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.
- steroid sulfatase (STS)
- breast cancer
- hormone-dependent diseases
Breast cancer (BC) is the most common malignant tumor in women (12%) worldwide and is the second leading cause of cancer mortality after lung cancer (26%) .
Approximately 95–97% of tumors are estrogen-dependent in the early stages of their development [2, 3] and more than 70% express very high levels of estrogen receptor alpha (ERα) . The fundamental difference of extragonadal estrogen synthesis is its autocrine nature—that an organ producing estrogens is a target organ at the same time. Thus, local concentration of estrogens in such organs may be markedly elevated. Peripheral estrogens formation is increased after menopause, and compensates estrogens deficiency in different organs and tissues . Extragonadal estrogens’ production may rise with the aging. Moreover, it was continually emphasized in the literature that the increased level of estrogens in the body is considered as a risk of the BC development [6, 7].
Biologically active hormones, in particular the most active estrogen estradiol (E2), play a critical role in the initiation and development of hormone-dependent breast cancer (HDBC). In premenopausal women, estrogens are mainly (75%) synthesized in the ovaries, and thus, a luteinizing hormone-releasing hormone (LH-RH) agonist [8, 9] is useful to suppress the function of pituitary hormone. In postmenopausal women estrogens are produced in peripheral tissues such as adipose tissues, skin, and mammary glands [10, 11].
Adrenal dehydroepiandrosterone sulfate (DHEAS), dehydroepiandrosterone (DHEA), and adrenal or ovarian androstenedione are also sources of E2 in peripheral tissues. In postmenopausal women, concentrations of DHEAS, DHEA, and androstenedione in plasma are relatively high; approximately 1.8, 6.6, and 1.9 nM, respectively. In contrast, plasma concentrations of estrone (E1) and (E2) are several-fold lower (70 and 30 pM, respectively) .
Another important steroid precursor for estrogen formation is E1-sulfate (E1S). It is the most important estrogen in the peripheral blood, with relatively high (0.6 nM) concentrations in postmenopausal women. E1S levels are associated with high body-mass index, which suggest that E1S originates from adipose tissue. Concentrations of E1S in plasma are 10–20 times higher than those of E1 and E2, as well as its half-life in the plasma is longer than the half-life of unconjugated estrogens.
Enzyme steroid sulfatase (STS) converts E1S to E1, followed by the reduction to the biologically active estrogen, E2, by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), which is overexpressed in many breast tumors.
In BC tissues estrogens can be locally produced
The level of estrogens in BC tissues of postmenopausal women can be 10–40 folds higher than in blood circulation and 5–10 times higher than in noncancerous breast tissues . Furthermore, the intratumoral E2/E1 ratio is significantly higher in postmenopausal BC than in premenopausal BC. High concentrations of estrogen in breast tissue increase the risk of BC development [14, 15].
Thus, inhibition of enzymatic synthesis of estrogens is an effective therapeutic strategy for postmenopausal women with estrogen receptor-positive (ER+) tumors [16, 17].
The scheme of estrogens formation in human body includes: (a) formation of E1 from androstenedione under the action of cytochrome P450 aromatase, (b) reduction of E1 by 17β-HSD1 leads to more active E2. Importantly, almost insoluble in aqueous media E1 is converted into water-soluble E1S under the action of sulfotransferase (STS). E1S does not possess hormonal activity, however it may be transported into various targets (Figure 1) [18, 19]. Several reviews focus on aspects of human steroidogenesis [18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29].
Free hormones are formed from sulfates of estrogen and androgens under action of steroid sulfatase. At high concentrations, androgens compete for binding with ERs. The activation of ERα under the action of androstenediol and DHEA in BC cells has been detected. It is confirmed by the inhibition of cell growth in the presence of antiestrogens. The evaluation of E1S level during diagnostic of various oncological diseases (for example, prostate cancer) is of high importance .
2. Approaches for the manipulation of estrogen level in tumors
2.1 Endocrine therapy
Hormonal (endocrine) therapy is effectively used for the treatment of HDBC. Most types of BCs are estrogen-dependent, with approximately 55% in premenopausal women and 75% in postmenopausal women [31, 32, 33, 34].
