Abstract
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.
Introduction
Many hormone-sensitive tumours depend upon the steroid 17β-oestradiol (E2) for growth (Dixon 2014) and about two-thirds of such breast tumours are oestrogen receptor α (ER) positive (ER+ status). Endocrine therapy in the postmenopausal breast cancer setting, with most ovarian oestrogen production having ceased, aims to target E2 reduction and is critical to underpin modern targeted cancer therapy (Rugo et al. 2016). For example, the newer mTOR and CDK4/6 inhibitors in breast cancer, such as everolimus and palbociclib respectively, are generally administered upon a background of endocrine therapy (Baselga et al. 2012, Finn et al. 2016). Access of oestrogens to the ER can be blocked clinically through a selective oestrogen receptor modulator (SERM) or down-regulator (SERD), for example, raloxifene, ospemifene, fulvestrant and so forth (Patel & Bihani 2018). The SERM tamoxifen has been the drug of choice for many years and enormously successful (Davies et al. 2013). In recent decades, however, inhibitors of oestrogen biosynthesis targeting the aromatase enzyme, aromatase inhibitors (AIs), have become the new gold standard for treatment of hormone-dependent breast cancer and the so-called ‘third generation aromatase inhibitors’ anastrozole (Arimidex), letrozole (Femara) or exemestane (Aromasin) are supplanting tamoxifen as first-line adjuvant endocrine therapy. AIs can reduce systemic oestrogen levels by as much as 98% and have been found to be superior to tamoxifen both in terms of disease-free survival and adverse side effects (Early Breast Cancer Trialists’ Collaborative Group 2015), although resistance will develop and ultimately tumour progression will occur (Dixon 2014, Reinert et al. 2017, Augusto et al. 2018). Although the use of AIs is clinically successful, and recent recommendations are to extend therapy for 5–10 years (Goss et al. 2016), such endocrine therapy is still not fully optimized and there is a need for new approaches and agents.
Dysregulation of sulphation and desulphation processes is associated with numerous pathologies (Reed et al. 2005, Mueller et al. 2015). The structural biology and enzymology of the enzymes of oestrogen metabolism have been recently reviewed (Thomas & Potter 2013). The in situ hydrolysis of the sulphated oestrone conjugate oestrone 3-O-sulphate (E1S) by steroid sulphatase (STS) has been proposed as an unexploited source of postmenopausal tumour oestrogen and was judged in the mid-1990s to be an area ripe for new drug discovery (Reed et al. 2005). In this setting, the half-life of E1S in plasma is long and levels of E1S formed via a sulphotransferase (SULT2A1) are much higher than those of E1 (Ruder et al. 1972, Noel et al. 1981, Reed et al. 2005). Circulatory E1S may thus act as a reservoir of E1 and can enter tumour cells via organic anion transporters (Mueller et al. 2015). STS converts E1S in situ in tumour cells to E1, which can then be converted to E2 by 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), thus producing the hormone in an intracrine fashion (Labrie 2015, McNamara & Sasano 2015). This may be the main route of oestrogen production in tumour cells, and levels of STS and 17β-HSD1 are reported to be higher in such tissues than in normal tissues (McNamara & Sasano 2015).
STS mRNA expression is significantly higher in malignant breast tissue and has prognostic significance, with high levels a predictor of reduced relapse-free survival (Utsumi et al. 1999, Miyoshi et al. 2003). Moreover androstenediol, a steroidal androgen with oestrogenic activity, is formed mostly via STS from androstenediol sulphate in postmenopausal women, derived from dehydroepiandrosterone sulphate (DHEAS). Although not an aromatic oestrogen, the high level of androstenediol synthesis in the postmenopausal setting nevertheless means that it can provide an additional oestrogenic stimulus at the ER (Poulin & Labrie 1986, Dauvois & Labrie 1989). Although the ER affinity of androstenediol is lower than that of E2, due to its higher serum and tissue concentrations, it may be of high importance in comparison to E2 in postmenopausal women (Spinola et al. 1986). Importantly, as there is only one STS enzyme (Purohit et al. 1994), STS inhibition, as a new therapeutic modality, should address both of these aromatase-independent pathways (Billich et al. 2000) and lead to a reduction in both the circulating androstenediol level and in in situ tumour cell E2 production, thus offering considerable new promise. Importantly, the use of AIs in postmenopausal women at risk of breast cancer, but not diagnosed, has been reported to have significant preventive action if given prophylactically (Cuzick et al. 2014). STS inhibitors might eventually also be found to exhibit such an effect.
A landmark 1994 paper (Howarth et al. 1994) reported the first, and picomolar-potent, steroidal STS inhibitor, one which possessed an aryl sulphamate pharmacophore. The overall strategic aim was to design a substituent group at the steroidal 3-phenolic position that resembled a sulphate group in structure, shape, electronics and so forth, but which would not be hydrolysed by the enzyme. Although early work explored such surrogates and indeed successfully generated useful prototype inhibitors the result was, not surprisingly, generation primarily of compounds with low micromolar and reversible inhibitory activity such as oestrone 3-O-methylphosphonothioate (Fig. 1A) (Thomas & Potter 2015a ). The key advance was the derivatization of the phenolic hydroxyl group of oestrone by a neutral sulphamate moiety, and this produced the first active site-directed irreversible inhibitors estrone 3-O-sulfamate (EMATE) (Howarth et al. 1994, Purohit et al. 1995a ) and estradiol 3-O-sulfamate (E2MATE) (Fig. 1) that were highly potent both in vitro and in vivo against STS (Purohit et al. 1995b ). The unique activity of this new compound class has since spawned hundreds of such sulphamate-based inhibitors based around the aryl sulphamate pharmacophore (Fig. 2) and founded new academic, industrial and clinical research areas. Oestrogen sulphamates also bind to carbonic anhydrase II (CA II) in red blood cells and transit the liver without first-pass metabolism (Ho et al. 2003). The underpinning pharmacophore has attractive properties, widely exploitable in drug discovery, imbuing excellent oral activity, bioavailability and pharmacokinetics (Ireson et al. 2004). A 2.6 Å X-ray crystal structure of human STS first became available in 2003 (Hernandez-Guzman et al. 2003, Ghosh 2007), illustrating a ‘mushroom-like’ structure (Fig. 1B) and confirming the presence of an active-site-hydrated formylglycine residue (FGly) that is known to be conserved across the STS family (Ghosh 2005, 2007). Mechanistically, while such phenol sulphamate ester drugs have been known to be time- and concentration-dependent inactivators of STS for some time now, and are thought to chemically modify the active site of the enzyme irreversibly (Fig. 1B for a pre-inhibition docking model with the clinical inhibitor Irosustat), the actual mechanism and site of inhibition have remained the subject of speculation (Thomas & Potter 2015a ). This review aims to survey briefly the developmental history of the STS inhibition concept and the clinical translation of the first STS inhibitors, summarize the most recent published clinical findings with future prospects and illuminate new mechanistic pointers that throw some preliminary light on how such sulphamate-based inhibitors function as irreversible STS active-site blockers.
