Estrone Sulfatase Inhibitors as New Anticancer Agents

Enzyme steroid sulfatase (STS) is considered as a promising therapeutic target for the treatment of hormone-dependent oncological diseases such as breast, endometrial, prostate cancers, and endometriosis. The discovery of potent and irreversible STS inhibitors stimulated huge efforts of preclinical and clinical work. Various STS inhibitors such as steroid sulfamate, steroid nonsulfamate, nonsteroidal sulfamate, and nonsteroidal nonsulfamate-based inhibitors have been developed. In the review known STS inhibitors from the point of view of their safety, side-effects and perspectives for clinical application are considered. Among STS inhibitors several dual (multitargeted) compounds have huge potential being nonestrogenic and acting in nanomolar levels on the targets. The dual aromatase-sulfatase inhibitors (DASI) approach has a great potential when a synergy between STS and aromatase inhibition is expected and, thus it could address acquired resistance mechanisms. Among STS inhibitors based on steroid skeleton 17α-benzyl-, 17β-arylsulfonamides, 17-diisopropylcarbamoyl-3-O-sulfamates exhibit the best properties, especially as dual anticancer potential drugs. The same modifications result in the increased activity against STS in 2-OMe-3-O-sulfamates as well as 2-OMe-3, 17β-bissulfamates, which are also active against triple negative breast cancer. 8α-Steroid estrogen analogs without estrogenic properties also possess high STS-inhibitory activity and block breast cancer cells growth with the activity comparable to tamoxifen

Novel inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) and steroid sulfatase (STS) with unique dual mode of action : potential drugs for the treatment of non-small cell lung cancer (NSCLC) and endometriosis

Estrogens, in particular estradiol‎ (E2)‎ play an important role in estrogen-dependent diseases (EDDs), such as non-small-cell lung cancer ‎‎(NSCLC) and endometriosis. 17β-Hydroxysteroid dehydrogenase type 1 (17β-HSD1) is frequently expressed in NSCLC tissues, leading to cancer development and progression. Thus, the first objective of this study (chapter 3.1) is the development of a novel series of highly potent non-steroidal, selective ‎‎17β-HSD1 inhibitors in order to enhance the treatment of NSCLC. ‎ This section of the study showed that 17β-HSD1 is a promising therapeutic target for NSCLC, providing new avenues for the treatment of this lethal cancer. Steroid sulfatase (STS) and 17β-HSD1 are promising targets for the treatment of endometriosis because they ‎limit estrogen formation mainly in the target cells, leading to fewer ‎side effects. ‎Thus, the second part of the study ‎(chapter 3.2) ‎aims at developing dual inhibitors of STS and 17β-HSD1, which provide a novel treatment option. The synthesized sulfamates should be drugs for inhibition of STS, and prodrugs for 17β-HSD1 inhibition. The most active compounds of this part showed nanomolar IC50 values for STS in cellular assays ‎and their corresponding phenols displayed potent 17β-HSD1 inhibition in cell-free and cellular ‎assays as well as high selectivity over 17β-HSD2. These findings suggest that the “drug-prodrug concept” ‎has been applied successfully ‎(chapter 3.2).Estrogene, insbesondere Estradiol (E2), spielen eine zentrale Rolle bei Estrogen-abhängigen Erkrankungen (estrogen-dependent diseases, EDD) wie nicht-kleinzellige Bronchialkarzinome (non-small-cell lung cancer, NSCLC) und Endometriose. 17β-Hydroxysteroid Dehydrogenase Typ 1 (17β-HSD1) ist in NSCLC-Gewebe häufig überexprimiert und trägt zu Tumorentstehung und -wachstum bei. Das erste Ziel dieser Arbeit war daher die Entwicklung von neuartigen und hochpotenten, nicht-steroidalen 17β-HSD1 Inhibitoren als potenzielle NSCLC-Therapeutika (Kapitel 3.1). Die Daten zeigen, dass 17β-HSD1 ein vielversprechendes Target darstellt, das neue Möglichkeiten in der NSCLC-Therapie eröffnen kann. Steroid Sulfatase (STS) und 17β-HSD1 sind vielversprechende Wirkstofftargets zur Behandlung der Endometriose, da sie die E2-Produktion lokal im erkrankten Gewebe reduzieren, was im Vergleich zu systemischen Therapien zu weniger Nebenwirkungen führen sollte. Gegenstand des zweiten Teils der Arbeit (Kapitel 3.2) war die Entwicklung von dualen Inhibitoren von STS und 17β-HSD1. Die so synthetisierten Sulfamate sollten Drugs für die Hemmung von STS und gleichzeitig Prodrugs für die Hemmung von 17β-HSD1 darstellen. Die aktivsten Verbindungen dieses Teils zeigten nanomolare IC50-Werte für STS in zellulären Assays und ihre entsprechenden Phenole zeigten eine starke 17β-HSD1-Hemmung in zellfreien und zellulären Assays sowie eine hohe Selektivität gegenüber 17β-HSD2. Die Daten belegen, dass das verfolgte “Drug-Prodrug-Konzept” der dualen Hemmstoffwirkung erfolgreich umgesetzt wurde (Kapitel 3.2)

