosterone and dihydrotestosterone, which implicates the androgenic disorders, including prostate cancer, hirsutism, and androgenic alopecia. Lack of single-crystal X-ray structures of 5α-reductase has hindered mechanistic understanding and delayed the discovery and development of an effective inhibitor. Herein, we illustrated a comparative structure of 5α-reductase type II that derived from the homology modeling, employing a membrane-bound protein, isoprenylcysteine carboxyl methyltransferase as a homologous template. A catalytic pocket and the transmembrane site were identified. The resulting in silico structure was validated via Ramachandran plot and confirmed by docking studies of 30 known 5α-reductase type I and type II inhibitors, including finasteride and dutasteride. The comparative docking study of the derived in silico 5α-reductase type II and 5β-reductase, a reported surrogate enzyme, was conducted. Our homology model approximately fitted to the membrane-associated character and showed the reasonable docking results, which depicted the well-defined comparative three-dimensional structure applicable for steroid reductase drug design.

Design of potent human steroid 5α-reductase inhibitors: 3D-QSAR CoMFA, CoMSIA and docking studies

3D-QSAR CoMFA, CoMSIA and docking studies were performed on a set of 4-azasteroidal human steroid 5α-reductase inhibitors. The models developed using maximal common substructure-based alignment was found to be reliable and significant with good predictive r2 value. CoMSIA model developed using combination of steric, electrostatic, hydrophobic, hydrogen bond donor and hydrogen bond acceptor features has shown rcv2 = 0.564 with six optimum components, rncv2 = 0.945, F value = 101.196, rPred2 = 0.693 and SEE = 0.209. The contour plots obtained has shown a favourable effect of bulkier groups at C-17 position. Docking studies indicates the importance of bulkier groups at C-17 position for favourable activity. The study further helps in design of potent inhibitors of the enzyme.

3D-QSAR CoMFA and CoMSIA studies for design of potent human steroid 5α-reductase inhibitors

3D-QSAR studies were performed on a set of sixty-one 6-azasteroidal human steroid 5α-reductase inhibitors. The models developed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methodologies employing atom and centroid based alignment were reliable and significant having good predictive r 2 value. CoMSIA model developed by a combination of steric, electrostatic, hydrophobic and hydrogen bond donor showed good relative and predictive properties. The predictive power of the models was assessed using an external test set of 10 compounds. The best model had shown cross validation r 2 (0.702) with 7 optimum components, non-cross-validation r 2 (0.956), F value (86.158), predicted r 2 (0.704), standard error of estimate (0.165). Further the CoMFA/CoMSIA contour plots show a preference toward the C-17 substituents and indicate that bulky group at C-17 is an important requirement for exhibiting 5α-reductase inhibition. These developed models could be used to design and optimise the more potent 6-aza steroidal inhibitors of 5α-reductase enzyme. The 3D-QSAR studies involving comparative molecular field analysis and comparative molecular similarity indices analysis on a series of 6-azasteroids as 5α-reductase inhibitors is reported.

ChemInform Abstract: Synthesis of 8-Chloro-benzo[c]quinolizin-3-ones as Potent and Selective Inhibitors of Human Steroid 5α-Reductase 1.

The synthesis of a series of differently substituted 8-chloro-benzo[c]quinolizin-3-ones, as potent and selective human steroid 5alpha-reductase type 1 inhibitors, has been accomplished by a four-step procedure based on the TiCl4-promoted tandem Mannich-Michael cyclization of 2-silyloxy-1,3-butadienes with N-t-Boc iminium ions from quinolin-2-ones. The presence on the benzo[c]quinolizinone nucleus of a methyl group and a double bond at positions 6 and 4-4a, respectively, as in compound 1d, gave rise to one of the most potent non-steroidal 5alphaR-1 inhibitors reported so far (IC50 = 14 nM).

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.

Novel 5α-Reductase Inhibitors: Synthesis, Structure−Activity Studies, and Pharmacokinetic Profile of Phenoxybenzoylphenyl Acetic Acids

Novel substituted benzoyl benzoic acids and phenylacetic acids 1-14 have been synthesized and evaluated for inhibition of rat and human steroid 5alpha-reductase isozymes 1 and 2. The compounds turned out to be potent and selective human type 2 enzyme inhibitors, exhibiting IC(50) values in the nanomolar range. The phenylacetic acid derivatives were more potent than the analogous benzoic acids. Bromination in the 4-position of the phenoxy moiety led to the strongest inhibitor in this class (12; IC(50) = 5 nM), which was equipotent to finasteride. Since oral absorption is essential for a potential drug, 12 was further examined. In the parallel artificial membrane permeation assay (PAMPA) it turned out to be a good permeator, whereas it was a medium permeator in Caco2 cells. After oral administration (40 mg/kg) to rats a high bioavailability and a biological half-life of 5.5 h were observed, making it a promising candidate for clinical evaluation.