(A) Structures of the first reversible steroidal STS inhibitor oestrone 3-O-methylthiophosphonate and the first irreversible inhibitors EMATE, oestrone 3-O-sulfamate and E2MATE estradiol 3-O-sulfamate, (B) Structure of human steroid sulphatase showing its mushroom-like shape (adapted from PDB.1P49); the arrow indicates the region of the active site and the expansion shows Irosustat (orange) docked into the active site, showing the Ca2+ ion depicted as a yellow sphere, FG75 as the gem-diol form of the catalytic FGly residue and illustrating the proximity of the sulphamate group to the FGly residue. Dotted line is a putative H-bond.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
(A) Structures of the first reversible steroidal STS inhibitor oestrone 3-O-methylthiophosphonate and the first irreversible inhibitors EMATE, oestrone 3-O-sulfamate and E2MATE estradiol 3-O-sulfamate, (B) Structure of human steroid sulphatase showing its mushroom-like shape (adapted from PDB.1P49); the arrow indicates the region of the active site and the expansion shows Irosustat (orange) docked into the active site, showing the Ca2+ ion depicted as a yellow sphere, FG75 as the gem-diol form of the catalytic FGly residue and illustrating the proximity of the sulphamate group to the FGly residue. Dotted line is a putative H-bond.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
(A) Structures of the first reversible steroidal STS inhibitor oestrone 3-O-methylthiophosphonate and the first irreversible inhibitors EMATE, oestrone 3-O-sulfamate and E2MATE estradiol 3-O-sulfamate, (B) Structure of human steroid sulphatase showing its mushroom-like shape (adapted from PDB.1P49); the arrow indicates the region of the active site and the expansion shows Irosustat (orange) docked into the active site, showing the Ca2+ ion depicted as a yellow sphere, FG75 as the gem-diol form of the catalytic FGly residue and illustrating the proximity of the sulphamate group to the FGly residue. Dotted line is a putative H-bond.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Structures of the aryl O-sulphamate pharmacophore (top, X, Y and Z are substituent groups), 4-methylcoumarin 7-O-sulphamate (R = H), 3,4-dimethylcoumarin 7-O-sulfamate (R = CH3) and the tricyclic series of coumarin-based STS inhibitors; n = 5 was chosen as optimal for the clinical candidate.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Structures of the aryl O-sulphamate pharmacophore (top, X, Y and Z are substituent groups), 4-methylcoumarin 7-O-sulphamate (R = H), 3,4-dimethylcoumarin 7-O-sulfamate (R = CH3) and the tricyclic series of coumarin-based STS inhibitors; n = 5 was chosen as optimal for the clinical candidate.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Structures of the aryl O-sulphamate pharmacophore (top, X, Y and Z are substituent groups), 4-methylcoumarin 7-O-sulphamate (R = H), 3,4-dimethylcoumarin 7-O-sulfamate (R = CH3) and the tricyclic series of coumarin-based STS inhibitors; n = 5 was chosen as optimal for the clinical candidate.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Clinical STS inhibitor development
With the discovery of EMATE and its 17-keto group-reduced form estradiol 3-O-sulfamate (E2MATE, also known as J995) (Fig. 1) as the first potent and irreversible STS inhibitors, the scene was apparently quickly set for the translation of such a steroidal sulphamate ester drug into clinical trials in hormone-dependent breast cancer. However, surprisingly, this class of compound was found to be highly oestrogenic in rodents and thus unsuitable as an anticancer therapeutic. This was thought to be because of its activity as a pro-drug for E1 or E2, respectively, with STS cleaving the sulphamate group (Elger et al. 1995) and stimulated the active search for a non-steroidal, non-oestrogenic STS inhibitor. Because this oestrogenicity of the steroidal sulphamate series effectively precluded applications in oncology (although it provided the stimulus for other clinical work vide infra), a search for an orally active, non-steroidal, non-oestrogenic, Lipinski rule-compliant STS inhibitor was undertaken, primarily based upon using steroidal A/B ring surrogates. A first lead was the two-ring coumarin aryl sulphamate, 4-methylcoumarin-7-O-sulphamate (Fig. 2), which was orally active in vivo and also like EMATE a highly potent time- and concentration-dependent STS inhibitor, but with no rodent estrogenic activity (Woo et al. 1996, Purohit et al. 1996). The related 3,4-dimethylcoumarin-7-O-sulphamate (Fig. 2) inhibited STS in MCF-7 cells with IC50 = 30 nM, and a series of derived tricyclic compounds was subsequently synthesized that proved even more potent (Woo et al. 2011a ) (Fig. 2). The synthetic route comprising only two steps was very straightforward; a Pechmann reaction between resorcinol and the corresponding cyclic β-ketoester, followed by a sulphamoylation of the coumarin phenol, made them attractive candidates for industrial scale-up. The greater activity of these non-steroidal compounds relative to the steroidal parent was attributed to the enhanced ‘sulphamoyl transfer potential’ as a result of the lower phenolic pKa (Woo et al. 2011a ), the idea being that STS is irreversibly modified through sulphamoylation of the active site. Of these, STX64 (667 COUMATE), also subsequently known as BN83495 and now more commonly known as Irosustat (Fig. 3), was selected for extensive in vivo studies.
Structures of the non-steroidal STS inhibitor Irosustat (top) and an example of a member of the tricyclic series in folded and ionized format (bottom right), showing its similarity to the steroidal oestrone 3-O-sulphate (left).
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Structures of the non-steroidal STS inhibitor Irosustat (top) and an example of a member of the tricyclic series in folded and ionized format (bottom right), showing its similarity to the steroidal oestrone 3-O-sulphate (left).
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Structures of the non-steroidal STS inhibitor Irosustat (top) and an example of a member of the tricyclic series in folded and ionized format (bottom right), showing its similarity to the steroidal oestrone 3-O-sulphate (left).
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
STX64/Irosustat was orally very active in vivo with a bioavailability of 95%. Single or multiple oral doses inhibited rat liver STS by >90%, prevented the growth of rodent nitrosomethyl-urea-induced mammary tumours and were very effective at inhibiting the growth of human breast cancer xenografts implanted in nude mice (Thomas & Potter 2015a ). The drug passed pre-clinical toxicological testing (Stanway et al. 2006). In plasma ex vivo, the drug was quickly degraded, but in vivo this is prevented by its sequestration inside red blood cells, where it binds to CA II (Ireson et al. 2004). Irosustat was co-crystallized with CA II (Lloyd et al. 2005, PDB 1TTM) and the complex was studied by X-ray crystallography. This stability and facilitation of transport to tissues may account for the high bioavailability of the drug. Moreover, the sequestration allows the drug to evade first-pass metabolism. Members of the non-steroidal tricyclic STX64/Irosustat drug class can be visualized with the aliphatic third ring in a C/D-like folded fashion to show how they could potentially mimic a steroid structure in binding to STS (Thomas & Potter 2015a ) (Fig. 3). Sulphamate esters, like sulphonamides, are weak acids with the first pKa in the 7–9 region, and there will thus be a significant amount present in the anionic form at physiological pH, thus making the drug a good mimic of the normally charged STS substrate. Importantly, however, a sulphamate ester can also be present as the neutral form which will aid permeability, tissue penetration and presumably its reversible sequestration into red blood cells. Thus, it is clear how Irosustat has all the structural properties to bind effectively to STS, an enzyme designed to bind a steroidal structure possessing a charged phenolic sulphate group. More extensive details of the background and development of Irosustat and the wider STS inhibitor classes are provided elsewhere (Thomas & Potter 2015a ,b ).
Clinical studies
STS inhibition has so far been studied using two sulphamate-based drugs in 19 international human clinical trials in 5 distinct pathologies. Positive clinical effects of Irosustat and E2MATE/J995 have been established with the underpinning academic science validated. These trials are summarized in the following section:
Hormone replacement therapy
Despite its unexpected oestrogenic activity, a steroidal sulphamate was nevertheless the first aryl-sulphamate-based drug to enter human clinical trials. Innovatively, the absence of hepatic oestrogenicity/production of clotting factors for this class of compound reduces risks of adverse events for applications in hormone replacement therapy/oral contraception. The orally active steroidal super-oestrogen E2MATE or J995 (Fig. 1), that was five times more oestrogenic than ethinylestradiol when administered orally in the rat (Elger et al. 1995), thus reached multiple clinical trials with >170 healthy women dosed in six phase I and II studies in a hormone replacement therapy setting. The drug was very safe and well tolerated, and the role of STS in activating the sulphamate derivative as a pro-drug was firmly established (Chander et al. 2004). However, oestrogenic activity was subsequently significantly limited in humans by metabolic deactivation at C-17, most likely in the gut, and this issue needs to be overcome for further development of the idea (Elger et al. 2017).
Breast cancer
With the overall rationale for STS inhibition established in pre-clinical studies (Reed et al. 2005, Thomas & Potter 2015a ) and the design of the tricyclic non-steroidal STS inhibitor STX64/Irosustat successfully achieved, the way was clear for the drug to enter a ‘first in class’ clinical trial, performed initially in an academic setting. Irosustat was administered orally to fourteen postmenopausal women with advanced breast cancer (9 patients at 5 mg and 5 at 20 mg dose) as an initial dose followed a week later by three 2-weekly cycles of 5-day dosing and 9 days off treatment. The drug had a pharmacokinetic profile suitable for daily dosing and was established as clinically potent and well tolerated with only minor adverse events. The median inhibition of STS activity was 98% in peripheral blood lymphocytes and 99% in biopsied breast tumour tissue at the end of the 5-day dosing period. Serum concentrations of steroids with oestrogenic properties, that is oestrone, oestradiol and especially androstenediol, as well as dehydroepiandrosterone (DHEA), all decreased significantly from their pre-treatment levels and, surprisingly, androstenedione and testosterone concentrations also decreased. Four patients, previously progressing on AIs, encouragingly showed evidence of stable disease for 2.75–7 months (Stanway et al. 2006).
