Sulphatase Inhibitors Research Articles

Dual Aromatase-Sulphatase Inhibitors (DASIs) for the Treatment of Hormone Dependent Breast Cancer.

Breast cancer is the most frequently diagnosed cancer in women and the second most common form of cancer, causing death after lung cancer, all across the globe at an alarming rate. The level of estrogens in breast cancer tissues of postmenopausal women is 10-40 folds higher than the non-carcinogenic breast tissues. As a result of this greater level of estrogen, breast tissue becomes more prone to develop breast cancer; mainly, estradiol plays a significant role in the initiation and development of hormone-dependent breast cancer. Androstenedione, Adrenal dehydroepiandrosterone sulfate, and estrone-sulfate also play an important role as precursors for estrogen biosynthesis. Estrogen deprivation exhibits an attractive phenomenon in the advancement of ideal therapeutics for the treatment of breast cancer. Inhibition of aromatase and sulphatase emerged as an attractive therapy for the treatment of hormone-dependent breast cancer via deprivation of estrogen by different pathways. The cocktail of aromatase and sulphatase inhibitors known as Dual Aromatase-sulphatase Inhibitors (DASIs) emerged as an attractive approach for effective estrogen deprivation. The present review article focused on the journey of dual aromatase-sulphatase inhibitors from the beginning to date (2020). Keeping in view the key observations, this review may be helpful for medicinal chemists to design and develop new and efficient dual aromatase-sulphatase inhibitors for the possible treatment of hormone- related 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 coumate analogues as estrogen receptor inhibitors for breast cancer therapy

A sulphatase inhibitor 667 COUMATE is in clinical trials for estrogen-positive breast cancer therapy for postmenopausal women, while there are a number of similar sulphatase inbitors are under development. Schiff's bases are versatile pharmacophores in which the N atom involves in hydrogen bonding with active cell centers interfering in normal cell biology. A library of novel coumate analogues with Schiff bases were designed, and structural based drug design was performed with human estrogen receptor (PDB ID: 2IOG) (Already reported). Based on the in-silico outcomes, seven coumate-schiff bases were synthesized. The compounds were obtained in good yield. The novelty had been ascertained by sci finder software. The synthesized molecules were consistent with their assigned spectra such as IR, Mass and NMR spectral data which confirmed their formation. The cytotoxicity study was performed by MTT assay for all the synthesized compounds. Most of the compounds have good IC50 values (below 100 µg/ml).Two of the synthesized compounds COU-2 and COU-5 have shown good IC50 values such as 19µg/ml and 39µg/ml, respectively. They suppressed the proliferation of estrogen receptor overexpressed MCF-7 cells.

SULFATION PATHWAYS: A role for steroid sulphatase in intracrine regulation of endometrial decidualisation.

In women, establishment of pregnancy is dependent upon ‘fine-tuning’ of the endometrial microenvironment, which is mediated by terminal differentiation (decidualisation) of endometrial stromal fibroblasts (ESFs). We have demonstrated that intracrine steroid metabolism plays a key role in regulating decidualisation and is essential for time-dependent expression of key factors required for endometrial receptivity. The primary aim of the current study was to determine whether sulphated steroids can act as precursors to bioactive sex steroids during decidualisation. We used primary human ESF and a robust in vitro model of decidualisation to assess the expression of genes associated with sulphation, desulphation and transport of sulphated steroids in human ESF as well as the impact of the steroid sulphatase (STS) inhibitor STX64 (Irosustat). We found evidence for an increase in both expression and activity of STS in response to a decidualisation stimulus with abrogation of oestrone biosynthesis and decreased secretion of the decidualisation marker IGFBP1 in the presence of STX64. These results provide novel insight into the contribution of STS to the intracrine regulation of decidualisation.

SULFATION PATHWAYS: Steroid sulphatase inhibition via aryl sulphamates: clinical progress, mechanism and future prospects.