Pharmacogenetic analysis of human steroid 5α reductase type II: comparison of finasteride and dutasteride

Human steroid 5 alpha-reductase type II is a prostate-specific, membrane-associated enzyme that catalyzes the conversion of testosterone to dihydrotestosterone, the most potent androgen in the prostate gland. Genetic variants of this enzyme have been associated with both the development and the progression of prostate cancer. Both finasteride and dutasteride are competitive inhibitors of the type II steroid 5 alpha-reductase that have been effectively used for the treatment of benign prostatic hyperplasia. Finasteride has also been successfully utilized for prostate cancer chemoprevention. We here investigate 5 alpha-reductase inhibition assays in vitro to measure the effect of incubation time on the apparent inhibition constant (Ki) for both constitutional and somatic (prostate cancer) enzyme variants. Our systematic pharmacogenetic analysis shows that both finasteride and dutasteride are slow, time-dependent inhibitors of steroid 5 alpha-reductase type II, and that the inhibition kinetics depend on the 5 alpha-reductase genotype. We also show that, overall, dutasteride is a more efficient steroid 5 alpha-reductase inhibitor than finasteride. Based on our data, we are able to map areas of the enzyme that are responsible for this time-dependent inhibition for either (or both) enzyme inhibitor(s). This comprehensive pharmacogenetic analysis of steroid 5 alpha-reductase variants unveiled significant pharmacogenetic variation for both finasteride and dutasteride and thus should be taken into account when designing protocols for treatment and/or chemoprevention of prostatic diseases with either one of these 5 alpha-reductase inhibitors since there is considerable pharmacogenetic variation for both drugs.

Steroidal nitrone inhibitors of 5α-reductase

This paper describes the rational design and synthesis of novel inhibitors of human steroid 5α-reductase. Steroidal nitrones were synthesised via an eight-step sequence from epiandrosterone and were tested for activity against type I and II 5α-reductase isozymes. Judicious placement of the nitrone into the steroid A-ring provided an effective and stable transition-state mimic of the postulated enolate intermediate involved in the conversion of testosterone into dihydrotestosterone.

Comparative study of human steroid 5α-reductase isoforms in prostate and female breast skin tissues: sensitivity to inhibition by finasteride and epristeride

Steroid 5α-reductase (5-AR) catalyses the reduction of testosterone (T) to dihydrotestosterone (DHT). The 5α-reductase found in human benign prostatic hyperplasia (BPH) has been compared with that found in human breast skin tissue in respect of sensitivity to inhibition by Finasteride and Epristeride. Kinetic studies showed the presence of two isoforms of 5α-reductase in benign prostatic hyperplasia indicated by low and high Km isoforms for testosterone, while female breast skin tissue contained only one isoform. The isoforms differ in their affinity for the inhibitors Finasteride and Epristeride, both compounds being more effective for the low Km 5α-reductase isoform than the high Km 5α-reductase of prostatic tissue, with Finasteride displaying competitive inhibition and Epristeride uncompetitive. Finasteride and Epristeride are also inhibitors of skin 5α-reductase, which possesses a comparable Ki for Finasteride to that of the low Km prostatic enzyme, but Epristeride was a less potent inhibitor of the skin enzyme relative to the prostate isoform. These results suggest that the inhibitors have therapeutic potential, other than for treatment of benign prostatic hyperplasia, for treating skin disorders influenced by the action of dihydrotestosterone and warrant further investigation.

Structure-function studies of human 5-alpha reductase type 2 using site directed mutagenesis

Site directed mutagenesis of human steroid 5α-reductase types 1 (5AR1) and 2 (5AR2) has been used to identify residues involved in inhibitor/substrate binding by 5AR2. Replacing residues 21–24 (GALA) in 5AR2 with the analogous residues 26–29 (AVFA) from 5AR1 did not significantly alter either the Km for testosterone or the Ki for the competitive inhibitor Finasteride. Replacement of AVFA in 5AR1 with GALA from 5AR2 however, significantly decreased the Km and increased the resistance to Finasteride. These findings confirm that 5AR1 residues 26–29 are involved in inhibitor/substrate binding but suggest residues 21–24 of 5AR2 are not. Replacing residues 20–29 (QCAVGCAVFA) of 5AR1 with the analogous residues 15–24 (ATLVALGALA) from 5AR2, changed the Km and Ki to values approaching those for wild type 5AR2. Replacing residues VAL in wild type 5AR2 with VGC from 5AR1 did not change Km or Ki but replacing ATL in 5AR2 with QCA from 5AR1 significantly decreased the Km and increased the resistance to Finasteride. Conversely, replacing QCA with ATL in 5AR1 containing GALA in place of AVFA, increased the Km and decreased resistance to Finasteride. These findings indicate residues 15–17 of human 5AR2 participate in inhibitor/substrate binding whereas residues 18–20 do not.

ChemInform Abstract: Simple Bi- and Tricyclic Inhibitors of Human Steroid 5α-Reductase.

Abstract A number of tricyclic thiolactams, bicyclic lactams, and bicyclic thiolactams have been prepared and evaluated in vitro as inhibitors of types 1 and 2 steroid 5α-reductase. The tricycles with an 8-chloro substituent in the C-ring are nM (IC 50 ) inhibitors of type 1 steroid 5α-reductase (SR). In all the cases studied, lactams are more potent than the corresponding thiolactams. Activity against type 2 SR is greatly enhanced by a styryl (or azo) substituent on the aryl ring of the tri- and bicycles and also a related tricyclic aryl acid.

Simple Bi- and tricyclic inhibitors of human steroid 5α-reductase

A number of tricyclic thiolactams, bicyclic lactams, and bicyclic thiolactams have been prepared and evaluated in vitro as inhibitors of types 1 and 2 steroid 5α-reductase. The tricycles with an 8-chloro substituent in the C-ring are nM (IC 50) inhibitors of type 1 steroid 5α-reductase (SR). In all the cases studied, lactams are more potent than the corresponding thiolactams. Activity against type 2 SR is greatly enhanced by a styryl (or azo) substituent on the aryl ring of the tri- and bicycles and also a related tricyclic aryl acid.