Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Wilms' tumor (WT1) (±KTS) variants decreases the progesterone secretion of bovine ovarian theca cells.

Wilms' tumor gene WT1 encodes a nuclear transcriptional factor, which has been shown to regulate granulosa cell steroidogenesis in bovine; however, it is not known whether the functions of theca cells are regulated by WT1. Here, we determined the effects of this gene on theca cell proliferation, apoptosis, and steroidogenesis in vitro. In cultured bovine theca cells, the downregulation of WT1 increased the secretion of progesterone but had no effect on proliferation and apoptosis. WT1 includes the variants WT1(+KTS) and WT1(-KTS), which differ by 3 amino acids KTS (lysine, threonine, and serine). WT1(±KTS) upregulation increased the messenger RNA (mRNA) expression of STAR and CYP17A1 and decreased the progesterone secretion and CYP11A1 mRNA expression. In contrast to WT1(+KTS), WT1(-KTS) upregulation also decreased the mRNA expression of 3ß-HSD. In both variants, WT1(-KTS) has more obvious effects. In conclusion, WT1 can decrease progesterone secretion, likely due in part to the inhibition of CYP11A1 and 3ß-HSD.

Microcystins and Microcystis aeruginosa PCC7806 extracts modulate steroidogenesis differentially in the human H295R adrenal model.

The aim of this study was to investigate the potential interference of cyanobacterial metabolites, in particular microcystins (MCs), with steroid hormone biosynthesis. Steroid hormones control many fundamental processes in an organism, thus alteration of their tissue concentrations may affect normal homeostasis. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to investigate the modulation of 14 hormones involved in the adrenal steroid biosynthesis pathway using forskolin-treated H295R cells, following exposure with either microcystin-LR (MC-LR) alone, a mixture made up of MC-LR together with eight other MCs and nodularin-R (NOD-R), or extracts from the MC-LR-producing Microcystis aeruginosa PCC7806 strain or its MC-deficient mutant PCC7806mcyB-. Production of 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) was increased in the presence of MC-LR in a dose-dependent manner, indicating an inhibitory effect on 3ß-hydroxysteroid dehydrogenase (3ß-HSD). This effect was not observed following exposure with a MCs/NOD-R mixture, and thus the effect of MC-LR on 3ß-HSD appears to be stronger than for other congeners. Exposure to extracts from both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- had an opposite effect on 3ß-HSD, i.e. concentrations of pregnenolone, 17-hydroxypregnenolone and DHEA were significantly decreased, showing that there are other cyanobacterial metabolites that outcompete the effect of MC-LR, and possibly result instead in net-induction. Another finding was a possible concentration-dependent inhibition of CYP21A2 or CYP11ß1, which catalyse oxidation reactions leading to cortisol and cortisone, by MC-LR and the MCs/NOD-R mixture. However, both M. aeruginosa PCC7806 and M. aeruginosa PCC7806mcyB- extracts had an opposite effect resulting in a substantial increase in cortisol levels. Our results suggest that MCs can modulate steroidogenesis, but the net effect of the M. aeruginosa metabolome on steroidogenesis is different from that of pure MC-LR and independent of MC production.

Attempts to downregulate ovarian function in the bitch by applying a GnRH agonist implant in combination with a 3ß-hydroxysteroid-dehydrogenase blocker; a pilot study.

Approaches to downregulate ovarian function in the sexually mature bitch by applying slow release GnRH agonist implants are hampered by the initial stimulation of folliculogenesis (flare up) and the resulting side effects. The present pilot study was designed to test to what extent these effects can be suppressed by simultaneous treatment with the 3ß-hydroxysteroid-dehydrogenase (HSD3B) blocker trilostane (T). Treatment with T in 6-h intervals completely blocked adrenal cortisol production. However, in parallel and concomitant with the increase of LH, progesterone and estradiol levels increased, ending up in pro-estric steroid levels in two of the three dogs. Hormonal changes were reflected in the respective clinical symptoms. During the whole observation period the course of LH concentrations did not indicate downregulation of pituitary function as a result of treatment with the GnRH-agonist Suprelorin®, 4.7 mg. The incomplete inhibitory effect of T on the follicular production of sex steroids could be explained by an insufficient transfer of T into the follicular compartment or the existence of a HSD3B isoform in the dog ovary different from the adrenal enzyme. Concerning the lack of downregulation and when accounting for published data different pharmacodynamics/pharmacokinetic activities of GnRH-agonists should be taken into account.

