Demethoxylation of curcumin enhances its inhibition on human and rat 17ß-hydroxysteroid dehydrogenase 3: QSAR structure-activity relationship and in silico docking analysis.

Curcuminoids have many pharmacological effects. They or their metabolites may have side effects by suppressing 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3). Herein, we investigated the inhibition of curcuminoids and their metabolites on human and rat 17ß-HSD3 and analyzed their structure-activity relationship (SAR) and performed in silico docking. Curcuminoids and their metabolites ranked in terms of IC50 values against human 17ß-HSD3 were bisdemethoxycurcumin (0.61 µM) > curcumin (8.63 µM) > demethoxycurcumin (9.59 µM) > tetrahydrocurcumin (22.04 µM) > cyclocurcumin (29.14 µM), and those against rat 17ß-HSD3 were bisdemethoxycurcumin (3.94 µM) > demethoxycurcumin (4.98 µM) > curcumin (9.62 µM) > tetrahydrocurcumin (45.82 µM) > cyclocurcumin (143.5 µM). The aforementioned chemicals were mixed inhibitors for both enzymes. Molecular docking analysis revealed that they bind to the domain between the androstenedione and NADPH active sites of 17ß-HSD3. Bivariate correlation analysis showed a positive correlation between LogP and pKa of curcumin derivatives with their IC50 values. Additionally, a 3D-QSAR analysis revealed that a pharmacophore model consisting of three hydrogen bond acceptor regions and one hydrogen bond donor region provided a better fit for bisdemethoxycurcumin compared to curcumin. In conclusion, curcuminoids and their metabolites possess the ability to inhibit androgen biosynthesis by directly targeting human and rat 17ß-HSD3. The inhibitory strength of these compounds is influenced by their lipophilicity and ionization characteristics.

Light-switchable diazocines as potential inhibitors of testosterone-synthesizing 17ß-hydroxysteroid dehydrogenase 3.

In patients with prostate carcinoma as well as in some other cancer types, the reduction of testosterone levels is desired because the hormone stimulates cancer cell growth. One molecular target for this goal is the inhibition of 17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3), which produces testosterone from its direct precursor androstenedione. Recent research in this field is trying to harness photopharmacological properties of certain compounds so that the inhibitory effect could be turned on and off by irradiation. Seven new light-switchable diazocines were investigated with regard to their inhibition of 17ßHSD3. For this purpose, transfected HEK-293 cells and isolated microsomes were treated with the substrate and the potential inhibitors with and without irradiation for an incubation period of 3 or 5 h. The amount of generated testosterone was measured by UHPLC and compared between samples and control as well as between irradiated and non-irradiated samples. There was no significant difference between samples with and without irradiation. However, four of the seven diazocines led to a significantly lower testosterone production both in cell and in microsome assays. In some of the irradiated samples, a partial destruction of the diazocines was observed, indicated by an additional UHPLC peak. However, the influence on the inhibition is negligible, because the majority of the substance remained intact. In conclusion, new inhibitors of 17ßHSD3 have been found, but so far without the feature of a light switch, since the configurational alteration of the diazocines by irradiation did not lead to a change in bioactivity. Further modification might help to find a light-switching molecule that inhibits only in one configuration.

Clinical, Hormonal, and Genetic Spectrum of 46 XY Disorders of Sexual Development (DSD) Patients.

OBJECTIVES: To evaluate the clinical, hormonal and genetic characteristics of 46XY disorders of sexual development (DSD) patients from South India. METHODS: 46XY DSD patients with a provisional diagnosis of 17ß-hydroxysteroid dehydrogenase 3 (17BHSD3) deficiency, 5 alpha-reductase type 2 deficiency (5ARD2) or partial androgen insensitivity syndrome (PAIS) based on clinical and hormonal analysis were included in this study. All the patients underwent detailed clinical and hormonal evaluations. Targeted next-generation sequencing for all three genes (AR, HSD17B3, and SRD5A2) in parallel was carried out for all the included patients and their parents. RESULTS: Based upon the clinical and hormonal analysis, among the 37 children with 46XY DSD in the present study, 21 children were diagnosed with 5ARD2, 10 with PAIS, and six with 17BHSD3 deficiency. However, genetic analysis revealed pathogenic mutations in nine patients - six in the AR gene, two in the SRD5A2 gene, and one in the HSD17B3 gene. The concordance rate between provisional hormonal and genetic diagnosis was only 22.2%. Two out of six subjects with AR gene variants were positive for somatic mosaicism. CONCLUSIONS: In the present study, a positive genetic diagnosis was detected in nine patients (24%), including five novel variants. In this study, mutations in the AR gene was the most reported. The authors did not find the testosterone: dihydrotestosterone (T: DHT) ratio to be an accurate hormonal diagnostic tool.

