Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC₅₀ below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC₅₀ below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 μM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.     

New potent steroid sulphatase inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives

In the present work, we report a new class of potent steroid sulphatase (STS) inhibitors based on 6-(1-phenyl-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate derivatives. Within the set of new STS inhibitors, 6-(1-(1,2,3-trifluorophenyl)-1H-1,2,3-triazol-4-yl)naphthalen-2-yl sulphamate 3L demonstrated the highest activity in the enzymatic assay inhibiting the STS activity to 7.98% at 0.5 µM concentration. Furthermore, to verify whether the obtained STS inhibitors are able to pass through the cellular membrane effectively, cell line experiments have been carried out. We found that the lowest STS activities were measured in the presence of compound 3L (remaining STS activity of 5.22%, 27.48% and 99.0% at 100, 10 and 1 nM concentrations, respectively). The measured STS activities for Irosustat (used as a reference) were 5.72%, 12.93% and 16.83% in the same concentration range. Moreover, a determined IC₅₀ value of 15.97 nM for 3L showed that this compound is a very promising candidate for further preclinical investigations.     

Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of potency against STS in two in vitro systems is observed for the derivatives: The 4-NO(2) > 2-halogens, 2-cyano > EMATE (unsubstituted)>17-deoxyEMATE > 2-NO(2) > 4-bromo>2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl)= 2,4-di-n-propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or N-unsubstituted sulfamate group (H(2)NSO(2)O-) is a prerequisite for potent and irreversible inhibition of STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-nitroEMATE (2), whose IC(50)s in placental microsomes and MCF-7 cells are respectively 0.8 nM and 0.01 nM.     

Inactivation of recombinant human steroid sulfatase by KW-2581

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC(50) of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner (k(inact), 0.439 min(-1); K(i, app), 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.     

Inhibition of carbonic anhydrase II by steroidal and non-steroidal sulphamates

Carbonic anhydrases (CAs) are expressed by many solid tumours where they may act to confer a growth advantage on malignant tissues. In this study we have examined the ability of a series of steroidal and non-steroidal sulphamates (originally developed as steroid sulphatase inhibitors) and related compounds to inhibit human CAII (hCAII) activity in vitro. Using a 96-well plate assay, oestrone-3-O-sulphamate (EMATE) and two coumarin-based sulphamate drugs (667 COUMATE and STX 118) were found to have IC(50) values of 25-59 nM for the inhibition of hCAII activity. These compounds therefore have a similar CAII inhibitory potency to that of acetazolamide (IC(50)=25 nM), a known hCAII inhibitor. Docking studies have been performed with selected compounds to the crystal structure of hCAII and excellent correlation of scores with biological activity was observed. This agrees with our recent observations when we were the first to report the inhibition of hCAII by STS inhibitors. These studies and initial results with docking to the crystal structure of the extracellular domain of hCAXII indicate that the STS sulphamate ester inhibitors should also be interesting candidates to pursue as inhibitors of CA isozymes that are over-expressed in human tumours.     

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17 alpha-Hydroxylase/C17-20-lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal-Retzius cells. Organic anion transporting proteins OATP-A, -B, -D, and -E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood-brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3beta-hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.     

The difluoromethylene group as a replacement for the labile oxygen in steroid sulfates: a new approach to steroid sulfatase inhibitors

Several estrone sulfate and estradiol sulfate analogues, in which the sulfate group was replaced with an alpha,alpha-difluoromethylenesulfonate group or an alpha,alpha-difluoromethylenetetrazole group, were examined as inhibitors of steroid sulfatase (STS). These compounds were 4.5-10.5 times more potent than their non-fluorinated analogues. Moreover, the presence of the fluorines changed the mode of inhibition from mixed to competitive. The inhibitor bearing the alpha,alpha-difluoromethylenetetrazole group exhibited an affinity for STS approaching that of the natural STS substrate, estrone sulfate. Possible reasons for the enhanced affinity of the fluorinated compounds compared to their non-fluorinated counterparts are discussed.     