Selective estrogen receptor modulators (SERMs) or down-regulators (SERD), such as tamoxifen, raloxifene, ospemifine, and fulvestrant are compounds that are currently used in clinical practice to treat BC [9, 35]. In breast tissues, SERMs effectively block the activation of ER(α) by endogenous ligands, preventing the transcription of genes mediated by estrogen response elements [36, 37]. SERMs have tissue-specific effects on ERα that results in antagonist activity in breast and uterus tissues as well as agonist activity in bone. Although tamoxifen and raloxifene possess the desired SERM activity, they also increase the risk of venous thromboembolism  and exhibit toxicity . Given that resistance (
2.2 Inhibitors of enzymes responsible for the estrogen formation in tumors
The aromatase enzyme is responsible for the conversion of testosterone and androstenedione to E2 and E1, respectively. Thus, inhibition of the aromatase enzyme is one of the approaches for the development of new drugs to treat BC [41, 42, 43].
Nonsteroidal third-generation aromatase inhibitors (AIs), such as anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin), are often used for postmenopausal hormone-dependent BC treatment in clinical practice. Despite the success of AIs in the clinic, numerous BC patients still progress after AI therapy due to the development of resistance to AIs and side-effects such as osteoporosis caused by whole-body deprivation of estrogen [44, 45]. Mechanisms of AI resistance include ligand-independent activation of the ER and signaling via other growth factor receptors; new insights about resistance are published recently .
The overall response rates for AIs (40–50%) suggest the presence of alternative sources of estrogens. The production of E1, DHEA and androstenediol is an important mechanism of resistance to AI treatment .
It was demonstrated that AIs used sequentially with tamoxifen had higher efficacy compared to tamoxifen alone, with an improvement in overall survival .
There are other factors involved in tumor growth . The enzymes STS and 17β-HSD1 have been identified as essential parts in E2 production and subsequent promotion of cancer growth. Recently it was shown that 17β-HSD7 also plays a key role in increasing the E2/E1 ratio in BC tumors . Very recently, some evaluations of the “sulfatase pathways” in tumor stroma have been carried out .
The STS is also responsible for the hydrolysis of DHEAS to DHEA, which is an immediate precursor of androstenediol, a potent estrogenic steroid , whose formation is not influenced by AIs. DHEAS stimulates proliferation of MCF-7 cells from BC, which could be blocked by an antiestrogen or STS inhibitor but by an AI. E1S and DHEAS are particularly abundant in blood circulation and could act as a reservoir of steroid precursors, specifically in BC [29, 52]. The formation of DHEA through the STS pathway accounts for the production of 90% of the androgen androstenediol , which possesses estrogenic properties, that are 100-times weaker than estradiol [13, 53]. Androstenediol is present at 100-fold higher concentrations than estradiol in the circulation, and may have estrogenic properties that are equal to estradiol . Thus, inhibition of STS has the dual property of reducing local androstenediol biosynthesis [55, 56].
2.3 Steroid sulfatase enzyme (STS)
The STS enzyme (EC 126.96.36.199, aryl sulfatase C, steryl-sulfatase) is widely distributed throughout the body and plays critical role in steroidogenesis . Publications in recent years indicate the role of STS activity in gynecological diseases , mentioning diminished endometriosis
According to the
The human STS is a protein, integrated in microsomal membrane. Its three-dimensional structure has been determined (PDBcode 1P49) . However, knowledge about regulation of its expression as well as activity is limited. The topology of the active site of the steroid sulfatase and the arylsulfatases A and B is similar .
Most of the STS inhibitors discovered to date, act as irreversible active-site-directed inhibitors. An aryl sulfamate group (ArOSO2NH2) is considered as the pharmacophore for irreversible inhibition of the enzyme. One of the first time-, pH-, and concentration-dependent irreversible active-site directed-steroidal inhibitor is estrone-3-O-sulfamate (EMATE), which inhibit STS in MCF-7 cells from BC by 99% at 0.1 μM and has an IC50 value of 65 pM (IC50 = 80 nM in placental microsomes). EMATE was evaluated in clinical trials . The highest effectiveness of EMATE has been demonstrated in rats (subcutaneous and oral administration). STS activity was also inhibited when EMATE was administered to humans in dose 0.5 mg/kg .