Flurbiprofen Inhibits Androgen Productions in Rat Immature Leydig Cells.

Flurbiprofen is one of the nonsteroidal anti-inflammatory drugs. Whether flurbiprofen affects androgen biosynthesis in Leydig cells is still unknown. Immature Leydig cells (ILCs) isolated from 35-day-old male Sprague-Dawley rats were cultured with 0-100 µM flurbiprofen for 24 h and medium androgen levels and Leydig cell mRNA levels were measured. Immature Leydig cells were also incubated with 100 µM flurbiprofen for 3 h in combination with luteinizing hormone (LH), 8bromo-cAMP, 22R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone, and dihydrotestosterone, respectively, and medium androgen levels were measured. The ROS generation and apoptosis rate were also investigated. The direct effects of flurbiprofen on androgen biosynthetic and metabolizing enzyme activities were measured. Flurbiprofen significantly inhibited basal, LH, and 8bromo-cAMP stimulated androgen production at 10 and 100 µM. Further study demonstrated that flurbiprofen competitively inhibited rat and human testis 3ß-hydroxysteroid dehydrogenase (HSD3B) activity with the half maximal inhibitory concentration (IC50) values of 0.95 µM for rat enzyme and 6.31 µM for human enzyme. In addition, flurbiprofen down-regulated the expression of Srd5a1 and Akr1c14 at 1, 10, and 100 µM. Flurbiprofen also down-regulated Lhcgr expression at 100 µM. Flurbiprofen at 10 and 100 µM increased ROS production and apoptosis rate of rat Leydig cells. In conclusion, flurbiprofen directly inhibits HSD3B activity and the expression levels of Srd5a1 and Akr1c14 in rat Leydig cells, thus leading to the reduction of androgen secretion.

A New Model for Adrenarche: Inhibition of 3ß-Hydroxysteroid Dehydrogenase Type 2 by Intra-Adrenal Cortisol.

We propose that the normal adrenarche-related rise in dehydroepiandrosterone (DHEA) secretion is ultimately caused by the rise in cortisol production occurring during childhood and adolescent growth, by the following mechanisms. (1) The onset of childhood growth leads to a slight fall in serum cortisol concentration due to growth-induced dilution and a decrease in the negative feedback of cortisol upon ACTH secretion. (2) In response, ACTH rises and stimulates increased cortisol synthesis and secretion in the growing body to restore the serum cortisol concentration to normal. (3) The cortisol concentration produced within and taken up by adrenocortical steroidogenic cells may rise during this time. (4) Cortisol competitively inhibits 3ß-hydroxysteroid dehydrogenase type 2 (3ßHSD2)-mediated conversion of 17αOH-pregnenolone to cortisol, causing a further fall in serum cortisol, a further decrease in the negative feedback of cortisol upon ACTH, a further rise in ACTH, and further stimulation of adrenal steroidogenesis. (5) The cortisol-mediated inhibition of 3ßHSD2 also blocks the conversion of DHEA to androstenedione, causing a rise in adrenal DHEA and DHEA sulfate relative to androstenedione secretion. Thus, the combination of normal body growth plus inhibition of 3ßHSD2 by intra-adrenal cortisol may cause normal adrenarche. Childhood obesity may hasten this process by causing a pathologic increase in body size that triggers these same processes at an earlier age, resulting in the premature onset of adrenarche.

Effects of antihistamines on the H295R steroidogenesis - Autocrine up-regulation following 3ß-HSD inhibition.

Millions of people of all ages suffer from allergies worldwide and as a consequence antihistamines are among the most commonly prescribed pharmaceuticals in the world. We investigated the disruptive effects of three antihistamines, promethazine (PMZ), cetirizine (CET) and fexofenadine (FEX) on the H295R steroidogenesis. A multi-steroid LC-MS/MS method was used to quantify 13 steroid hormones in the steroidogenesis. In addition, real-time RT-PCR was used to determine if exposure to antihistamines altered gene expression in the cell line. When exposing the H295R cells to PMZ and CET, significant increases in Δ5-steroids and significant decreases in Δ4-steroids were observed, indicating an inhibition of 3ß-hydroxysteroid dehydrogenase (3ß-HSD). A sequential decrease in corticosteroids, androgens and estrogens were also observed. Overall, FEX had no effect on the steroidogenesis even though minor effects were observed at the highest concentrations. Real-time RT-PCR showed that PMZ resulted in significant up-regulation of 3ß-HSD and 17ß-HSD, whereas CET only resulted in up-regulation of 3ß-HSD. This indicated that the decrease in steroids downstream from 3ß-HSD following PMZ and CT exposure induced a compensatory autocrine response in 3ß-HSD gene expression. The effects on the steroidogenesis were observed at concentrations 30-50 times higher than the therapeutic plasma concentrations. However, antihistamines are lipophilic and may accumulate in adrenals and gonads. Thus, disruptive effects of PMZ and CET on human steroidogenesis cannot be excluded.