The nature of inhibition of steroid sulphatase activity by tibolone and its metabolites

Tibolone is used for hormone replacement therapy and acts in a tissue-specific manner being oestrogenic on CNS and bone but not on breast tissues or endometrium. The ability of tibolone and its metabolites to inhibit steroid sulphatase (STS) activity has a crucial role in regulating its tissue-specific effects. In this study, we have examined the ability of tibolone and its non-sulphated and sulphated metabolites to inhibit STS activity in different enzyme preparations and in intact cells. For this, we have used an 'extracellular' method, which measures the amount of product released into culture medium, and an 'intracellular' method, which assesses the extent of product formation within cells. In addition, the nature by which tibolone and some of its metabolites inhibit STS activity was investigated using intact cells and an enzyme kinetic method. In MCF-7 and T47D breast cancer cells and JEG-3 choriocarcinoma cells, which have high STS activity, tibolone and its metabolites were relatively potent inhibitors of STS activity (33-57% inhibition at 10 microM) using the extracellular assay method. In HOS-TE-85 osteoblast-like cells, tibolone and its Delta-4 metabolite were relatively inactive whereas the 3alpha/3beta-hydroxy metabolites and their sulphated conjugates inhibited activity by 39-55%. When STS activity was assessed in HOS-TE-85 cells using an 'intracellular' method tibolone and its 3beta-hydroxy metabolite were inactive. Pre-treatment of breast cancer cells and JEG-3 cells, and removal of drugs prior to assaying for STS activity, revealed that in these cells tibolone and its metabolites were acting mainly as reversible inhibitors. This finding was confirmed in an enzyme kinetic study to measure concentration-dependent STS inhibition. In HOS-TE-85 cells, pre-treatment of cells and removal of compounds before assaying for remaining STS activity indicated that some tibolone metabolites appeared to stimulate STS activity. Possible mechanisms by which this might occur are discussed but, if confirmed, this could contribute to the positive oestrogenic effects that tibolone has on bone.     

Steroid sulphatase inhibitors for breast cancer therapy

In contrast to aromatase inhibitors, which are now in clinical use, the development of steroid sulphatase (STS) inhibitors for breast cancer therapy is still at an early stage. STS regulates the formation of oestrone from oestrone sulphate (E1S) but also controls the hydrolysis of dehydroepiandrosterone sulphate (DHEA-S). DHEA can be reduced to 5-androstenediol (Adiol), a steroid with potent oestrogenic properties. The active pharmacophore for potent STS inhibitors has now been identified, i.e. a sulphamate ester group linked to an aryl ring. This has led to the development of a number of STS inhibitors, some of which are due to enter Phase I trials in the near future. Such first generation inhibitors include the tricyclic coumarin-based 667 COUMATE. Aryl sulphamates, such as 667 COUMATE, are taken up by red blood cells (rbc), binding to carbonic anhydrase II (CA II), and transit the liver without undergoing first-pass inactivation. 667 COUMATE is also a potent inhibitor of CA II activity with an IC₅₀ of 17 nM. Second generation STS inhibitors, such as 2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE), in addition to inhibiting STS activity, also inhibit the growth of oestrogen receptor negative (ER⁻) tumours in mice and are anti-angiogenic. As the active pharmacaphores for the inhibition of aromatase and STS are now known it may be possible to develop third generation inhibitors that are capable of inhibiting the activities of both enzymes. Whilst exploring the potential of such a strategy it was discovered that 667 COUMATE possessed weak aromatase inhibitory properties with an IC₅₀ of 300 nM in JEG-3 cells. The identification of potent STS inhibitors will allow the therapeutic potential of this new class of drug to be explored in post-menopausal women with hormone-dependent breast cancer. Second generation inhibitors, such as 2-MeOE2bisMATE, which also inhibit the growth of ER⁻ tumours should be active against a wide range of cancers.     

Inhibition of aldosterone biosynthesis by staurosporine

Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50 = 11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 microg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p = 0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.     

Estrone 3-sulfate mimics,inhibitors of estrone sulfatase activity: homology model construction and docking studies