In a subsequent phase I clinical study (Coombes et al. 2013) using a new solid tablet formulation to determine the optimal biological dose, a three-part, open-label, multicentre, dose escalation study of Irosustat in ER+ breast cancer patients was performed. A single oral dose of the drug was administered with a 7-day observation period, followed by a daily dose for 28 days and an extension phase, in which dosing was continued discretionally if the patient was seen to benefit. Five doses of Irosustat were tested up to 80 mg in fifty patients. After 28 days of daily administration, all evaluated patients in the 5-, 20-, 40- and 80-mg cohorts achieved ≥95% STS inhibition in peripheral blood mononuclear cells and corresponding endocrine suppression. The maximum tolerated dose was not reached and the 40-mg dose was established as optimal. The median time to progression in the 40-mg cohort was 11.2 weeks. Biopsy-validated erythematous skin infiltration in one patient was no longer visible after one month of treatment. Disease stabilization is often taken to be a reliable indicator of effectiveness of a novel therapy, particularly endocrine therapy; five patients of this heavily pre-treated patient population (10%) remained progression-free for at least 24 weeks (33.1 weeks in one patient receiving 20 mg, 72.3, 28.4 and 27.1 weeks in three patients receiving 40 mg and 30.7 weeks in one patient receiving 80 mg), potentially indicative of drug activity. Dry skin, the most frequent adverse event, was easily managed. This study provided clinical proof of concept that STS is inhibited by Irosustat in patients with an effective suppression of peripheral steroid hormones. It was noted that a larger study is required to define an accurate response rate to the drug as a single agent and also whether co-administration with an aromatase inhibitor might potentially be more effective (vide infra). Further details of other phase II studies are yet to be reported.
In clinical trials, such as those mentioned earlier, the postmenopausal subjects have normally already been heavily pre-treated and thus may have various resistance pathways activated. One of the most recent clinical studies reported in breast cancer is the ‘IPET’ study, which was a pre-surgical window-of-opportunity study to assess Irosustat for the first time in ER+ early breast cancer patients (Palmieri et al. 2017a ). The aims of this trial were to assess the effect of Irosustat on tumour cell proliferation in treatment-naïve subjects, as measured by 3′-deoxy-3′-[18F] fluorothymidine (FLT) uptake monitored via positron emission tomography (PET) scanning (FLT-PET) and levels of the tumour proliferation marker Ki67. Postmenopausal women with untreated early breast cancer were recruited and treated orally for at least 2 weeks with Irosustat at 40 mg/day. Tumours were imaged with FLT-PET at baseline and after drug treatment, with the primary endpoint being changes in FLT uptake; secondary endpoints included safety and tolerability, changes in tumoural Ki67, circulating steroid hormone levels and expression of steroidogenic enzymes. Of thirteen women recruited, ten started Irosustat for 2 weeks, followed by repeat FLT-PET scans for eight women. The drug was generally well tolerated with all adverse events < Grade 2. STS decreases were seen in tumours with high basal STS expression and significant decreases were also noted in aromatase and 17β-HSD types 1 and 2. With the definition of a response being decreases of ≥20% in standardized uptake value or ≥30% in Ki67, one and three patients responded, respectively. Six out of seven patients had a Ki67 reduction with the median percentage difference being 52.3%. Irosustat treatment thus resulted in significant reductions in FLT uptake and Ki67 and was well tolerated. Baseline expression of STS was judged to be a potential biomarker of sensitivity to Irosustat and may facilitate future prior patient stratification. Importantly, these data are the first to demonstrate clinical activity of Irosustat in early breast cancer, albeit in a rather small patient population. Encouragingly also, data are broadly comparable to those from tamoxifen in the same setting and to phase III data for the well-established drugs exemestane and fulvestrant. However, patient recruitment in this single-centre pre-surgical population was challenging, and larger studies are now required to build upon these results.
Inhibition of aromatase lowers oestrogen levels and is an effective endocrine therapy for many breast cancers. However, it is to be expected that chronic treatment with an AI will lead to the development of compensatory mechanisms to increase E2 levels: indeed, a report (Chanplakorn et al. 2010) showed that the administration of an aromatase inhibitor to breast cancer patients increased levels of both STS and 17β-HSD1, leading to increased E2 levels. Moreover, a recent study also demonstrated the contribution of both STS and organic anion transporters in E1S metabolism to the proliferation of AI-resistant breast cancer cells and AI resistance (Higuchi et al. 2016). Letrozole-resistant cell lines were established that had higher levels both of STS mRNA and organic anion transporters. Such cells proliferated more in an E1S-supplemented medium, and this was effectively inhibited by Irosustat in combination with the AI Letrozole. This model was also supported by analysis of ER+ primary breast cancer tissues. The administration of an STS inhibitor should, as mentioned earlier, address the in situ intracrine production of oestrogen (and importantly including circulating androstenediol) from the sulphate by STS in tumour cells and, moreover, alongside an AI should lower oestrogen levels further in a multi-targeting fashion. In addition, the development of ESR1 mutations represent another resistance mechanism to AIs (Wang et al. 2016, Reinert et al. 2017). In some tumours, the rarer ESR1-mutant clones are enriched by endocrine therapy. Implications here for STS inhibition therapy are as yet unknown.
Another recent clinical study, reported in 2017, was the ‘IRIS’ study (Palmieri et al. 2017b ). This multicentre, open-label, phase II trial explored the clinical value of adding an STS inhibitor in addition to a first-line AI in patients with advanced breast cancer. This was both to evaluate the safety of the combination and to test the hypothesis that the addition of Irosustat to an AI may further suppress E2 levels and result in clinical benefit. Postmenopausal women with ER+ locally advanced or metastatic breast cancer who had benefited from a first-line AI but were subsequently progressing were enrolled. The first-line AI was continued and 40 mg/day oral Irosustat was added. The primary endpoint was clinical benefit rate (CBR) and secondary endpoints included safety, tolerability and pharmacodynamics. The study was judged to have met its pre-defined success criterion by both local and central radiological assessments. Twenty-seven women were recruited, although four discontinued treatment. Based on local reporting, the CBR was 18.5% on an intent to treat (ITT) basis, increasing to 21.7% by per-protocol analysis. In those five patients that achieved clinical benefit, the median duration was 9.4 months and the median progression-free survival time was 2.7 months in both the ITT and per-protocol analyses. The most frequent adverse effects were < grade 2, with the most common one being dry skin, as expected. Encouragingly, the addition of Irosustat to standard aromatase inhibitor therapy thus resulted in clinical benefit with an acceptable safety profile.
The objective response and CBRs of endocrine treatments used second-line are clearly limited, and results should be seen in this context. It is also important to emphasize that the newer targeted mTOR and CDK4/6 inhibitors that are showing current clinical success in breast cancer are used in concert with endocrine therapy (Hortobagyi et al. 2016); the efficacy of the former will be dependent upon optimization of the latter that could perhaps be enhanced further through, for example an STS inhibitor and AI combination. Further studies are, however, needed to explore the clinical activity of targeting both aromatase and STS, ideally as a first-line metastatic treatment in patients likely to be endocrine sensitive. Moreover, it would also be most desirable to enrich any future patient cohort in those that can be measurably selected for high tumour STS expression. Additionally, another innovative approach of future potential building on these results is that enshrined in the idea of dual aromatase-sulphatase inhibition, using a single agent (vide infra).
Endometrial cancer
Endometrial cancer, developing from the inner uterine lining, is the most common cancer found in the female reproductive system. Malignant endometrial tissue has a high STS activity and advanced/metastatic or recurrent endometrial cancer has a poor prognosis. STS was judged to be a good novel drug target for this indication (Foster et al. 2008a ). Despite progress in the treatment of early endometrial cancer, the advanced disease remains incurable and there is a strong need for new agents. STS was evaluated as a therapeutic target in patients with endometrial cancer via a phase II multicentre (11 European countries), randomized, 2-arm study of Irosustat in women with advanced/metastatic or recurrent oestrogen-receptor-positive endometrial cancer (Pautier et al. 2017). The optimal oral dose of 40 mg/day was used in comparison with the current standard of care, oral megestrol acetate (MA), at 160 mg/day. The primary endpoint was the proportion of patients without progression or death at 6 months and secondary endpoints included progression-free survival, time to progression, overall survival and safety. Thus, seventy-one postmenopausal patients with histologically verified oestrogen and/or progesterone-receptor-positive endometrial cancer with recurrent/advanced disease were treated; thirty-six received Irosustat and thirty-five MA. Results showed clinical activity and a good safety profile for Irosustat, with 36% of patients on Irosustat alive without progression at 6 months; 11% showed responses and there was more stable disease noted (47%) compared to the current therapy (32%). However, while overall there were no statistically significant differences between Irosustat and MA in response and survival rates, the study was terminated early after futility analysis. Overall, 36.1% and 54.1% of patients receiving Irosustat or MA had not progressed or died at 6 months, respectively. Irosustat patients had a median progression-free survival of 16 weeks vs 40 weeks for MA-treated patients. The drug was well tolerated. Mainly Grade 1 or 2 treatment-related adverse events occurred in 55.6% and 37.1% patients receiving Irosustat or MA, respectively.