Steroid sulphatase is an emerging drug target for the endocrine therapy of hormone-dependent diseases, catalysing oestrogen sulphate hydrolysis to oestrogen. Drug discovery, developing the core aryl O-sulphamate pharmacophore, has led to steroidal and non-steroidal drugs entering numerous clinical trials, with promising results in oncology and women's health. Steroidal oestrogen sulphamate derivatives were the first irreversible active-site-directed inhibitors and one was developed clinically as an oral oestradiol pro-drug and for endometriosis applications. This review summarizes work leading to the therapeutic concept of sulphatase inhibition, clinical trials executed to date and new insights into the mechanism of inhibition of steroid sulphatase. To date, the non-steroidal sulphatase inhibitor Irosustat has been evaluated clinically in breast cancer, alone and in combination, in endometrial cancer and in prostate cancer. The versatile core pharmacophore both imbues attractive pharmaceutical properties and functions via three distinct mechanisms of action, as a pro-drug, an enzyme active-site-modifying motif, likely through direct sulphamoyl group transfer, and as a structural component augmenting activity, for example by enhancing interactions at the colchicine binding site of tubulin. Preliminary new structural data on the Pseudomonas aeruginosa arylsulphatase enzyme suggest two possible sulphamate-based adducts with the active site formylglycine as candidates for the inhibition end product via sulphamoyl or sulphonylamine transfer, and a speculative choice is suggested. The clinical status of sulphatase inhibition is surveyed and how it might develop in the future. Also discussed are dual-targeting approaches, development of 2-substituted steroidal sulphamates and non-steroidal derivatives as multi-targeting agents for hormone-independent tumours, with other emerging directions.

X-ray crystal structure and mechanism of action of oestrone 3-O-sulphamate, a synthetic active site-directed inhibitor of oestrone sulphatase

Oestrone 3-O-sulphamate, wherein a sulphamate moiety is a sulphate group surrogate relative to oestrone sulphate, is the most potent oestrone sulphatase inhibitor developed to date and inhibits oestrone sulphatase in a time- and concentration-dependent manner, showing that it is acting as an active site-directed irreversible inhibitor. It also inhibits dehydroepiandrosterone sulphatase, the enzyme which regulates the synthesis of androstenediol. Oestrone 3-O-sulphamate has also been shown to inhibit these enzymes in-vivo. We report here its crystal structure and the synthesis and inhibitory activites of analogues in which the 3-O atom is replaced by other heteroatoms (N and S). Oestrone 3-N-sulphamate and oestrone 3-S-sulphamate were found to inhibit placental microsome oestrone sulphatase weakly, i.e. 53% and 12%, respectively, at 50 μM (compared with >99% for oestrone 3-O-sulphamate), but none of these compounds appears to behave as a time-dependent inhibitor.

Development of an Oral Formulation for Oestrone 3-O-Sulphamate, a Potent Sulphatase Inhibitor

Oestrone sulphatase is a key enzyme involved in oestrogen synthesis in breast tumours in postmenopausal women. As breast tumours can be endocrine-dependent, it is therefore feasible that blockage of this enzyme may be of value in the treatment of such tumours. Oestrone 3-O-sulphamate (EMATE) is the most potent inhibitor of oestrone sulphatase activity described to date. Here, we describe a preliminary examination of its physicochemical properties and the development of a self-emulsifying formulation for potential administration of EMATE by the oral route. A preliminary assessment of the absorption of the drug from the gut was undertaken, and pharmacological activity, determined as oestrone sulphatase inhibition, was studied following intravenous and oral administration. A high-performance liquid chromatography (HPLC) assay was developed to measure degradation of EMATE in the formulation and also its concentration in rat plasma. The drug was susceptible to degradation, but its stability in the formulation was satisfactory when stored at either 4°C or room-temperature over the timespan required for a clinical trial. Plasma EMATE concentrations after 2 h were a function of the dose of drug administered orally to rats over the 10–40 mg kg−1 range. After oral administration in the self-emulsifying system, EMATE appeared to be rapidly absorbed, with the peak plasma concentration being detected at 30 min, after which plasma concentrations rapidly decreased. After intravenous administration, a similar plasma EMATE concentration was detected at 1 h to that observed after oral administration. Rat liver sulphatase activity was almost completely inhibited (> 99%) within 30 min of oral or intravenous administration of EMATE.