Structure-function relationships for the selective inhibition of human 3ß-hydroxysteroid dehydrogenase type 1 by a novel androgen analog.

3ß-Hydroxysteroid dehydrogenase type 1 (3ß-HSD1) is selectively expressed in human placenta, mammary glands and breast tumors in women. Human 3ß-HSD2 is selectively expressed in adrenal glands and ovaries. Based on AutoDock 3 and 4 results, we have exploited key differences in the amino acid sequences of 3ß-HSD1 (Ser194, Arg195) and 3ß-HSD2 (Gly194, Pro195) by designing a selective inhibitor of 3ß-HSD1. 2,16-Dicyano-4,5-epoxy-androstane-3,17-dione (16-cyano-17-keto-trilostane or DiCN-AND) was synthesized in a 4-step procedure from androstenedione. In purified 3ß-HSD inhibition studies, DiCN-AND competitively inhibited 3ß- HSD1 with Ki=4.7µM and noncompetitively inhibited 3ß-HSD2 with a 6.5-fold higher Ki=30.7µM. We previously reported similar isoenzyme-specific inhibition profiles for trilostane. Based on our docking results, we created, expressed and purified the chimeric S194G-1 mutant of 3ß-HSD1. Trilostane inhibited S194G-1 (Ki=0.67µM) with a noncompetitive mode compared to its 6.7-fold higher affinity, competitive inhibition of 3ß-HSD1 (Ki=0.10µM). DiCN-AND inhibited S194G-1 with a 6.3-fold higher Ki (29.5µM) than measured for 3ß-HSD1 (Ki=4.7µM) but with the same competitive mode for both enzyme species. Since DiCN-AND noncompetitively inhibits 3ß-HSD2, which has the Gly194 and Pro195 of 3ß-HSD2 in place of the Ser194 and Arg195 in 3ß-HSD1, this suggests that Arg195 alone in 3ß-HSD1 or S194G-1 is required to bind DiCN-AND in the substrate binding site (competitive inhibition). However, both Ser194 and Arg195 are required to bind trilostane in the 3ß-HSD1 substrate site based on its noncompetitive inhibition of S194G-1 and 3ß-HSD2. In support of this hypothesis, DiCN-AND inhibited our chimeric R195P-1 mutant noncompetitively with a Ki=41.3µM (similar to the 3ß-HSD2 inhibition profile). Since DiCN-AND competitively inhibited S194G-1 that still contains R195 but noncompetitively inhibited R195P-1 that still contains S194, our data provides strong evidence that the Arg195 being mutated to Pro195 (as present in 3ß-HSD2) shifts the inhibition mode from competitive to noncompetitive in 3ß-HSD1. This supports the key role of Arg195 in 3ß-HSD1 for the high affinity, competitive binding of the trilostane analogs. Our new structure/function information for the design of targeted 3ß-HSD1 inhibitors may lead to important new treatments for the prevention of spontaneous premature birth.

Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors discovered by bioisosteric scaffold hopping approach.

The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide.

17beta-hydroxysteroid dehydrogenase type 5 is negatively correlated to apoptosis inhibitor GRP78 and tumor-secreted protein PGK1, and modulates breast cancer cell viability and proliferation.