Regulation of Steroid Sulfatase by Glucocorticoids in Human Breast Cancer and Bone Cancer Cell Lines

Steroid sulfatase is an enzyme involved in the conversion of active estrogens and androgens from inactive systemic precursors such as estrone sulfate (E1S) and dehydropiandrosterone sulfate (DHEAS). This protein is known to play a role in the fetal-adrenal endocrine axis during pregnancy and it is suspected to have additional physiological functions. Steroid sulfatase has also been implicated in stimulation of hormone-dependent cancers; consequently, a substantial amount of research has been undertaken to develop inhibitors of this enzyme. By comparison, relatively little is known about the regulation of steroid sulfatase. Previous work in our lab had suggested that glucocorticoids may affect steroid sulfatase activity in human breast and bone cells. The purpose of my research was to study the regulation of steroid sulfatase by glucocorticoids in two human cell lines, MDA-MB-231 hormone-independent breast cancer cells, and MG-63 osteosarcoma cells. MDA-MB-231 cells treated for 72 hours with 10 or 1.0µM cortisol showed a decrease in steroid sulfatase activity, as determined by a whole cell radiolabeled steroid conversion assay. Similarly, intact MDA-MB-231 cells treated with 10, 1.0 or 0.1µM dexamethasone showed significantly decreased steroid sulfatase activity. This reduction of steroid sulfatase activity was dose responsive. MDA-MB-231 cell lysates prepared after exposure to dexamethasone provided mixed results with regard to steroid sulfatase activity. The observed decrease in steroid sulfatase activity was not due to competitive inhibition by the glucocorticoids, as neither cortisol nor dexamethasone was found to be competitive inhibitors of steroid sulfatase activity in MDA-MB-231 microsomes. Qualitative analysis of steroid sulfatase mRNA using RT-PCR suggests levels are not affected by dexamethasone. However, quantitative assays must be performed to determine anything conclusive. In contrast to the MDA-MB-231 results, MG-63 bone precursor cells showed no change in steroid sulfatase activity after exposure to dexamethasone for 24 or 96 hours. However, MG-63 cell lysates demonstrated an upregulation in steroid sulfatase activity at the 96h time period. Data from this study indicate that glucocorticoids can influence steroid sulfatase activity in human breast and bone cells

Development of the first dual inhibitors for steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) : a novel treatment approach for endometriosis