Steroid sulfatase (STS) is a new target for the endocrine therapy of breast cancer. To ascertain some of the requirements for inhibition of estrone sulfatase activity, a number of novel analogues of estrone 3-O-sulfate possessing sulfate surrogates were synthesized and evaluated as inhibitors of estrone sulfatase (STS) in comparison to a lead inhibitor, estrone-3-O-methylthiophosphonate (E1-3-MTP). Using a selective enzyme digestion, one of the diastereoisomers of this compound, (R(p))-E1-3-MTP, could be prepared and evaluated. From structure-activity studies, we show that chirality at the phosphorus atom, hydrophobicity, basicity, size, and charge all influence the ability of a compound to inhibit estrone sulfatase activity. Of these, hydrophobicity seems to be the most important since simple, active nonsteroidal inhibitors, based on 5,6,7,8-tetrahydronaphth-2-ol (THN), can be prepared, provided that they are lipophilic enough to partition into a nonpolar environment. Also, a negatively charged group is favorable for optimal binding, although it appears that the presence of a potentially cleavable group can compensate for lack of charge in certain cases. A homology model of STS has been constructed from the STS sequence, and molecular docking studies of inhibitors have been performed to broaden the understanding of enzyme/inhibitor interactions. This model clearly shows the positions of the key amino acid residues His136, His290, Lys134, and Lys368 in the putative catalytic region of the formylglycine at position 75, with residues Asp35, Asp36, Asp342, and Gln343 as ligands in the coordination sphere of the magnesium ion. Docking studies using the substrate and estrone-3-sulfate mimics that are active inhibitors indicate they are positioned in the area of proposed catalysis, confirming the predictive power of the model.     

Inhibition of steroid sulfatase with 4-substituted estrone and estradiol derivatives

Steroid sulfatase (STS) catalyzes the desulfation of biologically inactive sulfated steroids to yield biologically active desulfated steroids and is currently being examined as a target for therapeutic intervention for the treatment of breast cancer. We previously demonstrated that 4-formyl estrone is a time- and concentration-dependent inhibitor of STS. We have prepared a series of 4-formylated estrogens and examined them as irreversible STS inhibitors. Introducing a formyl, bromo or nitro group at the 2-position of 4-formylestrone resulted in loss of concentration and time-dependent inhibition and a considerable decrease in binding affinity. An estradiol derivative bearing a formyl group at the 4-position and a benzyl group at the 17β-position yielded a potent concentration and time-dependent STS inhibitor with a K(I) of 85 nM and a k(inact) of 0.021 min(-1) (k(inact)/K(I) of 2.3 × 10(5)M(-1)min(-1)). Studies with estrone or estradiol substituted at the 4-position with groups other than a formyl group revealed that good reversible inhibitors can be obtained by introducing small electron withdrawing groups at this position. An estradiol derivative with fluorine at the 4-position and a benzyl group at the 17β-position yielded a potent, reversible inhibitor of STS with an IC(50) of 40 nM. The introduction of relatively small electron withdrawing groups at the 4-position of estrogens and their derivatives may prove to be a general approach to enhancing the potency of estrogen-derived STS inhibitors.     

N-Acyl arylsulfonamides as novel, reversible inhibitors of human steroid sulfatase

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl-arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest K(i)=76 nM), they showed only weak inhibition of STS activity in cells (lowest IC(50) around 2 microM). Extended structure-activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC(50)=270 nM).     

Microtiter plate cellular assay for human steroid sulfatase with fluorescence readout

Steroid sulfatase (STS; E.C. 3.1.6.2) is an enzyme involved in the local production of estrogens and androgens in target organs. Inhibitors of steroid sulfatase activity are considered novel therapeutic agents for the treatment of different pathologic conditions, including cancers of breast, endometrium, and prostate and disorders of the pilosebaceous unit. Evaluation of steroid sulfatase inhibition in cells up to now has been a cumbersome process, involving the extraction of a radioactive cleavage product into organic solvents. Here, we describe a rapid, nonradioactive cellular assay in microtiter plate format, using 4-methylumbelliferyl sulfate as a substrate. The reaction product, 4-methylumbelliferone, is read in a fluorescence microtiter plate reader. Several cell lines were assayed for sulfatase activity. To increase the sensitivity of the assay, we developed a Chinese hamster ovary (CHO) cell line stably transfected with a cDNA encoding the human steroid sulfatase. The steroid sulfatase activity in transfected cells correlated with the presence of the enzyme in these cells, as determined by immunofluorescence. For most STS inhibitors tested, including estrone-3-O-sulfamate, the results from the CHO cellular assay were in good agreement with those from a standard cell-free assay.     

4,4'-Benzophenone-O,O'-disulfamate: a potent inhibitor of steroid sulfatase

We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors. While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC(50) values between 5 and 7 microM irrespective of additional hydroxy and methoxy substituents at the second phenyl ring, benzophenone-O,O'-disulfamates show increased activity. With an IC(50) value of 190 nM the 4,4'-derivative is the first small monocyclic STS inhibitor coming close to the potency of the steroidal standard estrone sulfamate.     