Despite the exceptional potency of the EMATE [67, 68], it is not used in clinical practice to treat hormone-dependent BC because metabolic conversion of EMATE by STS releases estrone, which act via estrogen receptors, and can directly promote tumor growth . Nevertheless, EMATE is now the prototypical inhibitor, and used as standard during evaluation of other potential STS inhibitors .
2.4 Mechanisms of inactivation of steroid sulfatase
Several research groups made attempts to establish the mechanism(s) of sulfatase inactivation. However, the precise mechanism of inhibition is still uncertain. In 2010, Spillane and Malaubier have established that the hydrolysis of EMATE occurs by two different mechanisms: an SN2 mechanism below pH 9.5 and E1cB mechanisms involving N-sulfonylamines at higher pHs . Detailed presumable mechanisms have been discussed in recent reviews [71, 72, 73].
Based on the mechanisms, the result of the hydrolysis is free estrone. Moreover, under per os administration, the activity of EMATE is several times higher than the activity of estrone, due slow metabolism of EMATE in liver . EMATE is not subjected to metabolic inactivation in red blood cells. Thus, consideration of hormonal activity and side-effects of steroids with free phenolic group is important in the modeling of sulfatase inhibitors for therapeutic use [54, 74, 75].
The knowledge of the crystal structure opens the rational drug design of molecules for the inactivation of steroid sulfatase.
2.5 Nonsteroidal STS inhibitors
Many investigations have been carried out to develop nonsteroidal STS inhibitors, because nonsteroidal drugs and their metabolites may have less undesirable effects.
A search of an orally active, nonestrogenic, nonsteroidal STS inhibitors among tricyclic compounds based around the coumarin core resulted in the discovery of Irosustat (667-coumate, STX64, BN83495) , which is the first-in-class irreversible time- and concentration-dependent STS inhibitor for the treatment of hormone-dependent BC in postmenopausal women that has been clinically evaluated in breast, endometrial, and prostate cancers  and there is potential for innovative dual-targeting approaches [78, 79], with an IC50 value of 8 nM in placental microsomes. The inhibitor (
The optimum dose of 40 mg/day was estimated in phase I/II trials . Efficiency of Irosustat has also been demonstrated in a phase II study in (ER+) endometrial cancer in women with advanced or recurrent disease . The high bioavailability of Irosustat is explained by the prevention of degradation by sequestration inside red blood cells where it, similarly to EMATE, binds to (and inhibits) carbonic anhydrase II (IC50 = 22 nM) . The inactivation mechanism suggests that a sulfamate group is transferred to the gem-diol form of formylglycine 75 of steroid sulfatase due to a facile E1cB elimination of sulfamate anion to give the corresponding coumarin, which has a long half-life in blood . However, the further development of Irosustat in monotherapy was stopped in the phase I/II clinical studies, because Irosustat does not possess superior properties to the current standard of care megesterol acetate, and its relative bioavailability decreases with increasing dose. The study of its combination with other hormonal therapies (for example, with the aromatase inhibitor anastrozole) is underway . Metabolism of Irosustat has been investigated . Irosustat also inhibits skin and liver STS .
2.5.1 Dual selective estrogen receptor modulators/STS inhibitors
One of the first examples of the dual SERM/STS inhibitor was published by Duquesne University . 4-Hydroxytamoxifen is a metabolite of main drug tamoxifen used as endocrine therapy in (ER+) BCs . This metabolite is a SERM and has antiestrogen effects in breast tissues, however, acts as an estrogen agonist in other tissues such as bone marrow. The sulfamate derivative
Surprisingly, among silicon-containing derivatives compound
Poirier with colleagues, among tetrahydroisoquinoline-N-substituted derivatives , found second-generation dual-action compounds that inhibit STS and act as a SERM. These compounds are devoid of estrogenic activity and toxicity. Their sulfamate derivatives possess high inhibitory activity toward STS (IC50 of 3.9, 8.9, and 16.6 nM). Both phenolic and their sulfamate derivatives show no estrogenic activity and moderate antiestrogenic properties. All compounds significantly stimulate osteoblast-like Saos-2 cell proliferation, thus suggesting a SERM activity. The results of molecular docking experiments suggest that the most active compounds
2.5.2 Dual aromatase/STS inhibitors
Another approach for the treatment of hormone-dependent BC is the development of DASIs, which may have an additive or synergistic antitumor effect. The potential advantages of a single chemical agent with the ability to interact with multiple biological targets were highlighted previously . In the case of DASIs, this goal is being pursued by the introduction of the critical sulfamate unit in structures with known aromatase-inhibiting properties [93, 94]. All DASIs are still in preclinical investigations .