Endometriosis is an estrogen dependent disease (EDD) that has no satisfying treatment option, as the existing ones mainly comprise endocrine treatments that lead to severe systemic hypo-estrogenic side effects. Steroid sulfatase (STS) and 17β hydroxysteroid dehydrogenase type 1 (17β HSD1) are attractive new targets for the treatment of EDDs. Their inhibition leads to blockage of the local biosynthesis of estrogen without significantly affecting the circulating estrogen. The simultaneous inhibition of both enzymes appears to be more promising than the blockage of only one protein. The main aim of this study is the development of dual inhibitors of STS and 17β-HSD1 (DSHIs) that offers a novel treatment option for endometriosis without severe side effects. Using a designed multiple ligand (DML) approach, the first DSHIs were identified. Upon structural optimizations, highly potent inhibitors in cell-free and cellular assays were achieved that are characterized by high selectivity over 17β-HSD2 which plays a protective role in endometriosis. The DSHIs were able to efficiently reverse the E1-S and E1- induced T47D cell proliferation. The most interesting inhibitor described in this work is characterized by high metabolic stability in human and mouse hepatic S9 fraction, along with good physicochemical properties and high safety margins in cytotoxicity assays. Furthermore, this DSHI is considered a suitable candidate for in vivo proof of principle studies based on its pharmacokinetic profile.Endometriose ist eine Estrogen-abhängige Erkrankung für die bislang keine zufriedenstellende Therapieoption existiert. Zum Einsatz kommen hauptsächlich endokrine Behandlungen, die systemisch zu stark hypoestrogenen Zuständen und damit zusammenhängenden, ernsthaften Nebenwirkungen führen. Die Enzyme Steroid Sulfatase (STS) and 17β-Hydroxysteroid Dehydrogenase Typ 1 (17β-HSD1) sind attraktive, neuartige Targets zur Behandlung Estrogen-abhängiger Erkrankung. Ihre Inhibierung führt zur Hemmung lokaler Estrogen-Biosynthese, ohne starke Beeinflussung systemischer Estrogen-Konzentrationen. Die gleichzeitige Hemmung beider Enzyme erscheint vielversprechender als die Blockade eines einzelnen Proteins. Ein Gegenstand der vorliegenden Arbeit ist die Entwicklung dualer Inhibitoren von STS und 17β-HSD1 (DSHIs). Solche Wirkstoffe sind eine neuartige Therapieoption für Endometriose, die nicht zu den erwähnten Nebenwirkungen führt. Unter Anwendung eines Ansatzes zum gezielten Design von Liganden mehrerer biologischer Targets wurden die ersten DSHIs identifiziert. Anschließende Strukturoptimierungen führten zu Wirkstoffen, die in Zell-freien und zellbasierten Assays beide Targetenzyme hochpotent hemmten. Darüberhinaus waren die DSHIs in der Lage, die Estronsulfat- und Estron-induzierte Proliferation von T47D Zellen vollständig aufzuheben. Die vielversprechendste Verbindung zeigt hohe metabolische Stabilität in den S9-Lebermikrosomenfraktionen von Mensch und Maus, vorteilhafte physiko-chemische Eigenschaften und geringe Cytotoxizität. Darüberhinaus zeigte sie günstige pharmakokinetische Eigenschaften in der Maus, was sie zu einem geeigneten Kandidaten für eine proof-of-principle Studie macht

Substrate inhibition of 17 beta-hydroxysteroid dehydrogenase type 1 in living cells and regulation among the steroid-converting enzymes in breast cancers