Diagnostic approach in 46, XY DSD: an endocrine society of bengal (ESB) consensus statement.

OBJECTIVES: 46, XY difference/disorder of sex development (DSD) is a relatively uncommon group of heterogeneous disorders with varying degree of underandrogenization of male genitalia. Such patients should be approached systematically to reach an aetiological diagnosis. However, we lack, at present, a clinical practice guideline on diagnostic approach in 46, XY DSD from this part of the globe. Moreover, debate persists regarding the timing and cut-offs of different hormonal tests, performed in these cases. The consensus committee consisting of 34 highly experienced endocrinologists with interest and experience in managing DSD discussed and drafted a consensus statement on the diagnostic approach to 46, XY DSD focussing on relevant history, clinical examination, biochemical evaluation, imaging and genetic analysis. CONTENT: The consensus was guided by systematic reviews of existing literature followed by discussion. An initial draft was prepared and distributed among the members. The members provided their scientific inputs, and all the relevant suggestions were incorporated. The final draft was approved by the committee members. SUMMARY: The diagnostic approach in 46, XY DSD should be multidisciplinary although coordinated by an experienced endocrinologist. We recommend formal Karyotyping, even if Y chromosome material has been detected by other methods. Meticulous history taking and thorough head-to-toe examination should initially be performed with focus on external genitalia, including location of gonads. Decision regarding hormonal and other biochemical investigations should be made according to the age and interpreted according to age-appropriate norms Although LC-MS/MS is the preferred mode of steroid hormone measurements, immunoassays, which are widely available and less expensive, are acceptable alternatives. All patients with 46, XY DSD should undergo abdominopelvic ultrasonography by a trained radiologist. MRI of the abdomen and/or laparoscopy may be used to demonstrate the Mullerian structure and/or to localize the gonads. Genetic studies, which include copy number variation (CNV) or molecular testing of a candidate gene or next generation sequencing then should be ordered in a stepwise manner depending on the clinical, biochemical, hormonal, and radiological findings. OUTLOOK: The members of the committee believe that patients with 46, XY DSD need to be approached systematically. The proposed diagnostic algorithm, provided in the consensus statement, is cost effective and when supplemented with appropriate genetic studies, may help to reach an aetiological diagnosis in majority of such cases.

Reduction of photoswitched, nitrogen bridged N-acetyl diazocines limits inhibition of 17ßHSD3 activity in transfected human embryonic kidney 293 cells.

Testosterone depletion is a common aim in the treatment of hormone-dependent prostate cancer, since the steroid boosts the tumor's proliferation. Therefore, inhibition of 17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3), which catalyzes the carbonyl reduction of androstenedione to testosterone, represents an expedient therapeutic drug target. Among the compounds targeting 17ßHSD3, tetrahydrodibenzazocines have been reported to be highly potent inhibitors. Thus, we hypothesized that structural analogs to the tetrahydrodibenzazocine scaffold, namely diazocines, which contain an azo group instead of the ethylene moiety, are also able to inhibit 17ßHSD3. Diazocines consist of a photoresponsive core and can be isomerized from Z into E configuration by irradiation with a specific wavelength. In the present study, 17ßHSD3 inhibition by diazocine photoisomers was examined in transfected human embryonic kidney 293 cells (HEK-293) and isolated microsomes. For this purpose, cells or microsomes were treated with androstenedione and incubated for 2 or 24 h in the presence or absence of irradiated and non-irradiated diazocines. Testosterone formation was determined by uHPLC. We report a weak inhibition of 17ßHSD3 activity by diazocines in HEK-293 cells and microsomes. Furthermore, we found no significant difference between samples treated with irradiated and non-irradiated diazocines in terms of inhibition. However, we detected a new compound by HPLC analysis, which only appeared in light-treated samples, indicating a chemical modification of the photoswitched diazocines, presumably rendering them ineffective. Further investigations revealed that this modification occurs in the presence of reducing agents like dithiothreitol and glutathione. A preliminary mass-spectrometric analysis suggests that the N-N double bond is reduced, resulting in a dianiline derivative. Nevertheless, optimized photoswitchable diazocine derivatives, which are stable in a cellular environment, might serve as potent 17ßHSD3 inhibitors, effective only in irradiated tissue.