Thus, while Irosustat monotherapy did not demonstrate activity sufficient for further commercial development in patients with advanced/recurrent endometrial cancer, the study confirmed the clinical benefit of the drug and further validated the underpinning scientific rationale of targeting STS. However, in a recent study to determine STS expression in endometrial cancer patients and its potential prognostic significance (Lee et al. 2016), it was found that STS expression is not significantly associated with disease-free survival and overall survival, despite positive STS expression in 27% of endometrial cancer patients. Thus, STS as a prognostic factor in endometrial cancer patients requires more analysis.
Prostate cancer
Androgen-dependent prostate cancer represents a very large unmet medical need. STS activity has been detected in prostate tissue and in LNCaP prostate cancer cells, suggesting that, like in the breast, STS could facilitate intracrine production of hormones to stimulate tumour growth from a precursor source (Day et al. 2009) and this was also explored with the steroidal STS inhibitor EM-213 (Roy et al. 2013). DHEAS is present at plasma concentrations up to 500 times higher than testosterone and can potentially be delivered into prostate cancer cells via organic anion transporters and converted via endogenous STS into desulphated DHEA and then into testosterone by hydroxysteroid dehydrogenases and into dihydrotestosterone (DHT) via 5α reductase. In the prostate, adrenal androgens can be converted into testosterone and DHT, driving tumour growth, analogously to the in situ cleavage of E1S in breast tumours. The effect of Irosustat on hormone levels in patients suffering from castration-resistant prostate cancer with ongoing androgen deprivation therapy was studied in the first clinical trial of an STS inhibitor in men through a US phase I dose escalation study (Denmeade et al. 2011). This was conducted in seventeen chemo-naïve, castration-resistant patients with evidence of disease progression following 28 days of oral Irosustat administration. The aims were evaluation of safety, tolerability, pharmacokinetic, pharmacodynamic and endocrine parameters (plasma DHEA: DHEAS ratio, inhibition of androstenediol, androstenedione and testosterone) consequent upon STS inhibition, with dosing cohorts at 20, 40 and 60 mg. Pharmacodynamic proof of concept was demonstrated and in all patients; there was notable suppression of the non-sulphated androgens testosterone, androstenediol and DHEA. The DHEA:DHEAS ratio was significantly decreased. Effects were slightly better at the two higher doses. Importantly, Irosustat effected nearly complete STS inhibition at all three doses, and the drug was well tolerated at all doses, with dry skin being the most common related adverse event. No peer-reviewed publication has, however, yet appeared from this study. The results also provide a basis for potential future trials in those patients who have demonstrated resistance to the CYP17A1 inhibitor Abiraterone (Zytiga) and who may potentially be employing STS-mediated conversion of sulphated androgens such as DHEAS to contribute to the resistance phenotype and drive tumour growth.
Since prostate cancer may also be associated with high plasma oestrogen exposure, an in vivo rat study examined the effects of STX/64/Irosustat to explore whether E1S uptake in the prostate increases during ageing. If applicability to humans is valid, the protective results seen with the drug support the idea in principle of reducing STS activity in older men with high E1S plasma levels (Giton et al. 2015).
Endometriosis
Another clinical target, the poorly understood gynaecological disease endometriosis, is also hormone-driven and leads to chronic pelvic pain and infertility. This benign disease is characterized by the presence of endometrial tissue outside the uterus. With an estimated 80 million patients worldwide, the disease is still poorly understood; most treatments have unpleasant side effects and current therapies are inadequate (Vercellini et al. 2014). Discovery of new drugs is thus a pressing unmet need. In addition to production of oestrogens in the ovaries, there is compelling evidence that local synthesis of oestrogens in endometriotic lesions promotes progression of the disease and resistance to endocrine therapy (Tosti et al. 2016). The first rationale for STS inhibition in endometriosis was devised (Purohit et al. 2008) and led directly to a phase I study using the steroidal drug E2MATE (PGL2001) to explore a new treatment option for this disease. In such an application not directed at the postmenopausal setting, it is necessary to employ combination of an STS inhibitor with a progestin such as norethindrone acetate (NETA) to reduce ovarian oestrogen production and the STS inhibitor would also decrease local oestrogen production. A randomized, double-blind and placebo-controlled phase I study was used to investigate the pharmacodynamics, pharmacokinetics and safety of the E2MATE (PGL2001) and NETA combination (Pohl et al. 2014). Twenty-four healthy women of reproductive age were treated with weekly doses of the STS inhibitor or daily doses of NETA or a combination of both compounds for 4 weeks. Such treatment reduced STS activity in the endometrium by 91% and 96%, respectively, with comparable values observed 1 month after the cessation of treatment. The combined drug treatment led to significantly higher STS inhibition at both times.
This study showed that administration of the sulphatase inhibitor either alone at 4 mg/week or with the progestin to healthy pre-menopausal women led to STS inhibition and changes in functional endometrium STS biomarkers, resulting in synergistic effects of the combination on STS activity. The proof-of-principle combination was thus well tolerated. Double-blind, multicentre, placebo-controlled, phase II clinical trials have been in progress since 2012 to investigate efficacy, safety, pharmacokinetics and pharmacodynamics in Hungary, Poland and Romania. One potential disadvantage of these current studies is however, that, as described earlier, E2MATE possesses residual oestrogenic activity which, although reduced in humans, would still seem non-applicable to this indication, with the ideal overriding preference being for use of a pure STS inhibitor such as Irosustat, which should be a future aim. Publication of detailed clinical results is awaited.
Other potential clinical targets for STS inhibition are emerging. For example, oestrogens affect the incidence and progression of colorectal cancer (CRC) that exhibits dysregulated oestrogen metabolism with E2 synthesis favoured, but molecular mechanisms are still unclear. In a recent study (Gilligan et al. 2017), STS activity was shown to be significantly elevated in human CRC and STS over-expression accelerated CRC proliferation in vitro and in vivo. STS inhibition using Irosustat can block this effectively, and future clinical studies may be indicated. Ovarian cancer is also an attractive possibility as a clinical target, and Irosustat was shown to inhibit the growth of the ER+ ovarian cancer OVCAR-3 cell line (Day et al. 2009). In a study of specimens from thirty-seven patients with advanced stage ovarian cancer, it was investigated whether a clinical correlation existed between STS activity and survival (Chura et al. 2009). STS activity is widely present in specimens of ovarian cancer. Median progression-free survival was 23.5 months for patients with low STS activity compared to 6.9 months for patients with high STS activity. Increased STS activity is associated with worse progression-free survival in patients with advanced stage disease, and the STS pathway is thus a potential therapeutic target in the treatment of ovarian cancer. A recent study has suggested that in the most frequent and lethal type of the disease, advanced high-grade serous epithelial ovarian cancer, SULT1EI expression is significantly associated with better survival and targeting the STS pathway should show clinical benefit (Mungenast et al. 2017). Intracrine activation of precursors and the use of more stratified patient population as well as combination therapy, including dual inhibitors (vide infra) in, for example, ovarian cancer was stressed in a recent comprehensive review (Rižner et al. 2017). There are also interesting preliminary implications for STS involvement in bladder cancer (Eri et al. 2014).
Mechanism of action
Sulphatase enzymes are derived from an evolutionarily highly conserved gene family. While they catalyse sulphate group cleavage from a wide variety of substrates including glucosaminoglycans, glycolipids, amino acids and hydroxysteroids, the various enzymes have similar folds, active-site architecture, mechanisms of action and bivalent metal ion binding sites (Ghosh 2005, 2007). In 2003, the 3D structure of human STS became available (Hernandez-Guzman et al. 2003). Here, as in eukaryotic sulphatases, a critical formylglycine FGly residue is generated by post-translational modification of an active-site cysteine residue; in some bacteria, this is by modification of a serine residue (Marquordt et al. 2003). This FGly, in its hydrated form as a gem-diol, is thought to be generally involved in the catalytic mechanism of all such sulphatases and catalytic mechanisms have been developed with the aid of structural biology (Lukatela et al. 1998, Boltes et al. 2001, von Bülow et al. 2001). While a mechanism for STS-mediated cleavage is now generally well established, with nucleophilic attack on the sulphated phenolic substrate by a hydroxyl of the FGly gem-diol leading to an intermediate pro-R gem-diol sulphate adduct, that can sometimes be observed crystallographically in the resting state, this does not yet hold for STS inhibition by aryl sulphamates, the mechanism of which is still controversial. It was shown some time ago that aryl sulphamates act as irreversible active-site-directed STS inhibitors, working in a time- and concentration-dependent fashion (Purohit et al. 1995a). However, little progress has been made over the years in firming up an inhibitory mechanism, even though proposals have been made and compounds such as Irosustat have now proceeded to many clinical trials (Thomas & Potter 2015a ).