Cette étude a permis de démontrer les fonctions et les mécanismes de la 17bêtahydroxystéroïde déshydrogénase de type 1 (17β-HSD1) et de la stéroïde sulfatase (STS) au niveau du cancer du sein, y compris la cinétique moléculaire et cellulaire, la liaison du ligand étudiée par la titration de fluorescence, la régulation des stéroïdes et la régulation mutuelle entre les enzymes stéroïdiennes et les cellules cancéreuses du sein. 1), L’inhibition de la 17β-HSD1 par son substrat a été démontrée par la cinétique enzymatique au niveau cellulaire pour la première fois, soutenant ainsi la fonction biologique de l’inhibition produite par le substrat. 2), En tant qu’inhibiteur, la dihydrotestostérone (DHT) n’a pas affecté la concentration du substrat estrone (E1) à laquelle l’activité enzymatique a commencé à diminuer, mais certaines augmentations de vitesse ont été observées, suggérant une diminution significative de l’inhibition par le substrat. 3), Les résultats de la modulation de l’ARNm ont démontré que la transcription du gène codant la 17β-HSD7 diminuait en réponse à l’inhibition de la 17β-HSD1 ou au knockdown dans les cellules du cancer du sein par la modification estradiol (E2). 4), L’expression de la STS est stimulée par E2 de manière à générer une rétroaction positive, ce qui favorise la biosynthèse de E2 dans les cellules de cancer du sein. 5), L’inhibition conjointe de la STS et de la 17β-HSD7 pourrait bloquer leurs activités enzymatiques, diminuant ainsi la formation de E2, mais rétablissant la formation de DHT, réduisant de façon synergique la prolifération cellulaire et induisant l’arrêt du cycle cellulaire en G0 / G1. 6), Les 17β-HSD7 et STS synthétisent E2 et sont toutes deux régulées par E2. Ainsi, elles forment un groupe fonctionnel d’enzymes mutuellement positivement corrélées, l’inhibition de l’une peut réduire l’expression d’une autre, amplifiant ainsi potentiellement les traitements inhibiteurs. 7), Le recepteur estrogenique α ERα a été non seulement régulés à la baisse par E2, mais également réduits par la DHT grâce à l’activation des récepteurs aux androgènes (AR). En conclusion, la 17β-HSD1 et la 17β-HSD7 jouent des rôles essentiels dans la conversion et la régulation des hormones sexuelles, et l’inhibition conjointe de la STS et de la 17β-HSD7 constitue une nouvelle stratégie pour le traitement hormonal des cancers du sein sensibles aux estrogènes.Human 17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1), 17betahydroxysteroid dehydrogenase type 7 (17β-HSD7) and steroid sulfatase (STS) play a crucial role in regulating estrogen synthesis for breast cancer (BC). However, mutual regulation of enzymes and the interaction of these steroids (estrogens, androgens and their precursor dehydroepiandrosterone (DHEA)) are not clear. This study demonstrated the functions and mechanisms including kinetics at molecular level and in cells, ligand binding using fluorescence titration, regulation of steroids and mutual regulation between steroid enzymes in BC cells: 1) Substrate inhibition of 17β-HSD1 was shown for the first time by enzyme kinetics at the cell level, supporting the biological function of substrate inhibition. 2) As an inhibitor, dihydrotestosterone (DHT) did not affect the estrone (E1) substrate concentration at which the enzyme activity started to decrease, but some increases in velocity were observed, suggesting a corresponding decrease in substrate inhibition 3) The mRNA modulation results demonstrated that 17β-HSD7 transcription decreased in response to 17β-HSD1 inhibition or knockdown in BC cells due to estradiol (E2) concentration decrease. 4) The expression of STS is stimulated by E2 in a positive-feedback manner which finally promotes E2 biosynthesis within BC cells. 5) The joint inhibition of STS and 17β-HSD7 could block the activities of these enzymes, thus decreasing E2 formation but restoring DHT formation, to synergistically reduce cell proliferation and induce G0/G1 cell cycle arrest. 6) 17β-HSD7 and STS can synthesize E2 and are all regulated by E2. Thus, they form a functional group of enzymes mutually positively correlated, inhibition of one can reduce the expression of the other, thereby potentially amplifying the inhibitory effects. 7) Estrogen Receptor α (ERα) is not only down-regulated by E2, but also reduced by DHT though androgen receptor (AR) activation. In conclusion, 17β-HSD1 and 17β-HSD7 play essential roles in sex-hormone conversion and regulation, and the joint inhibition of STS and 17β-HSD7 constitutes a novel strategy for hormonal treatment of estrogen-receptor positive B

Effect of Steroids and Sulfated Steroids on Growth of the Human MG-63 Osteoblast-like Cell Line