4- and 6-( p-Sulphamoylphenyl)androstenediones: Studies of aromatase inhibitor-based oestrone sulphatase inhibition

4-( p-Sulphamoylphenyl)androstenedione ( 3) and 6α- p-sulphamoylphenyl analogues 12– 14 were synthesised and tested as aromatase inhibitors as well as oestrone sulphatase inhibitors in human placental microsomes. All of the p-sulphamoylphenyl compounds synthesised were powerful inhibitors of aromatase with apparent K i values ranging between 30 and 97 nM. In addition, the aromatase inhibitory activities of 6α- p-hydroxyphenyl compounds 9– 11, which may be produced from their respective sulphamoylphenyl compounds by action of oestrone sulphatase, were also high in a range of 23 and 75 nM of the K i values. On the other hand, all of the sulphamoylphenyl compounds were poor inhibitors of oestrone sulphatase with more than about 200 μM of IC 25 values. Although the present findings of the oestrone sulphatase inhibition are disappointing, such attempts may be valuable to develop a new class of drugs having a dual function, aromatase inhibitor and oestrone sulphatase inhibitor, for the treatment of oestrogen-dependent breast cancer.

A letrozole-based dual aromatase–sulphatase inhibitor with in vivo activity

The role of aromatase inhibitors in the treatment of hormone-dependent breast cancer is well established. However, it is now recognised that steroid sulphatase (STS) inhibitors represent a new form of endocrine therapy. To explore the potential advantage of dual inhibition by a single agent, we recently developed a series of dual aromatase–sulphatase inhibitors (DASIs) based on the aromatase inhibitor YM511. We report here a new structural class of DASI obtained by obtained introducing the pharmacophore for STS inhibition, i.e. a phenol sulphamate ester into another established aromatase inhibitor letrozole. Hence, the bis-sulphamate 9 was synthesised which exhibited IC 50 values of 3044 nM for aromatase and >10 μM for STS in JEG-3 cells. However, at a single oral dose of 10 mg/kg, 9 inhibited aromatase and rat liver STS by 60% and 88%, respectively, 24 h after administration. A proposed metabolite of 9, carbinol 10, was synthesised. Despite also showing weak STS inhibition in JEG-3 cells, 10 inhibited rat liver STS activity to the same extent as 9 at a single oral dose of 10 mg/kg. Thus, the concept of a letrozole-based DASI has been validated and could be further developed and modified for therapeutic exploitation.

Pharmacokinetics of the nonsteroidal steroid sulphatase inhibitor 667 COUMATE and its sequestration into red blood cells in rats.

Breast cancer is a major cause of mortality in Western countries and there is an urgent requirement for novel treatment strategies. The nonsteroidal sulphatase inhibitor 667 COUMATE inhibits hepatic steroid sulphatase and growth of oestrone sulphate stimulated tumours in the nitrosomethylurea-induced rat mammary model. Other compounds that contain an aryl sulphamate moiety, for example, oestrone-3-O-sulphamate, are sequestered into red blood cells (RBCs). The aims of this study were to determine the pharmacokinetics of 667 COUMATE and to investigate its sequestration into RBCs. We administered a single p.o. or i.v. dose (10 mg kg−1) of 667 COUMATE to rats and used a high-performance liquid chromatography method to measure the levels of the agent and its putative metabolites in plasma. 667 COUMATE had a bioavailability of 95% and could be detected in plasma for up to 8 h. Using two independent analytical methods, we demonstrated that 667 COUMATE is sequestered by RBCs both ex vivo and in vivo. Previous investigations have revealed that 667 COUMATE is rapidly degraded in plasma ex vivo. In this study, we demonstrate that 667 COUMATE is stabilised due to its sequestration into RBCs. In conclusion, the pharmacological efficacy and high oral bioavailability of 667 COUMATE may be partly a consequence of the ability of RBCs to both protect the agent from metabolic degradation and facilitate its transport to tissues. These data support the further clinical evaluation of this novel endocrine therapeutic agent.