17beta-hydroxysteroid dehydrogenase type 5 (17ß-HSD5) is an important enzyme associated with sex steroid metabolism in hormone-dependent cancer. However, reports on its expression and its prognostic value in breast cancer are inconsistent. Here, we demonstrate the impact of 17ß-HSD5 expression modulation on the proteome of estrogen receptor-positive (ER+) breast cancer cells. RNA interference technique (siRNA) was used to knock down 17ß-HSD5 gene expression in the ER+ breast cancer cell line MCF-7 and the proteome of the 17ß-HSD5-knockdown cells was compared to that of MCF-7 cells using two-dimensional (2-D) gel electrophoresis followed by mass spectrometry analysis. Ingenuity pathway analysis (IPA) was additionally used to assess functional enrichment analyses of the proteomic dataset, including protein network and canonical pathways. Our proteomic analysis revealed only four differentially expressed protein spots (fold change > 2, p<0.05) between the two cell lines. The four spots were up-regulated in 17ß-HSD5-knockdown MCF-7 cells, and comprised 21 proteins involved in two networks and in functions that include apoptosis inhibition, regulation of cell growth and differentiation, signal transduction and tumor metastasis. Among the proteins are nucleoside diphosphate kinase A (NME1), 78kDa glucose-regulated protein (GRP78) and phosphoglycerate kinase 1 (PGK1). We also showed that expression of 17ß-HSD5 and that of the apoptosis inhibitor GRP78 are strongly but negatively correlated. Consistent with their opposite regulation, GRP78 knockdown decreased MCF-7 cell viability whereas 17ß-HSD5 knockdown or inhibition increased cell viability and proliferation. Besides, IPA analysis revealed that ubiquitination pathway is significantly affected by 17ß-HSD5 knockdown. Furthermore, IPA predicted the proto-oncogene c-Myc as an upstream regulator linked to the tumor-secreted protein PGK1. The latter is over-expressed in invasive ductal breast carcinoma as compared with normal breast tissue and its expression increased following 17ß-HSD5 knockdown. Our present results indicate a 17ß-HSD5 role in down-regulating breast cancer development. We thus propose that 17ß-HSD5 may not be a potent target for breast cancer treatment but its low expression could represent a poor prognosis factor.

Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity.

Resistance to anticancer medications often leads to poor outcomes. The present study explored an effective approach for enhancing chemotherapy targeted against human cancer cells. Real-time quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed overexpression of members of aldo-keto reductase (AKR) 1C family, AKR1C1, AKR1C2, AKR1C3, and AKR1C4, in cisplatin, cis-diamminedichloroplatinum (II) (CDDP)-resistant human cancer cell lines, HeLa (cervical cancer cells) and Sa3 (oral squamous cell carcinoma cells). The genes were downregulated using small-interfering RNA (siRNA) transfection, and the sensitivity to CDDP or 5-fluorouracil (5-FU) was investigated. When the genes were knocked down, sensitivity to CDDP and 5-FU was restored. Furthermore, we found that administration of mefenamic acid, a widely used non-steroidal anti-inflammatory drug (NSAID) and a known inhibitor of AKR1Cs, enhanced sensitivity to CDDP and 5-FU. The present study suggests that AKR1C family is closely associated with drug resistance to CDDP and 5-FU, and mefenamic acid enhances their sensitivity through its inhibitory activity in drug-resistant human cancer cells. Thus, the use of mefenamic acid to control biological function of AKR1C may lead to effective clinical outcomes by overcoming anticancer drug resistance.

The protective effects of zinc in lead-induced testicular and epididymal toxicity in Wistar rats.

The aim of this study was to investigate the beneficial effects of zinc (Zn) in preventing lead (Pb)-induced reproductive toxicity in Wistar rats. The rats were divided into four groups, namely, control group, Pb group, Zn group, and Pb + Zn group. Animals were exposed to Pb (819 mg of Pb/L) or Zn (71 mg of Zn/L) or both through drinking water for 65 days. Rats exposed to Pb showed decreased weights of testes and accessory sex organs. Significant decrease in the testicular daily sperm production, epididymal sperm count, motility, viability, and number of hypoosmotic tail coiled sperm was observed in Pb-exposed rats. Testicular 3ß- and 17ß-hydroxysteroid dehydrogenase activity levels and circulatory testosterone levels were also decreased significantly in Pb-exposed rats. A significant increase in the lipid peroxidation products with a significant decrease in the activities of catalase and superoxide dismutase were observed in the testes and epididymis of Pb-exposed rats. Moreover, the testicular architecture showed lumens devoid of sperm in Pb-exposed rats. Supplementation of Zn mitigated Pb-induced oxidative stress and restored the spermatogenesis and steroidogenesis in Pb-exposed rats. In conclusion, cotreatment of Zn is effective for recovering suppressed spermatogenesis, steroidogenesis, elevated oxidative status, and histological damage in the testis of rats treated with Pb.

Inhibition of AKR1C3 Activation Overcomes Resistance to Abiraterone in Advanced Prostate Cancer.