Whole exome sequencing and functional characterization increase diagnostic yield in siblings with a 46, XY difference of sexual development (DSD).

Pathogenic biallelic variants in HSD17B3 result in 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3) deficiency, variable disruption of testosterone production, and phenotypic diversity among 46, XY individuals with differences of sexual development (DSDs). We performed quad whole exome sequencing (WES) on two male siblings with microphallus, perineal hypospadias, and bifid scrotum and their unaffected parents. Both male siblings were compound heterozygous for a rare pathogenic HSD17B3 variant (c.239 G > A, p.R80Q) previously identified among individuals with 17ß-HSD3 deficiency and a HSD17B3 variant (c.641A > G, p.E214 G) of uncertain significance. Following WES, the siblings underwent hCG stimulation testing with measurement of testosterone, androstenedione, and dihydrotestosterone which was non-diagnostic. To confirm pathogenicity of the HSD17B3 variants, we performed transient transfection of HEK-293 cells and measured conversion of radiolabeled androstenedione to testosterone. Both HSD17B3 variants decreased conversion of radiolabeled androstenedione to testosterone. As pathogenic HSD17B3 variants are rare causes of 46, XY DSD and hCG stimulation testing may not be diagnostic for 17ß-HSD3 deficiency, WES in 46, XY individuals with DSDs can increase diagnostic yield and identify genomic variants for functional characterization of disruption of testosterone production.

17ß-Hydroxysteroid dehydrogenase 3 deficiency: Three case reports and a systematic review.

17ß-Hydroxysteroid dehydrogenase 3 deficiency is a rare autosomal recessive cause of 46, XY disorders of sex development resulting from HSD17B3 gene mutations, however, no case has been reported in East Asia. The aim of this study was to report three Chinese 46, XY females with 17ß-HSD3 deficiency in a single center and perform a systematic review of the literature. Clinical examination, endocrine evaluation and HSD17B3 gene sequencing were performed in the three Chinese phenotypically females (two sisters and one unrelated patient). Relevant articles were searched by using the term "HSD17B3" OR "17beta-HSD3 gene" with restrictions on language (English) and species (human) in Pubmed and Embase. All the three phenotypically female subjects showed 46, XY karyotype, inguinal masses, decreased testosterone and increased androstenedione. Two novel homozygous mutations (W284X and c.124_127delTCTT) in HSD17B3 gene were identified. A systematic review found a total of 121 pedigrees/158 patients, with 78.5% (124/158) of patients assigned as females, 15.2% (24/158) from females to males, and 5.1% (8/158) raised as males. The most common mutation was c.277+4C>T (allele frequency: 25/72) for patients from Europe, and R80Q (allele frequency: 21/54) for patients from West Asia. The testicular histology showed normal infantile testicular tissue in 100% (9/9) infantile patients, normal quantity germ cells in 44.4% (8/18) prepubertal patients and 19.0% (4/21) pubertal and adult patients. We reported the first East Asian 17ß-hydroxysteroid dehydrogenase 3 deficiency cases. Additional literature reviews found founder effects among patients with different ethnic background and early orchiopexy may benefit fertility in patients assigned as males. These findings may significantly expand the clinical, ethnic and genetic spectrum of 17ß-hydroxysteroid dehydrogenase 3 deficiency.

46,XY disorder of sex development (DSD) due to 17ß-hydroxysteroid dehydrogenase type 3 deficiency.