A feasible working hypothesis adopted for the mechanism of action of aryl sulphamates in STS inactivation is generally that the sulphamoyl group is transferred to the enzyme active site in some fashion and that this destroys activity irreversibly. Several mechanisms have been postulated, although none is conclusively proven. Early work suggested, given the several active-site lysine and histidine residues flanking the FGly residue that lysine or histidine sulphamide-type adducts might be the end product of inhibition (Woo et al. 2000). FGly, both in its hydrated or aldehydic form is clearly another obvious target for irreversible modification. Using the STS crystal structure docking experiments indicated that the likely orientation of the sulphamate group of Irosustat and related tricyclic sulphamates in the active site is in close proximity to the crucial hydrated FGly residue (Fig. 1B), further suggesting that, as a good steric and electronic sulphate mimic, it might be transferred to this residue during inhibition (Woo et al. 2011a ). Indeed, it has been noted that there is a qualitative similarity in the reaction coordinates for bimolecular sulphamoyl and sulphuryl transfer reactions of sulphamate and sulphate monoesters and thus a close mechanistic relationship for sulphamoyl and sulphuryl group transfer (Denehy et al. 2006). The idea that inhibition of STS is facilitated by some kind of sulphamoyl group transfer to the active site is still the currently accepted mode of action for such drugs, even if the precise transfer mechanism and any subsequent consequences for the active-site machinery are as yet unknown.
Working with human STS is difficult for mechanistic purposes; it is membrane bound and can be heterogeneously glycosylated. By contrast, the soluble bacterial Pseudomonas aeruginosa arylsulphatase (PaAtsA) also catalyses the cleavage of aryl sulphates and is an excellent model for human oestrone sulphatase and the enzyme has been crystallised (Boltes et al. 2001, PDB 1HDH). Valuable experimental insight into the mechanism of sulphatase inactivation by sulphamates was provided via this model. The inactivation of Pseudomonas aeruginosa arylsulphatase A by a range of aryl sulphamates was studied, including the clinical Irosustat (Bojarová et al. 2008). Inactivation was found to be similarly time-dependent, irreversible and active-site-directed, consistent with a covalent modification at the active site. The transition state for the first irreversible chemical step of inactivation was shown to involve a high degree of charge transfer and cleavage of the ArO-S bond. Interestingly, however, the stoichiometry of inactivation was established as in the range of 3–6, with the highest value found for the most effective inactivators. Thus, it was concluded that multiple sulphamoylation events may occur during the inactivation process. In a recent extensive physical-organic extension of this work (Williams et al. 2014), reversible sulphamoylation of the active-site lysines and histidines was invoked to explain this surprising stoichiometry. Attempts to shed light on the irreversible adduct(s) using mass spectrometry were reported to be unsuccessful, but an N-imine sulphate FGly adduct was nevertheless proposed to be the most likely end product of inhibition.
In more recent work in our group, it has proved possible to inactivate PaAtsA with Irosustat until there is no more activity measurable and subsequently to crystallize the inactivated protein, that could then be studied using structural biological techniques (G Cozier, C Martinez-Fleites, G Davies & B V L Potter, unpublished work). The preliminary results are shown in Fig. 4. Interestingly, it can be seen that there is new covalent electron density apparently at the pro-R hydroxyl of the hydrated FGly gem-diol (but see also the figure legend for a stereochemistry caveat). While this density cannot be unambiguously assigned as yet at the resolution achieved, it nevertheless seems to be located at the pro-R hydrated FGlyS site seen generally to be sulphated in the resting form of sulphatase enzymes (Fig. 5), and which provides some encouragement in favour of a sulphate-mimicking sulphamoyl transfer inhibitory mechanism. Importantly, however, there are no covalent His or Lys modifications as previously suggested, both originally (Woo et al. 2000) and in the later inactivation experiments (Bojarová et al. 2008), but just an apparently covalently-modified FGly residue. With this result, it is possible to construct tentatively theoretical inactivation possibilities with both direct sulphamoyl group transfer leading to either an O-linked hydrated FGly product I (and possible interference blocking the next catalytic step perhaps as a result of new H-bonding possibilities provided by the amino group of the sulphamate or disturbed metal ion interactions) or potentially to an N-linked product II formed via interaction of the sulphamate nitrogen with an unhydrated FGly, analogous to the synthetic reaction noted of a sulphamate ester anion with dimethylformamide (Woo et al. 1998, Williams et al. 2014). The involvement of an electrophilic sulphonylamine in this process (Thea et al. 1986, McCaw & Spillane 2006), either singly or doubly charged, and ‘free’ or otherwise, could be invoked in both possibilities and is a compelling mechanistic prospect.
The X-Ray crystal structure of the active site region of inactivated PaAtsA. Active Pseudomonas aeruginosa arylsulphatase A (PaAtsA) was inactivated by incubating the enzyme at room temperature for ca. 10 min with a ca. 10-fold molar excess of Irosustat/STX64, as monitored by using a 4-nitrocatechol sulphate assay. The enzyme was shown to be completely inactivated. The hanging drop vapour diffusion method was used for protein crystallization. Inactivated enzyme was mixed with well buffer and after ca. 2 weeks at room temperature, diamond-shaped crystals formed in the C2 space group, which diffracted to 2.0 Å. Two subunits were observed in the unit cell with differences at FGly between them. The structure for the Irosustat-inactivated enzyme showed only a single, apparently covalent, modification at FGly in both subunits, with no modification of nearby flanking Lys or His residues. The structure showed an area of new density (marked by the yellow arrow) in the active site close to the hydrated FGly and calcium in one subunit that was best modelled as a tetrahedral covalent attachment to the pro-R hydroxyl group of FGly (possibly with a small degree of dual occupancy); and in the other subunit (not shown), the density seemed to be more planar in character, perhaps suggestive of a potential two-step inactivation process (Fig. 6A). In the former case, while both pro-R and pro-S stereochemistry for FGly (I) can be modelled into the density, the pro-R stereochemistry had the best fit, as illustrated.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
The X-Ray crystal structure of the active site region of inactivated PaAtsA. Active Pseudomonas aeruginosa arylsulphatase A (PaAtsA) was inactivated by incubating the enzyme at room temperature for ca. 10 min with a ca. 10-fold molar excess of Irosustat/STX64, as monitored by using a 4-nitrocatechol sulphate assay. The enzyme was shown to be completely inactivated. The hanging drop vapour diffusion method was used for protein crystallization. Inactivated enzyme was mixed with well buffer and after ca. 2 weeks at room temperature, diamond-shaped crystals formed in the C2 space group, which diffracted to 2.0 Å. Two subunits were observed in the unit cell with differences at FGly between them. The structure for the Irosustat-inactivated enzyme showed only a single, apparently covalent, modification at FGly in both subunits, with no modification of nearby flanking Lys or His residues. The structure showed an area of new density (marked by the yellow arrow) in the active site close to the hydrated FGly and calcium in one subunit that was best modelled as a tetrahedral covalent attachment to the pro-R hydroxyl group of FGly (possibly with a small degree of dual occupancy); and in the other subunit (not shown), the density seemed to be more planar in character, perhaps suggestive of a potential two-step inactivation process (Fig. 6A). In the former case, while both pro-R and pro-S stereochemistry for FGly (I) can be modelled into the density, the pro-R stereochemistry had the best fit, as illustrated.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
The X-Ray crystal structure of the active site region of inactivated PaAtsA. Active Pseudomonas aeruginosa arylsulphatase A (PaAtsA) was inactivated by incubating the enzyme at room temperature for ca. 10 min with a ca. 10-fold molar excess of Irosustat/STX64, as monitored by using a 4-nitrocatechol sulphate assay. The enzyme was shown to be completely inactivated. The hanging drop vapour diffusion method was used for protein crystallization. Inactivated enzyme was mixed with well buffer and after ca. 2 weeks at room temperature, diamond-shaped crystals formed in the C2 space group, which diffracted to 2.0 Å. Two subunits were observed in the unit cell with differences at FGly between them. The structure for the Irosustat-inactivated enzyme showed only a single, apparently covalent, modification at FGly in both subunits, with no modification of nearby flanking Lys or His residues. The structure showed an area of new density (marked by the yellow arrow) in the active site close to the hydrated FGly and calcium in one subunit that was best modelled as a tetrahedral covalent attachment to the pro-R hydroxyl group of FGly (possibly with a small degree of dual occupancy); and in the other subunit (not shown), the density seemed to be more planar in character, perhaps suggestive of a potential two-step inactivation process (Fig. 6A). In the former case, while both pro-R and pro-S stereochemistry for FGly (I) can be modelled into the density, the pro-R stereochemistry had the best fit, as illustrated.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Top, STS active–site-hydrated formylglycine FGly residue (left) and proposed N-imine sulphate end product III (right); bottom, the sulphated hydrated FGlyS resting form (left) and the suggested pro-R O- I and N-linked FGly II modifications (middle and right, respectively) derived from the preliminary crystallographic study of Pseudomonas aeruginosa arylsulphatase inactivated by Irosustat.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Top, STS active–site-hydrated formylglycine FGly residue (left) and proposed N-imine sulphate end product III (right); bottom, the sulphated hydrated FGlyS resting form (left) and the suggested pro-R O- I and N-linked FGly II modifications (middle and right, respectively) derived from the preliminary crystallographic study of Pseudomonas aeruginosa arylsulphatase inactivated by Irosustat.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Top, STS active–site-hydrated formylglycine FGly residue (left) and proposed N-imine sulphate end product III (right); bottom, the sulphated hydrated FGlyS resting form (left) and the suggested pro-R O- I and N-linked FGly II modifications (middle and right, respectively) derived from the preliminary crystallographic study of Pseudomonas aeruginosa arylsulphatase inactivated by Irosustat.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