The role of steroid sulphatase in regulating the oestrogenicity of oestrogen sulphamates

Oestrogen sulphamates have increased systemic, but reduced hepatic oestrogenicity which results from their sequestration and transport through the liver by red blood cells. Oestrogen sulphamates act as prodrugs for the release of natural oestrogens but, as yet, there is little information as to how the sulphamoyl moiety is cleaved from the steroid nucleus. In the present investigation we have used the potent steroid sulphatase (STS) inhibitor, 667 COUMATE, to explore the possibility that STS might be responsible for the hydrolysis of oestrogen sulphamates. Administration of oestrone sulphamate (10 μg/day, s.c., for 5 days) to ovariectomised rats resulted in a 3.5-fold increase in the uterine weights of treated animals. Co-administration of oestrone sulphamate and 667 COUMATE (2 mg/kg) completely blocked STS activity in treated animals and completely abrogated the ability of oestrone sulphamate to stimulate uterine growth. In vitro studies, using [ 3H]oestrone sulphamate or [ 3H]oestrone, revealed that the uptake of the sulphamate derivative (95.9 ± 2.4%) by red blood cells was considerably higher than that for the non-sulphamoylated oestrogen (25.1 ± 1.9%). Results from these studies demonstrate convincingly that STS is the enzyme responsible for the removal of the sulphamoyl group from oestrogen sulphamates. This enzyme therefore has a crucial role in regulating the oestrogenicity associated with this class of drug.

2-Difluoromethyloestrone 3- O-sulphamate, a highly potent steroid sulphatase inhibitor

Steroid sulphatase is a target enzyme of growing therapeutic importance. The synthesis and in vitro biological evaluation of three novel 2-substituted analogues of oestrone 3- O-sulphamate (EMATE), an established steroid sulphatase inhibitor, are described. One inhibitor, 2-difluoromethyloestrone 3- O-sulphamate ( 6), was found to have an IC 50 of 100 pM and be some 90-fold more potent than EMATE in inhibiting steroid sulphatase activity in a placental microsomal preparation, rendering this agent the most potent steroidal STS inhibitor in vitro reported to date. Lowering of the p K a value of the leaving parent steroid phenol by the 2-difluoromethyl group during irreversible enzyme sulphamoylation most likely facilitates the potent inactivation of steroid sulphatase by ( 6). However, our preliminary molecular docking studies using the X-ray crystal structure of steroid sulphatase suggest that F⋯⋯H interactions between the 2-difluoromethyl group of ( 6) and hydrogen bond donor residues lining the catalytic site of STS might also contribute to the high potency observed for ( 6).

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 IC 50 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 IC 50 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 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.

Docking studies of sulphamate inhibitors of estrone sulphatase in human carbonic anhydrase II

We describe the docking of selected steroidal and non-steroidal estrone sulphatase inhibitors, including the Phase I clinical trial candidate 667COUMATE ( 6), into the active site of human carbonic anhydrase II (hCA II). The docking scores are compared with the inhibition of hCA II and show good correlation with biological activity.

Development of a sensitive high-performance liquid chromatography method for the detection of 667 COUMATE in vivo

Steroid sulphatase inhibitors which decrease or prevent the biosynthesis of oestrogens, potentially have an important role in the treatment of breast cancer in postmenopausal women. The non-steroidal sulphatase inhibitor 667 COUMATE has been shown to be active both in vitro and in vivo. The pharmacokinetics of this drug have not been investigated. In preparation for the clinical evaluation of this agent, a sensitive and robust reversed phase high-performance liquid chromatography (HPLC) method was developed for the detection of 667 COUMATE in biological fluids. The sulphatase inhibitor was extracted from plasma with diethyl ether and separated from putative metabolites and endogenous plasma components with a C3-phenyl column. Using this method an extraction efficiency of 76±5% and a limit of detection of less than 0.1 ng/ml was achieved. The stability of this agent was investigated under different pH conditions and during storage in plasma at room temperature or −20 °C. 667 COUMATE was found to be stable when stored in acidified plasma (pH 4.5) at −20 °C. In conclusion, the HPLC method developed is a reproducible and sensitive assay that will enable quantitation of the potent non-steroidal sulphatase inhibitor 667 COUMATE in biological fluids in the forthcoming Phase I clinical trial.