The importance of estrogen in bone regulation is exemplified by the rapid loss of bone density at the onset of menopause. Post-menopausal women have low levels of estrogens, but high levels of inactive sulfated steroids. These can be converted to active steroids by steroid sulfatase. While it is known that estrogens can stimulate the growth and maintenance of bone cells, it is not known if sulfated steroids can induce a similar response. The purpose of this study was to determine if sulfated steroids can induce proliferation of a human osteoblast-like cell line MG-63, and if steroid sulfatase inhibitors were capable of blocking that response. A growth assay was developed to assess proliferation of MG-63 cells in the presence of various steroids. The results of an initial experiment with a number of steroids indicated differences in growth, with estradiol, estrone sulfate, and dehydroepiandrosterone sulfate showing increased proliferation. A follow up experiment with estradiol, estrone sulfate, and dehydroepiandrosterone sulfate showed that proliferation was increased in the presence of estradiol and estrone sulfate. A dose-response testing proliferation of MG-63 cells to estradiol and estrone sulfate, resulted in estradiol stimulating growth above baseline at 10μM and 1μM, and estrone sulfate increasing growth only at 10μM. A steroid sulfatase inhibitor, DU-14, was able to block estrone sulfate-stimulated growth at 10μM, but not at 1μM. Another steroid sulfatase inhibitor, EMATE, actually stimulated growth at 1μM; however, this inhibitor is known to be estrogenic. The estrogen receptor antagonist ICI 182,780 inhibited estrone sulfate- and estradiol-stimulated growth at 100nM. Steroid sulfatase activity was assessed in MG-63 microsomes and whole cells in the presence of DU-14 and EMATE. It was found that steroid sulfatase activity in the presence of the inhibitors was virtually eliminated. These data demonstrate that growth in the human MG-63 osteoblast-like cells is stimulated by estrogens and by sulfated estrogens. This supports the concept that sulfated steroids are important in maintaining bone density

Characterization and Regulation of Steroid Sulfatase in the Human MG-63 Pre-osteoblastic Cell Line

The importance of estrogen in bone regulation is exemplified by the reduction in bone density at the onset of menopause. Post-menopausal women have low levels of estrogens, but high levels of inactive sulfated steroids. These can be converted to active steroids by steroid sulfatase (STS), which is a microsomal enzyme found in many tissues. STS desulfates common steroids such as dehydroepiandrosterone sulfate and estrone sulfate, the products of which can serve as precursors for active estrogens. We sought to characterize the activity and expression of STS in human bone cells with the idea that increasing STS expression in bone could offset low bone density. STS activity was found to be relatively high in MG-63 pre-osteosarcoma cells, indicating that these cells can produce active estrogens from precursors. STS activity was blocked by the known inhibitors EMATE and 667 Coumate. In addition, cell growth was stimulated by addition of sulfated steroids. STS activity and expression were examined in pre-osteoblastic and differentiated MG-63 cells over a 21-day period. STS activity and expression were higher in pre-osteoblastic cells than in differentiated cells. The STS decline was found to be due to the presence of the glucocorticoid dexamethasone in the differentiation medium. Inhibition of the glucocorticoid receptor with RU-486 blocked the decline in STS activity. Results from a collaborator suggested that NF-κB might regulate STS transcription. The NF-κB activators LPS and PMA increased STS expression, which was lowered in the presence of the NF-κB inhibitor BAY. Glucocorticoids and NF-κB are antagonistic to each other with regard to immune responses. Thus, steroid sulfatase appears to be regulated like an immune response protein in pre-osteoblastic cells. The significance of this for bone physiology is unclear. Our data indicate that steroid sulfatase is present in bone cells and that it can influence bone cell growth by converting inactive sulfated steroids to estrogenic forms. Furthermore, STS expression is regulated by glucocorticoid and NF-κB pathways. Manipulation of STS expression via these pathways may lead to a potential treatment for osteoporosis