The new crystallographic results, although only preliminary, have implications for the recent inactivation model proposed (Williams et al. 2014). Here, a range of potential end products of STS inactivation by aryl sulphamates was evaluated theoretically, employing physical-organic considerations. The most favoured irreversible adduct was proposed to be an imine N-sulphate III (Fig. 5). While it is certainly not possible from the present preliminary crystallographic data to distinguish between N- and O-linked FGly covalent adducts I and II, respectively (Fig. 5), it seems nevertheless apparent from the new electron density observed that the steroidal phenol motif is absent, which is not too unexpected, and that the adduct comprising the new electron density is likely linked to the pro-R hydroxyl group. The other pro-S hydroxyl group, however, is still clearly present and this therefore seems to rule out the possibility that the adduct is the proposed imine N-sulphate III. Therefore, it is proposed on this basis that the best candidates for the final irreversible adduct are the two O- and N-linked FGly adducts (Fig. 5). One should note, of course, that an N-imine sulphate moiety could be reached by dehydration of the N-linked FGly adduct II proposed. With no evidence for retention of the parent phenolic group in the adduct, a dead-end sulfonimine, however, formed (Hanson et al. 2006, Woo et al. 2000), or any other putative adducts with the phenolic group still in place (Woo et al. 2000, Williams et al. 2014), a double-sulphamoylated hydrated FGly (Bojarová et al. 2008, Williams et al. 2014) or even an irreversible internal Schiff base (Hanson et al. 2006) can all now essentially be excluded.
Here is, not the place, however, for extensive discussion of the various mechanistic possibilities that are viable to reach either of these irreversible adducts and that have to some extent already been rehearsed elsewhere, but this new emphasis on the two most likely candidates should now at least make it possible to refine and test the possibilities in a more focused fashion experimentally. Nevertheless, given the lack of progress in this area over very many years, some outline suggestions are worthy of mention (Fig. 6) with brief discussion. In mechanism A, the sulphamate drug is deemed to bind to the catalytically non-productive FGly aldehyde form of the enzyme. Potential electrophilic activation of the phenol-sulphur bond by the enzyme leads to a well-precedented elimination of sulphonylamine (Thea et al. 1986, McCaw & Spillane 2006), that is more stable than the corresponding sulphur trioxide in sulphuryl transfer (Williams et al. 2014) and that then can attack the FGly keto group, in principle from either of its two faces. The resulting planar and electrophilic adduct is trapped by water ingress to give the N-linked FGly sulphamate covalent adduct II. Note the ambiguous stereochemistry at the adduct; such a possibility, to the best of our knowledge, has not been discussed before and could potentially contribute to the irreversibility of the process enzymatically. In an alternative pathway B, with ligand binding to the active hydrated form of the enzyme, the sulphamoyl group is transferred from the substrate to FGly, akin to a sulphate group to give the O-linked FGly sulphamate adduct I, analogous to the crystallographically observed resting pro-R sulphate intermediate FGlyS (Fig. 5) for sulphatases. Note that this could also occur in principle via the simple capture of sulphonylamine in A as above by the hydrated FGly diol.
Putative mechanisms to produce the two candidates for irreversibly inhibited enzyme adduct. Route A: Sulphamate ester binds to the catalytically non-productive formyl form of STS and the phenol-S bond is possibly electrophilically activated (by group X) to facilitate elimination of a reactive sulphonylamine that then attacks FGly (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is not fixed). The planar highly electrophilic adduct is then trapped by water ingress to generate adduct II (note again indeterminate stereochemistry). Route B: The pro-R hydroxyl of hydrated FGly of STS attacks the sulphamate ester analogously to normal attack on a sulphate ester to generate adduct I, a candidate for an irreversible adduct if the rest of the catalytic mechanism leading normally to sulphate extrusion is impeded by the presence of the amino group. If not, however, and the next stage of the reaction proceeds, then sulphamic acid rather than sulphate is released, as in the normal catalytic cycle for a pro-R sulphated intermediate, and can then either be extruded like sulphate or could tumble and rearrange internally, with its nitrogen moiety attacking the FGly formyl form (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is again not fixed) to generate adduct II.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Putative mechanisms to produce the two candidates for irreversibly inhibited enzyme adduct. Route A: Sulphamate ester binds to the catalytically non-productive formyl form of STS and the phenol-S bond is possibly electrophilically activated (by group X) to facilitate elimination of a reactive sulphonylamine that then attacks FGly (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is not fixed). The planar highly electrophilic adduct is then trapped by water ingress to generate adduct II (note again indeterminate stereochemistry). Route B: The pro-R hydroxyl of hydrated FGly of STS attacks the sulphamate ester analogously to normal attack on a sulphate ester to generate adduct I, a candidate for an irreversible adduct if the rest of the catalytic mechanism leading normally to sulphate extrusion is impeded by the presence of the amino group. If not, however, and the next stage of the reaction proceeds, then sulphamic acid rather than sulphate is released, as in the normal catalytic cycle for a pro-R sulphated intermediate, and can then either be extruded like sulphate or could tumble and rearrange internally, with its nitrogen moiety attacking the FGly formyl form (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is again not fixed) to generate adduct II.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Putative mechanisms to produce the two candidates for irreversibly inhibited enzyme adduct. Route A: Sulphamate ester binds to the catalytically non-productive formyl form of STS and the phenol-S bond is possibly electrophilically activated (by group X) to facilitate elimination of a reactive sulphonylamine that then attacks FGly (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is not fixed). The planar highly electrophilic adduct is then trapped by water ingress to generate adduct II (note again indeterminate stereochemistry). Route B: The pro-R hydroxyl of hydrated FGly of STS attacks the sulphamate ester analogously to normal attack on a sulphate ester to generate adduct I, a candidate for an irreversible adduct if the rest of the catalytic mechanism leading normally to sulphate extrusion is impeded by the presence of the amino group. If not, however, and the next stage of the reaction proceeds, then sulphamic acid rather than sulphate is released, as in the normal catalytic cycle for a pro-R sulphated intermediate, and can then either be extruded like sulphate or could tumble and rearrange internally, with its nitrogen moiety attacking the FGly formyl form (note in principle, this can be from either face of the keto group so the stereochemistry of the adduct is again not fixed) to generate adduct II.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
As discussed earlier, adduct I could in principle also be a dead-end in nature, but such an adduct has been deemed to be potentially reversible from energetic considerations (Williams et al. 2014). However, noting that in the normal catalysed process sulphate is most likely eliminated from the sulphated FGly diol by an energetically favourable E2 process (Williams et al. 2014), thus regenerating the FGly aldehyde, it is possible that this next enzymatic step could also proceed with sulphamic acid elimination instead. The sulphamic acid anion thus released could be extruded from the enzyme, parallel to sulphate, or it could potentially tumble and attack the FGly aldehyde so generated (in principle also from one of the two faces, although this is likely to be severely restricted within the confines of an active site) to generate again the final adduct II, again with potentially indeterminate stereochemistry. The new preliminary crystallographic work cannot explain the surprising stoichiometry reported in the inactivation of PaAtsA (Bojarová et al. 2008), but perhaps this could be the result of enzyme inactivation not being the product of a single substrate turnover in principle in either of A or B; especially if, as in Fig. 6, this proceeds via the non-catalytically active FGly formyl form of the enzyme via route A and the sulphonylamine is not always successful at inactivation, sometimes being quenched by, for example water before it or any resulting sulphamic acid can reach the FGly.