One of the best dual inhibitors is compound
One of the most potent dual inhibitor is compound
Among compounds on letrozole and vorozole templates, the most potent inhibitors were compounds
Potter with coauthors published the successful realization of the strategy when the core components of the two leading DASIs resulted in the hybrid structures that exhibit a very high level of dual inhibition against aromatase and STS
2.6 Steroidal STS inhibitors
2.6.1 Steroid-based STS inhibitors without sulfamate group
Nonsulfamated STS inhibitors based on estrogens are weaker than EMATE. Most active STS inhibitors without sulfamate group with highest activity are represented by compounds
In the series of 4-substituted 17β-arylsulfonamides of 17β-aminoestra-1,3,5(10)-trien-3-ol, compounds
2.6.2 Steroid-based STS inhibitors with sulfamate group
The estrogenicity of EMATE and estradiol-3-O-sulfamate (
Simple modifications of the D-ring have led to dramatic variations in estrogenicity. Thus, the conversion of EMATE to the oxime results in a super-estrogen. From the other hand, D-ring heterocyclic derivatives exhibit reduced estrogenicity [121, 122].
The replacement of ring D with N-substituted piperidinedione moiety results in the loss of estrogenic properties and greater STS inhibitory activity
Among various 2- and 4-substituted and 2,4-disubstituted EMATE derivatives, most active compounds are 2-(2-prop-2-enyl)-EMATE (
2.6.3 Dual 17β-HSD1/STS inhibitors
17βHSD converts E1 to E2 and DHEA to androstanediol . Several inhibitors based on steroidal skeleton have been successfully developed [133, 134]. Few dual inhibitors of 17β-HSD and STS for the treatment of steroid hormone-dependent diseases are patented . The example of such inhibitors is represented by the compound
A-ring-modified steroidal sulfamates, for example, series of 2-OMe-estradiol sulfamates and analogs have been investigated as nonestrogenic STS inhibitors [136, 137]. 2-MeO-EMATE
It is known that 2-methoxyestradiol, a metabolite of E2, possesses antiangiogenic properties and prevents tumor growth through disrupting tubulin polymerization by binding at the colchicine-binding site [142, 143]. 2-Methoxyestradiol is considered as the perspective compound for the treatment of endometriosis .
The anticancer effects of the 2-substituted sulfamate estrogen derivatives arise from disruption of tubulin polymerization, and the compounds also binding at the colchicine site . 3,17β-Bissulfamates of estrogens are other representatives of multitargeted antitumor agents, acting as STS inhibitors with antiproliferative activity (IC50 = 18–250 nM) . Such bissulfamates compete with colchicine for tubulin binding and disrupt microtubules resulting into cell cycle arrest just by apoptosis
Coordination of the 17-sulfamate residue to the zinc in active site of the complex of STX140 with human carbonic anhydrase II is revealed .
STX140 depolarizes mitochondrial bioenergetics, activates caspase 3/7 causing apoptosis through the intrinsic mitochondrial pathway, and downregulates the expression of caspase inhibitors . The activity of such compounds is also explained by their ability to disrupt the tubulin-microtubule equilibrium in cells as being central to their antitumor activity. STX140 and STX243 bind with the colchicines binding site of tubulin. 2-(11C)Methoxy-3,17β-OO-bis(sulfamoyl)estradiol has been proposed as a new potential PET agent for imaging of steroid sulfatase in cancers .