A-ring analogues of oestrone 3-O-sulphamate as potent steroid sulphatase inhibitors and potential anti-cancer agents

A-ring analogues of oestrone 3-O-sulphamate as potent steroid sulphatase inhibitors and potential anti-cancer agents

D-ring modified steroids as potent oestrone sulphatase inhibitors

D-ring modified steroids as potent oestrone sulphatase inhibitors

Synthesis and steroid sulphatase inhibitory activity of C19- and C21-steroidal derivatives bearing a benzyl-inhibiting group

Two series of compounds, benzyl alkylated at position 17α and 20 of androstane and pregnane, respectively, were synthesised and tested for steroid sulphatase inhibition. We compared the ability of the compounds to inhibit steroid sulphatase obtained from two different sources (homogenates of transfected HEK-293 cells and Jeg-3 cells) and with two types of substrate (DHEAS or E 1S). The inhibitory activity of 17α-benzyl-5α-androstane-3β,17β-diol ( 7), 17α-benzyl-5-androstene-3β,17β-diol ( 9), 17α-benzyl-4,17β-dihydroxy-4-androsten-3-one ( 15) and 20-benzyl-5-pregnene-3β,20α-diol ( 16) has proven to be superior to that of danazol, the first steroid sulphatase inhibitor to be reported, but still lower than that of the potent inhibitor estrone-3- O-sulphamate. The inhibitory activity of compound 7 was as potent as that of its previously reported estrane analogue, 17α-benzyl estradiol. Benzyl alkylated compounds with no OH group on the A-ring (with a 4-OCH 3, 4-Cl, or 4-H and their precursor epoxides), as well as a series of basic steroids without a benzyl group (ADT, epi-ADT, 3α-diol, 3β-diol, DHEA, Δ 5-diol, DHT, T, Preg and Prog), did not show steroid sulphatase inhibition. We have thus demonstrated that the steroid sulphatase inhibitory effect of a benzyl group, previously observed for an estrane nucleus, can be extended to certain androstane and pregnane nuclei bearing a 3β-OH or a 4-OH group. Inhibitors 7, 9, 15 and 16 did not induce any proliferative effect on androgen-sensitive Shionogi cells. However, when tested on oestrogen-sensitive ZR-75-1 cells, a proliferative effect was observed for 7 and 9, but not for 15 and 16.

Human granulosa cells are a site of sulphatase activity and are able to utilize dehydroepiandrosterone sulphate as a precursor for oestradiol production.

Dehydroepiandrosterone sulphate (DHEAS) is the most abundant androgen in the circulation and in ovarian follicular fluid. A steroid sulphatase accepting DHEAS as a substrate has been identified in the follicle, but the cellular location has not been determined. As DHEAS is also a potential source of oestrogen for endocrine-dependent tumours, a potent steroid sulphatase inhibitor oestrone-3-O-sulphamate (EMATE) has been developed which inhibits this activity in rat liver and mammary tumour. The aim of this study was to investigate human granulosa cells as a site of steroid sulphatase activity, to determine whether DHEAS can be utilized as a precursor for oestrogen synthesis and to investigate the inhibitory capacity of EMATE in these cells. Conversion of DHEAS to DHEA was assessed in luteinized granulosa cells by tritiated steroid assay following incubation with or without LH or insulin and steroid accumulation in the medium measured by RIA. The effects of EMATE were assessed by addition of a range of doses during the measurement of conversion of DHEAS to DHEA. Cells from three sizes of small follicles from an unstimulated ovary were also assessed for their ability to produce oestradiol from DHEAS. Sulphatase enzyme activity was present in all cells; the mean conversion of tritiated DHEAS to DHEA was 50% (range 4-65%). LH and EMATE inhibited and insulin stimulated this activity. Addition of DHEAS to granulosa cells caused a dose-dependent increase in oestradiol and androstenedione production with no change in progesterone concentration. LH increased the accumulation of oestradiol in the medium. DHEAS also stimulated oestradiol production by granulosa cells from small follicles. This is the first demonstration that granulosa cells are a site of sulphatase activity and that DHEAS can be utilized as a substrate for androstenedione and oestrogen production. This may be of physiological importance for both normal folliculogenesis and oestrogen-dependent tumour growth.