In any case, much higher-resolution crystallographic structures are required to move on definitively from these preliminary data and to identify unambiguously the true form of covalent modification. To distinguish between either the O- and N-linked FGly adducts crystallographically, however, is likely to be a very tough prospect indeed, requiring very high resolution. Most interestingly, although highly preliminary and still very speculative in nature, is the observation that the data shown in Fig. 4 implying a tetrahedral adduct at FGly were obtained for only one of the two subunits of PaAtsA. While the other subunit was indeed also modified at the same FGly site, this new density seemed to show characteristics of a more ‘planar-like’ adduct (see also legend to Fig. 4). These results are to be viewed as guidelines only supporting directions for future work and should not be over-interpreted, but it is nevertheless tantalizing to speculate that such data are in principle in line with mechanism A outlined in Fig. 6, with an initial planar adduct (at sulphur) being converted to a tetrahedral N-linked final structure II. Taken together with all other evidence to date, it is therefore tempting to de-prioritize adduct I of the two possibilities and be most drawn to adduct II. Given the well-precedented physical-organic mechanistic data for sulphonylamine generation from aryl sulphamates by elimination, together with the preliminary crystallographic observations discussed here, a serious candidate for the inactivation mechanism must thus surely be A of Fig. 6, with adduct II as the end product. Chemically, adduct II might also in principle be reversible, but perhaps this is not feasible within the STS active site because of possibly modified FGly stereochemistry, or simply that the presence of an N–H substitution replacing O in the modified but standard stereochemistry of the FGly residue might offer, for example new active-site H-bonding possibilities that could interfere with the normal catalytic machinery.
Future developments
Dual aromatase-sulphatase inhibitors (DASIs)
As mentioned already, an STS inhibitor given alongside an AI should, in a multi-targeting fashion, possess significant advantages. An alternative to administering two drugs to hit each enzyme is to develop a single molecule to inhibit both aromatase and STS, that is a Dual Aromatase-Sulfatase Inhibitor (DASI). Pharmaceutical arguments in favour of using single molecule multi-targeted agents have been made (Morphy & Rankovic 2006). Moreover, there are now numerous reports of dual- and even triple-targeted agents in clinical pipelines, particularly to address the development of resistance. An attractive approach to imbue dual activity in a DASI prototype is to take an advanced or clinically used AI with its existing heterocyclic CYP19 binding motif and to build in the aryl sulphamate pharmacophore (Woo et al. 2007) (Fig. 7A). Lead DASI compounds have been developed possessing IC50 values as AIs that are at least comparable to, or even better than, those AIs in widespread clinical use. Such compounds are effective in vivo against both enzymes validating the concept (Foster et al. 2008b ). Two examples of optimized sulphamate-based compounds with excellent in vivo activity against both targets, for example STX681 and the biphenyl-based STX1983, have been published (Woo et al. 2007, 2010) (Fig. 7) and the overall concept and wider structures have been discussed in detail (Thomas & Potter 2015a ). Using a ‘merged pharmacophore’ strategy, single compounds were even designed with dual picomolar multi-targeting activities in vitro (Woo et al. 2011b ), for example the four-ringed structures (Fig. 7A). It should be noted that in such a ‘double warhead’ DASI, the low nM AI activity is of a reversible nature, whereas the low nM STS inhibitory activity is of an irreversible nature. Thus, clinically, there should only effectively be a need to titrate the dose required for aromatase inhibition, since STS will always be rapidly and irreversibly inhibited in a time-dependent fashion. Since AIs alone have major preventive effects in healthy women with high breast cancer risk and this might also extend eventually to STS inhibitors, it is conceivable that DASIs could also prove valuable in this regard in the longer term.
(A) Dual inhibitors: top, dual inhibitory DASI compounds, for example STX681 and STX1983 (showing two pharmacophoric components) and bottom (left), the merged pharmacophore template of the pM-active dual inhibitors and (right) the first example of a dual STS and 17β-HSD1 inhibitor; (B) multi-targeting agents: structures of 2-methoxyestradiol bis-sulphamate STX140 and a typical non-steroidal derivative STX2484.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
(A) Dual inhibitors: top, dual inhibitory DASI compounds, for example STX681 and STX1983 (showing two pharmacophoric components) and bottom (left), the merged pharmacophore template of the pM-active dual inhibitors and (right) the first example of a dual STS and 17β-HSD1 inhibitor; (B) multi-targeting agents: structures of 2-methoxyestradiol bis-sulphamate STX140 and a typical non-steroidal derivative STX2484.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
(A) Dual inhibitors: top, dual inhibitory DASI compounds, for example STX681 and STX1983 (showing two pharmacophoric components) and bottom (left), the merged pharmacophore template of the pM-active dual inhibitors and (right) the first example of a dual STS and 17β-HSD1 inhibitor; (B) multi-targeting agents: structures of 2-methoxyestradiol bis-sulphamate STX140 and a typical non-steroidal derivative STX2484.
Citation: Journal of Molecular Endocrinology 61, 2; 10.1530/JME-18-0045
Development of enzyme-based PET cancer imaging agents has also been pursued using the DASI concept. New carbon-11-labelled sulphamate derivatives were designed and synthesized as potential PET radiotracers for imaging of aromatase and STS expression in breast cancer (Wang et al. 2009). Also interestingly and expanding on the DASI idea, a recent report has provided the first single molecule dual inhibitors of STS and 17β-HSD1 (Salah et al. 2017) (Fig. 7). Since 17β-HSD1 is also implicated in controlling levels of E2 and is also upregulated in tumour tissue of patients treated with AIs, it is also an attractive idea to combine these two activities for exploitation in oestrogen-dependent diseases.
Aryl sulphamates for hormone-independent tumours
Hormone-receptor-negative subtypes of breast tumours remain an unmet clinical challenge, with a high rate of recurrence and poor survival in patients following treatment. 2-Methoxyestradiol (2ME) is an endogenous non-oestrogenic anticancer steroid with multiple activities relevant to hormone-receptor-negative tumours (Parada-Bustamante et al. 2015) and also anti-inflammatory activity (Duncan et al. 2012), but has the big disadvantage of very low oral bioavailability (ca 1%), poor solubility and extensive metabolism in vivo. These very poor pharmaceutical properties are a significant barrier to further progression and although numerous oncology clinical trials have been carried out up to phase II, with the compound developed under the trade name Panzem, and with some promising results, severe limitations are apparent (Tevaarwerk et al. 2009, Parada-Bustamante et al. 2015). 2ME has been evaluated in patients with, for example different refractory solid tumours, ovarian cancer, breast cancer, metastatic prostate cancer, primary peritoneal carcinomatosis, metastatic kidney cancer and advanced carcinoid tumours. For one clinical trial, the phase II dosing regimen was 1000 mg orally every 6 h (Tevaarwerk et al. 2009) and one trial of 2ME2 had to be cancelled because plasma drug concentrations after oral administration were lower than the effective dose. Structure-activity relationships and synthetic design strategies in this area have been reviewed (Peyrat et al. 2012).
A new sulphamate-based drug candidate STX140 was designed (Leese et al. 2006) as a derivative of 2ME, an anti-tumour agent with excellent oral activity, improved potency, low metabolism and good pharmacokinetic properties (Thomas & Potter 2015a ,b ) (Fig. 7B). As well as being a potent inhibitor of STS, it exhibits a range of interesting additional properties, anti-angiogenic activity, induction of cell cycle arrest and apoptosis in human tumour xenografts, and has major clinical potential for the therapy of hormone-independent tumours. Some of this activity is thought to stem from tubulin binding at the colchicine site and disruption of interphase microtubules. STX140 is also highly active in tumours that have become resistant to chemotherapy (Newman et al. 2008).
In pre-clinical mouse xenograft models using STX140, complete cures were achieved after oral treatment in models of breast and prostate cancer and drug-resistant tumours also shrank in size after oral treatment (Thomas & Potter 2015b ). Conventional treatments for hormone-independent cancers targeting tubulin are associated with major side effects, including neurotoxicity, and can only be given infrequently and intravenously (Bates & Eastman 2017). STX140 is more effective on the same tumours, blocks metastatic spread and with no signs of the peripheral neuropathy associated with current clinical anticancer drugs (Meyer-Losic et al. 2013). STX140 thus offers, in a uniquely competitive fashion, not only a highly innovative solution to the problems faced by 2ME, but a new bioavailable drug with additional activities and properties that enhance its overall potential. While targeted therapies are offering new approaches in oncology, ultimately they rarely overcome acquired resistance mechanisms and they are still most attractive in combination with other drugs. STX140 offers, in one molecule and with highly attractive pharmaceutical properties, two novel ‘first-in-class’ targeted therapeutic approaches: inhibition of STS and also of another novel target carbonic anhydrase IX (CAIX), in concert with other anticancer activities of established clinical potential (Thomas & Potter 2015b ). Like the labelled DASI sulphamate derivative 2-[11C]methoxy-3,17β-O,O-bis(sulfamoyl)estradiol ([11C]-STX140) was designed and synthesized as a new potential imaging agent for the PET imaging of STS in cancers (Wang et al. 2012).