Abiraterone suppresses intracrine androgen synthesis via inhibition of CYP17A1. However, clinical evidence suggests that androgen synthesis is not fully inhibited by abiraterone and the sustained androgen production may lead to disease relapse. In the present study, we identified AKR1C3, an important enzyme in the steroidogenesis pathway, as a critical mechanism driving resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. We found that overexpression of AKR1C3 confers resistance to abiraterone while downregulation of AKR1C3 resensitizes resistant cells to abiraterone treatment. In abiraterone-resistant prostate cancer cells, AKR1C3 is overexpressed and the levels of intracrine androgens are elevated. In addition, AKR1C3 activation increases intracrine androgen synthesis and enhances androgen receptor (AR) signaling via activating AR transcriptional activity. Treatment of abiraterone-resistant cells with indomethacin, an AKR1C3 inhibitor, overcomes resistance and enhances abiraterone therapy both in vitro and in vivo by reducing the levels of intracrine androgens and diminishing AR transcriptional activity. These results demonstrate that AKR1C3 activation is a critical mechanism of resistance to abiraterone through increasing intracrine androgen synthesis and enhancing androgen signaling. Furthermore, this study provides a preclinical proof-of-principle for clinical trials investigating the combination of targeting AKR1C3 using indomethacin with abiraterone for advanced prostate cancer. Mol Cancer Ther; 16(1); 35-44. ©2016 AACR.

3D-QSAR studies of 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids as AKR1C3 inhibitors: Highlight the importance of molecular docking in conformation generation.

AKR1C3 is a promising drug target for castration-resistant prostate cancer (CRPC). Here, 3D-QSAR analysis were performed on 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids to correlate their chemical structures with their observed AKR1C3 inhibitory activity. Three structural alignment methods employing various conformers were used to scrutinize the effect of conformation selection on the predictive accuracy of QSAR models. Using docked conformation, the best CoMFA and CoMSIA models were developed and validated with a training set of 61 molecules and a test set of 7 molecules. Detailed analysis of contour maps provided helpful structural insights to rational design of AKR1C3 inhibitors with enhanced potency.

Dihydrotestosterone synthesis pathways from inactive androgen 5α-androstane-3ß,17ß-diol in prostate cancer cells: Inhibition of intratumoural 3ß-hydroxysteroid dehydrogenase activities by abiraterone.

Intratumoural dihydrotestosterone (DHT) synthesis could be an explanation for castration resistance in prostate cancer (PC). By using liquid chromatography-mass spectrometry, we evaluated the intratumoral DHT synthesis from 5α-androstane-3ß,17ß-diol (3ß-diol), which is inactive androgen metabolized from DHT. 3ß-diol had biochemical potential to be converted to DHT via three metabolic pathways and could stimulate PC cell growth. Especially, 3ß-diol was not only converted back to upstream androgens such as dehydroepiandrosterone (DHEA) or Δ5-androstenediol but also converted directly to DHT which is the main pathway from 3ß-diol to DHT. Abiraterone had a significant influence on the metabolism of DHEA, epiandrosterone and 3ß-diol, by the inhibition of the intratumoural 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activities which is one of key catalysts in androgen metabolic pathway. The direct-conversion of 3ß-diol to DHT was catalysed by 3ß-HSD and abiraterone could inhibit this activity of 3ß-HSD. These results suggest that PC had a mechanism of intratumoural androgen metabolism to return inactive androgen to active androgen and intratumoural DHT synthesis from 3ß-diol is important as one of the mechanisms of castration resistance in PC. Additionally, the inhibition of intratumoural 3ß-HSD activity could be a new approach to castration-resistant prostate cancer treatment.

Discovery of (R)-2-(6-Methoxynaphthalen-2-yl)butanoic Acid as a Potent and Selective Aldo-keto Reductase 1C3 Inhibitor.

Type 5 17ß-hydroxysteroid dehydrogenase, aldo-keto reductase 1C3 (AKR1C3) converts Δ(4)-androstene-3,17-dione and 5α-androstane-3,17-dione to testosterone (T) and 5α-dihydrotestosterone, respectively, in castration resistant prostate cancer (CRPC). In CRPC, AKR1C3 is implicated in drug resistance, and enzalutamide drug resistance can be surmounted by indomethacin a potent inhibitor of AKR1C3. We examined a series of naproxen analogues and find that (R)-2-(6-methoxynaphthalen-2-yl)butanoic acid (in which the methyl group of R-naproxen was replaced by an ethyl group) acts as a potent AKR1C3 inhibitor that displays selectivity for AKR1C3 over other AKR1C enzymes. This compound was devoid of inhibitory activity on COX isozymes and blocked AKR1C3 mediated production of T and induction of PSA in LNCaP-AKR1C3 cells as a model of a CRPC cell line. R-Profens are substrate selective COX-2 inhibitors and block the oxygenation of endocannabinoids and in the context of advanced prostate cancer R-profens could inhibit intratumoral androgen synthesis and act as analgesics for metastatic disease.