The Steroid Sulfatase Inhibitor (p-O-Sulfamoyl) - Tetradecanoyl Tyramine (DU-14) Enhances Memory Retention in Passive Avoidance in Rats with Hippocampal Cholinergic Deficit

Neurosteroids, such as dehydroepiandrosterone sulfate, (DHEAS) are steroidal compounds synthesized within the central nervous system that modulate its function. Among the effects associated with sulfated neurosteroids is the enhancement of memory. Previous studies have demonstrated that altering the metabolism of neurosteroids via inhibition of the enzyme steroid sulfatase could reverse scopolamine-induced amnesia. p-O-(Sulfamoyl) - tetradecanoyl tyramine (DU-14) is a steroid sulfatase inhibitor (SSI) that prevents the conversion of DHEAS to DHEA thereby increasing the concentration of sulfated neurosteroids to potentiate the effect of endogenous DHEAS. To determine whether DU-14 could enhance memory retention for footshock in rats with hippocampal cholinergic hypofunction, male Sprague-Dawley rats were infused with the immunotoxin 192 IgG-saporin (SAP) into the medial septum, and tested using a step-through passive avoidance paradigm. There was a greater than 80% decrease in ChAT activity in the hippocampi of animals treated with SAP (p \u3c 0.0001). There was also a 24.1% decrease in ChAT activity of in the frontal cortex of SAP animals administered DU-14. However, this small but significant decrease did not affect locomotor behavior in rats. Administration of DU-14 enhanced memory retention for 1.0 mA and not 1.25 mA footshock in animals with SAP lesion compared to vehicle treated SAP animals (p\u3c 0.05). The effect of DU-14 on locomotor activity was also investigated. DU-14 did not affect locomotor activity during acclimation or retention. These results suggest that DU-14 may enhance memory retention in rats with hippocampal cholinergic lesion

Characterization of Steroid Sulfatase in Human and Mouse Tissues and in the Mouse MC3T3-E1 Pre-Osteoblastic Cell Line

Steroid sulfatase cleaves the sulfate group from 3Β-hydroxysteroid sulfates. This enzyme is widely distributed among mammalian tissues, and it has been suggested to play a role in the in situ conversion of inactive steroids to active forms in a variety of tissues. Two tissues in which steroid sulfatase may be particularly important are breast cancer and bone. Our laboratory is interested in the role of steroid sulfatase; consequently, I worked on four projects characterizing steroid sulfatase in human and mouse tissues and investigating the role of this enzyme in mouse bone cells. Recent evidence indicates that steroid sulfatase could be a prognostic indicator in breast cancer. Our lab generated a steroid sulfatase antibody to examine the presence of steroid sulfatase in a variety of tissues, including breast carcinomas. I performed immunohistochemical analyses on human tissue slides and a human tissue microarray. The findings confirm previous reports that steroid sulfatase is higher in hormone-dependent breast cancers, suggesting that this enzyme could serve as a useful prognostic indicator of breast cancer. I next sought to determine activity and presence of steroid sulfatase and the presence of steroid sulfatase mRNA in a variety of mouse tissues. Using enzyme assays, Western blotting and RT-PCR, it was determined to each of six mouse tissues (liver, lung, kidney, muscle, ovary and uterus) contained steroid sulfatase protein and mRNA. Our results show that mouse tissues have the ability to convert inactive steroids into active steroids. The mouse model may prove useful in future studies of steroid sulfatase physiology. Steroid sulfatase may provide a local mechanism for 17Β-estradiol needed to maintain bone health, as estrogens play a central role in the regulation of bone density. I characterized steroid sulfatase in the MC3T3-E1 mouse pre-osteoblastic cell line, as these cells are a model for bone differentiation. I then developed a 21-day protocol to measure steroid sulfatase throughout differentiation. Steroid sulfatase showed changes in activity during development, depending on the treatment; however steroid sulfatase mRNA was constant throughout development. These data provide a baseline for future studies of the role of steroid sulfatase in bone development