Because of its multi-targeting activities, STX140 can function as an STS inhibitor in hormone-dependent settings, with its extra activities or it can be utilized for the more intractable hormone-independent solid tumours. Importantly, with its aryl sulphamate pharmacophore, it also benefits from the red blood cell transport mechanisms of other STS inhibitors and enhanced stability. Moreover, a range of small molecule non-steroidal STX140 mimics have been developed (Leese et al. 2010) such as STX 2484 (Fig. 7B) that exhibit similar activities and development potential, but are pharmaceutically even more versatile and synthetically more tractable. These show a range of interesting biological activities (Thomas & Potter 2015a ,b , Stengel et al. 2014, Shen et al. 2015) and are now ripe for wider development. Interestingly, some of these analogues, despite possessing an aryl sulphamate pharmacophore, are only very weak STS inhibitors. Very recently, an example of such a non-steroidal sulphamate analogue from a quinazolinone-based series was shown in atomic detail to bind to the colchicine binding site on tubulin, as the first example of any sulphamate-based ligand (Dohle et al. 2018). Unlike the previous pro-drug and sulphamoyl transfer activities discussed, in this setting, the sulphamate group is simply acting as an additional binding element to enhance interaction of ligand with target. These classes of compounds continue to illustrate the potential of the aryl sulphamate pharmacophore.
Wnt signalling pathway activity
While STS is now well established as a therapeutic target for oestrogen-dependent diseases, potential wider cellular functions of STS are still unclear. A recent report (Shin et al. 2017) showed that STS induces Wnt/β-catenin signalling in PC-3 and HeLa cells and STS knockdown results in downregulation of Wnt/β-catenin signalling. Treatment with an STS inhibitor prevented STS-mediated Wnt/β-catenin signalling and Twist1 expression. Importantly, cancer cell migration, invasion and matrix metalloproteinase expression induced by STS could also be inhibited by an STS inhibitor. The ability of STS to induce the Wnt/β-catenin signalling and an epithelial-mesenchymal transition may have interesting implications for oestrogen-mediated carcinogenesis in the human cancer cell and opens up a whole new area of interest.
Conclusions
AIs are clinically successful and their prophylactic properties in breast cancer add to their utility. The recent striking clinical success exemplified by combining androgen deprivation therapy with the 17α-hydroxy lyase/C17, 20 lyase inhibitor Abiraterone (Zytiga) in prostate cancer (James et al. 2017) is also highly notable. Application of new mechanisms for endocrine therapy involving intracrine biosynthetic principles is both an attractive academic and clinical pursuit. Pharmacological intervention in the emerging STS-mediated pathways is clearly worthy of clinical exploration and the last 10–15 years have seen that come gradually to greater fruition in the clinic. There is now a greater appreciation of the intracrine mechanisms whereby circulating precursors can be converted in situ in tumour cells, for example via mechanisms involving the import of E1S locally into tumours via organic anion transporters followed by further in situ enzymatic transformations. Of the various enzymes implicated in such processes, the STS-mediated pathway has emerged as a significant player, not only in breast cancer but also in other hormone-dependent disease states.
The aryl-O-sulphamate pharmacophore of the most successful STS inhibitors seems to function in three different and distinct roles: it can be a pro-drug with the sulphamate group cleavable by STS; an STS inhibitor where the sulphamate moiety is almost certainly transferred in some fashion in a similar way to sulphuryl transfer to a residue in the STS active site – this now at last appears to be clarified tentatively as the catalytic FGly, with the end product not a previously proposed N-imine sulphate, but a sulphamoylated adduct with the linking atom being either O- or N- and most likely N; finally, the sulphamate group can be viewed simply as supplying structural augmentation to aid potency in, for example, the multi-targeting series of steroidal and non-steroidal microtubule disruptors. In this latter case, it has been shown to augment ligand binding to the colchicine site at the αβ dimer interface of tubulin. The pharmaceutical properties intrinsic to such sulphamate esters could possibly be widely exploited more generally in drug design. The sulphamate group not only offers versatility and high potency, but by virtue of facilitating sequestration in red blood cells to CA II also confers in vivo drug stabilization, resistance to first-pass metabolism and excellent pharmacokinetic and bioavailability properties. Sulphamate-based STS inhibitors have yet to be surpassed in terms of potency and their other attractive pharmaceutical properties.
The steroidal E2MATE was originally developed as the oestradiol pro-drug J995 and reached phase II trials. Now, as PGL2001, it is being actively developed in endometriosis employing its STS inhibitory properties. Such aryl-O-sulphamates, as exemplifiers of the first potent and irreversible inhibitors of STS have stimulated a large body of pre-clinical and clinical work through the ‘first-in-class’ drugs E2MATE (J995, PGL2001) and Irosustat and early clinical studies have substantially validated the scientific and therapeutic concepts originally proposed. While only one non-steroidal sulphamate-based drug Irosustat has been explored clinically in three types of hormone-dependent cancer to date, there are wider emerging targets becoming available. Some, such as ovarian cancer, are already well predicated, while others, such as colorectal cancer and bladder cancer, are just emerging as possibilities. Measurement of tumour STS expression can be a helpful prognostic biomarker and this should almost certainly be targeted for patient stratification in future clinical studies.
No aryl-sulphamate-based drug targeting hormone-dependent disease has yet progressed beyond phase II clinical trials. It remains to be seen whether this will be achieved in some indication as a monotherapy and the limited patient cohort of the recent IPET clinical trial offers the attraction to pursue this in early breast cancer with a larger patient cohort; or whether it might better be achieved via a combination approach, for example with an AI, where the first such potential has been demonstrated recently in the IRIS clinical trial. In terms of such a multi-targeting strategy, it seems clear that the DASI approach also offers great potential in cases where there is expected to be a synergy between STS and aromatase inhibition and that such an approach could address some acquired resistance mechanisms that have now been established. Additionally, compounds such as STX140 or their non-steroidal counterparts offer huge potential as multi-targeting agents for either hormone-independent tumours, where the pre-clinical potential is already strongly proven, or perhaps in a hormone-dependent tumour setting even as a still highly potent STS inhibitor with added cytotoxic/anti-angiogenic activity and activity against CAIX. Compared with the parent endogenous 2ME that is very poorly bioavailable and extensively metabolized, the bis-sulphamate STX140 is highly bioavailable, protected against metabolism, whilst maintaining and even enhancing the core activities of the parent steroid. However, it must be noted that the STS inhibitory activity of the non-steroidal variants cannot always be taken for granted.
After some 20 years of discovery and development, it seems that while the clinical promise of aryl-O-sulphamates as STS inhibitors are now established, they have yet to demonstrate their full potential. The optimal setting for use of an STS inhibitor still needs to be found, both in terms of pathology and whether as monotherapy, classical combination therapy with an AI or, in certain relevant applications, using a single-molecule dual agent such as a DASI or even as a combined STS-17β-HSD1 inhibitor. Exploitation in hormone-independent settings is predicated by excellent pre-clinical data, for example for 2-substituted oestradiol derivatives such as STX140 and non-steroidal relatives, but clinical entry has yet to be achieved. In the context of this particular review, support for the continuing development and optimization of new types of endocrine therapy is also provided by a recently reported study (Roswall et al. 2018) that used resistant ERα-negative breast tumours and showed that they can be sensitized to hormone therapy. Genetic or pharmacological targeting of platelet-derived growth factor-CC isoform activity converted basal-like tumours in mouse cancer models into a hormone receptor-positive state. This and the emergence of new roles for STS, for example in Wnt/β-catenin signalling, will be sure to maintain levels of excitement and motivation to apply further the concepts and the related drugs discussed here, both in endocrine therapy applications and beyond.
Declaration of interest
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review. He is a co-inventor on patents for some of the agents reported here, was a co-founder of Sterix Limited and holds stock in EstryX Pharma Limited.
Funding
B V L P was funded for some of the work discussed here through Sterix Limited, a member of the Ipsen Group, which received research grants from Ipsen.
Acknowledgement
We thank the numerous collaborators who have contributed to much of the work reported here and whose names appear in references and in the text. We also acknowledge Dr G Cozier whose preliminary unpublished enzymology and crystallographic work has enabled the outline mechanistic discussions presented and Dr M P Thomas for help with the modelling in Figure 1.
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