One more example of 2-MeO-derivatives as effective STS inhibitors is illustrated by compound
The level of STS inhibition for 17β-(N-alkylcarbamoyl)-estra-1,3,5(10)-trien-3-O-sulfamates (
Among a series of C17-ketone and amide-modified estrone-derived sulfamates, compound KW-2581 (
Compound EM-1913 (
17α-Benzyl substituent yields reversible STS inhibitors in the absence of a sulfamate group, and incorporation of an aryl sulfamate onto the A-ring results in a potent time-dependent irreversible inhibitor. The IC50 of the tert-butylbenzyl derivative
In the series of A-ring thioether-modified sulfamates, the steroid
2.6.4 Dual STS/SERM inhibitors
Maximum estrogen blockade in the treatment of (ER+) BC may be achieved using dual ERα antagonists and STS inhibitors, which might cause osteoporosis as a side effect . In this case, the compound possessing SERM properties is needed.
Thus, a novel orally available irreversible dual STS/SERM inhibitor SR16157 (NSC 732011) (
Desulfamoylation of SR16157 (
Manipulation of hormone biosynthesis in tumors by enzymes inhibitors is a very attractive approach for the treatment of hormone-dependent tumors such as breast, prostate cancer, and endometriosis.
The importance of STS in human body has been underlined by many investigations. Thus, STS-catalyzed hydrolysis of pregnolone-3-sulfate and dehydroepiandro-sterone-3-sulfate in the brain regulates neurosteroid synthesis and influences memory. STS inhibition for the potentiation of memory in sufferers of neurological diseases such as Alzheimer’s disease and dementia has been postulated . The role of STS inhibitors as agents to reveal beneficial endogenous glucocorticoid effects was also claimed. The use of STS inhibitors in combination with the immunosuppressive ascomycin for the treatment of acne, seborrhoea, androgenetic alopecia, and hirsutism is patented. The administration of an estrogen (including norgestimate and norelgestromin), in combination with a progestogen in hormone-replacement therapy act by inhibiting STS, thus reduce estrogen production and protect the endometrium and breast from hormone-dependent cancers . STS inhibitors prevent ovarian cycle disturbance, prolonged unopposed secretion of estrogens, and ovarian follicular cyst formation in premenopausal women, as well as prevent premature uterine contractions, particularly for preterm labor .
The importance of STS inhibition in endometriosis, prostate cancer, as well as latest discussions about mechanism of inhibition is well considered in the review of Prof. Potter . The significance of steroid sulfatase and sulfotransferases in gynecological diseases are summarized in the review .
As far as estrogenic compounds may stimulate tumor cells growth, the main requirement for STS inhibitors and their metabolites is the absence of estrogenicity. Among nonsteroidal STS inhibitors only one nonestrogenic compound-Irosustate was evaluated in clinical trials with excellent properties, however its further development was stopped. Currently, the action of Irosustate in the combination with AIs is investigated.
The discovery of dual (multitargeted) inhibitors is the most promising nowadays. For example, several DASIs based on anastrazole, letrozole, and vorozole templates inhibit both STS and aromatase in nanomolar concentrations, being nonestrogenic; and have a chance to be introduced in clinical trials.
Among STS inhibitors based on steroid skeleton 17α-benzyl-derivatives, 17β-arylsulfonamides, and 17-diisopro-pylcarbomoyl-3-O-sulfamates exhibit the best properties, especially as multitargeted (dual) anticancer potential drugs. The same modifications result in the increased activity against STS in the case of 2-OMe-3-O-sulfamates as well as 2-OMe-3,17β-bissulfamates. The latter also possess activity against most aggressive form—triple negative BC.
Additionally, 8α-steroid estrogen analogs without estrogenic properties possess high STS activity and block BC cells growth with the activity comparable to standard of care for BC treatment tamoxifen.
The authors greatly appreciate Dr. Anna S. Chentsova for careful reading of the manuscript and her valuable comments. The reported study was funded by RFBR according to the Research Project No. 16-54-76024.
Conflict of interest
The authors confirm that this article content has no conflicts of interest.
|DASI||dual aromatase-sulfatase inhibitor|
|GPER, GPR30||G-protein-coupled estrogen receptor|
|HDBC||hormone-dependent breast cancer|
|LH-RH||luteinizing hormone-releasing hormone|
|SERD||selective estrogen receptor down-regulators|
|SERM||selective estrogen receptors modulator|