Rational design of an AKR1C3-resistant analog of PR-104 for enzyme-prodrug therapy.

The clinical stage anti-cancer agent PR-104 has potential utility as a cytotoxic prodrug for exogenous bacterial nitroreductases expressed from replicating vector platforms. However substrate selectivity is compromised due to metabolism by the human one- and two-electron oxidoreductases cytochrome P450 oxidoreductase (POR) and aldo-keto reductase 1C3 (AKR1C3). Using rational drug design we developed a novel mono-nitro analog of PR-104A that is essentially free of this off-target activity in vitro and in vivo. Unlike PR-104A, there was no biologically relevant cytotoxicity in cells engineered to express AKR1C3 or POR, under aerobic or anoxic conditions, respectively. We screened this inert prodrug analog, SN34507, against a type I bacterial nitroreductase library and identified E. coli NfsA as an efficient bioactivator using a DNA damage response assay and recombinant enzyme kinetics. Expression of E. coli NfsA in human colorectal cancer cells led to selective cytotoxicity to SN34507 that was associated with cell cycle arrest and generated a robust 'bystander effect' at tissue-like cell densities when only 3% of cells were NfsA positive. Anti-tumor activity of SN35539, the phosphate pre-prodrug of SN34507, was established in 'mixed' tumors harboring a minority of NfsA-positive cells and demonstrated marked tumor control following heterogeneous suicide gene expression. These experiments demonstrate that off-target metabolism of PR-104 can be avoided and identify the suicide gene/prodrug partnership of E. coli NfsA/SN35539 as a promising combination for development in armed vectors.

In vitro CAPE inhibitory activity towards human AKR1C3 and the molecular basis.

AKR1C3 is a critical enzyme for producing testosterone and 5α-DHT in the human body. Inhibiting AKR1C3 is a potential target for treating castration-resistant prostate cancer (CRPC). To find AKR1C3 inhibitors with a new molecular skeleton and binding mode, we analyzed the in vitro inhibitory activity of caffeic acid phenethyl ester (CAPE) and eight other phenolic acid analogues towards AKR1C3 and six other human AKR1 enzymes. We analyzed CAPE and octyl gallate interactions with AKR1C3 using X-ray crystallography, which provided a molecular basis for understanding the phenolic acid inhibitory activity and selectivity towards human AKR1s.

Bioconcentration and endocrine disruption effects of diazepam in channel catfish, Ictalurus punctatus.

Recently, the detection of pharmaceuticals in surface waters has increased worldwide. Pharmaceuticals are typically found in the environment at concentrations well below therapeutic levels in humans; however, their mechanisms of action may be largely unknown in non-target organisms, such as teleost species. Thus, chronic exposure to these types of compounds warrants further investigation. The goal of this study was to examine the potential for diazepam, a model benzodiazepine drug, to bioconcentrate in tissues of channel catfish and to examine its ability to interact with the endocrine system through modulation of steroid hormones and/or steroidogenic genes. To investigate the bioconcentration potential of diazepam, channel catfish (Ictalurus punctatus) were exposed to 1 ng/mL diazepam for seven days, followed by clean water for another seven days, using an abbreviated OECD 305 Fish Bioconcentration Test study design. This concentration of diazepam is well below environmentally relevant concentrations of diazepam (ng/L). To evaluate steroidogenic effects, fish were exposed to 1 ng/mL diazepam for seven days only. Steroid hormone concentrations were analyzed for various tissues, as well as expression of selected steroidogenic genes. Calculated bioconcentration factors for diazepam were well below regulatory threshold values in all tissues analyzed. No changes in steroid hormone concentration were detected in any tissue analyzed; however, the steroidogenic gene cytochrome P450 side chain cleavage (P450scc) was significantly down-regulated at day 5 and 3ß-hydroxy steroid dehydrogenase (3ß-HSD) was significantly down-regulated at day 7 in the gonad. These results indicate that although diazepam does not significantly bioconcentrate, low-level chronic exposure to diazepam may have the potential to interact with endocrine function by altering gene expression.