6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate, a steroid sulfatase inhibitor for the treatment of androgen- and estrogen-dependent diseases

Steroid sulfatase (STS) offers a new target for the treatment of steroid hormone-dependent diseases, such as breast and prostate cancer and androgen-dependent skin diseases. We here characterize a novel non-estrogenic inhibitor of the enzyme, namely 6-[2-(adamantylidene)-hydroxybenzoxazole]-O-sulfamate (AHBS), with special attention to its potential use in the treatment of acne. The compound blocks STS activity in homogenates of human skin with IC(50)=16 nM. Following a single oral dose (5 mg/kg) in rats, the compound blocks STS in the skin by 95% at 8 h, followed by recovery of activity over 5 days. Following topical application to the skin, both in vitro and in vivo, AHBS passes through the stratum corneum leading to inhibition of STS activity in the dermal compartment with rapid onset and long duration. Topical application of AHBS to G?ttingen minipigs for a period of 2 weeks does not induce symptoms of ichthyosis as seen in STS-deficient human subjects, but leads to a reduction of sebum secretion to the skin surface. Based on these data, clinical studies with AHBS in acne patients are warranted, in order to verify the hypothesis on the importance of the sulfatase pathway in androgen-dependent skin diseases.     

Nortropinyl-arylsulfonylureas as novel, reversible inhibitors of human steroid sulfatase

Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases. Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput screening. A series of analogues was prepared in order to explore the essential structural elements for STS inhibition, and first structure-activity relationships were established. Mechanistic investigations revealed that the compounds are reversible, competitive inhibitors of STS.     

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes,membrane tethers) in human neutrophils

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm. The extensions were filled with cytoplasm and covered with membrane, as they included fluorescent cytoplasmic and lipid dyes. Neither protein kinase C inhibitors H-7 and bisindolylmaleimide VII, nor tyrosine protein kinase inhibitors tyrphostin AG 82 and genistein caused such extensions formation. Supposedly, STS induces membrane tubular extension formation promoting actin cytoskeleton depolymerization or affecting NO synthesis.     

Fine mapping of the distal short arm of the human X chromosome using X/Y translocations.

The loci for steroid sulfatase (STS), the deficiency of which causes X-linked ichthyosis, the cell surface antigen 12E7 (MIC2X), and the blood group antigen Xg (Xg) have been mapped to Xp22.3. These loci are of particular interest since they do not appear to undergo X-chromosome inactivation. In an attempt to establish the relative order of STS and MIC2X, fibroblasts from carriers of four different X/Y translocations and an X/10 translocation were obtained and fused with mouse cell lines deficient in hypoxanthine phosphoribosyltransferase. The breakpoints on the X chromosome in these five translocations are in Xp22. Several independent clones from each fusion were isolated in HAT medium. The clones were examined cytogenetically, and in each case at least two independent clones were identified that have an active X/Y or X/10 translocation chromosome in the absence of other X or Y material. These clones were then tested for STS and 12E7 expression. In two of the X/Y translocations, the markers, STS and 12E7, were both absent. In the X/10 and a third X/Y translocation, both markers were retained. In each of three clones containing the fourth X/Y translocation, STS activity was retained but 12E7 antigenicity was lost. Assuming that this is a simple translocation and does not represent a more complex rearrangement, these results suggest that MIC2X is distal to STS.     

Steroid sulfatase activity in homogenates, microsomes and purified Leydig cells from adult rat testis

Steroid sulfatase (STS) activity was studied in the Long-Evans rat testis. The rate of dehydroepiandrosterone sulfate (DHA-S) hydrolysis determined in whole testis homogenates was low compared to that of the corresponding microsomal fractions, which was, in contrast, as high as that expressed in homogenates from purified Leydig cells. Such an increment in STS activity between total homogenates and the corresponding microsomes was not observed for the seminiferous tubules. The STS affinity reported for total testicular microsomes (Km = 3.47 +/- 0.54 microM; mean +/- SEM) was of the same magnitude as that previously reported for Leydig cells, but was about 3 times higher than that measured for whole testis homogenate (Km = 10.11 +/- 0.92 microM). In vivo hCG treatment decreased the STS affinity in total testicular microsomes without affecting this kinetic parameter in whole testis homogenate. These data suggest that the steroid sulfatase expressed in total testicular microsomes (activity and regulation by hCG) could be considered as a good index of Leydig cell STS activity.