17ß-hydroxysteroid dehydrogenase 3 deficiency consists of a defect in the last phase of steroidogenesis, in which androstenedione is converted into testosterone and estrone into estradiol. External genitalia range from female-like to atypical genitalia and most affected males are raised as females. Virilization in subjects with 17ß-HSD3 deficiency occurs at the time of puberty and several of them change to male social sex. In male social sex patients, testes can be safely maintained, as long as they are positioned inside the scrotum The phenotype of 46,XY DSD due to 17ß-HSD3 deficiency is extremely variable and clinically indistinguishable from other causes of 46,XY DSD such as partial androgen insensitivity syndrome and 5α-reductase 2 deficiency. Laboratory diagnosis is based on a low testosterone/androstenedione ratio due to high serum levels of androstenedione and low levels of testosterone. The disorder is caused by a homozygous or compound heterozygous mutations in the HSD17B3 gene that encodes the 17ß-HSD3 isoenzyme leading to an impairment of the conversion of 17-keto into 17-hydroxysteroids. Molecular genetic testing confirms the diagnosis and provides the orientation for genetic counseling. Our proposal in this article is to review the previously reported cases of 17ß-HSD3 deficiency adding our own cases.

Suppression of rat and human androgen biosynthetic enzymes by apigenin: Possible use for the treatment of prostate cancer.

Apigenin is a natural flavone. It has recently been used as a chemopreventive agent. It may also have some beneficial effects to treat prostate cancer by inhibiting androgen production. The objective of the present study was to investigate the effects of apigenin on the steroidogenesis of rat immature Leydig cells and some human testosterone biosynthetic enzyme activities. Rat immature Leydig cells were incubated for 3h with 100µM apigenin without (basal) or with 1ng/ml luteinizing hormone (LH), 10mM 8-bromoadenosine 3',5'-cyclic monophosphate (8BR), and 20µM of the following steroid substrates: 22R-hydroxychloesterol (22R), pregnenolone (P5), progesterone (P4), and androstenedione (D4). The medium levels of 5α-androstane-3α, 17ß-diol (DIOL), the primary androgen produced by rat immature Leydig cells, were measured. Apigenin significantly inhibited basal, 8BR, 22R, PREG, P4, and D4 stimulated DIOL production in rat immature Leydig cells. Further study showed that apigenin inhibited rat 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase/17, 20-lyase, and 17ß-hydroxysteroid dehydrogenase 3 with IC50 values of 11.41±0.7, 8.98±0.10, and 9.37±0.07µM, respectively. Apigenin inhibited human 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase 3 with IC50 values of 2.17±0.04 and 1.31±0.09µM, respectively. Apigenin is a potent inhibitor of rat and human steroidogenic enzymes, being possible use for the treatment of prostate cancer.

Disorders of sexual development in a cultural context.

BACKGROUND: Disorders of sexual development (DSD) are congenital conditions in which the development of the chromosomal, gonadal or anatomical sex can be deemed atypical. The external genitalia should appear 'normal' in size and shape from birth, with no question of abnormality, and the individual must receive appropriate social-environmental feedback in the course of the sexual maturation process. METHODS: We review regional differences in the variables considered important for gender assignment in individuals with DSD. Various approaches to certain forms of DSD are analysed within their cultural context. RESULTS: The decision to leave the sex of rearing undisturbed or to change it is difficult. It depends on the patient's age and the extent to which the gender identity has been established with parental gender preference, social, cultural and religious factors. Severe forms of genetically female congenital adrenal hyperplasia, androgen insensitivity syndrome, 17ß-hydroxysteroid dehydrogenase-3, 5α-reductase and cytochrome P450 oxidoreductase deficiencies are found to be the most difficult cases to diagnose and/or manage. CONCLUSION: Gender assignment in children with DSD is a subject of intense debate. Each case of DSD must be evaluated individually and on its merits and potentials. Although early admission and appropriate diagnostic facilities could provide the correct diagnosis, this is not the case in some cultures. It is seen that 'gender panic', social and religious concepts affect the decision-making process in gender assignment, especially in delayed cases.