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Jon Wolf Mueller and Paul A Foster
Douglas A Gibson, Paul A Foster, Ioannis Simitsidellis, Hilary O D Critchley, Olympia Kelepouri, Frances Collins and Philippa T K Saunders
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.
Oscar J Pozo, Josep Marcos, Olha Khymenets, Andy Pranata, Christopher C Fitzgerald, Malcolm D McLeod and Cedric Shackleton
The steroid disulfates (aka bis-sulfates) are a significant but minor fraction of the urinary steroid metabolome that have not been widely studied because major components are not hydrolyzed by the commercial sulfatases commonly used in steroid metabolomics. In early studies, conjugate fractionation followed by hydrolysis using acidified solvent (solvolysis) was used for the indirect detection of this fraction by GC–MS. This paper describes the application of a specific LC–MS/MS method for the direct identification of disulfates in urine, and their use as markers for the prenatal diagnosis of disorders causing reduced estriol production: STSD (steroid sulfatase deficiency), SLOS (Smith-Lemli-Opitz syndrome) and PORD (P450 oxidoreductase deficiency). Disulfates were detected by monitoring a constant ion loss (CIL) from the molecular di-anion. While focused on disulfates, our methodology included an analysis of intact steroid glucuronides and monosulfates because steroidogenic disorder diagnosis usually requires an examination of the complete steroid profile. In the disorders studied, a few individual steroids (as disulfates) were found particularly informative: pregn-5-ene-3β,20S-diol, pregn-5-ene-3β,21-diol (STSD, neonatal PORD) and 5α-pregnane-3β,20S-diol (pregnancy PORD). Authentic steroid disulfates were synthesized for use in this study as aid to characterization. Tentative identification of 5ξ-pregn-7-ene-3ξ,20S-diol and 5ξ-pregn-7-ene-3ξ,17,20S-triol disulfates was also obtained in samples from SLOS affected pregnancies. Seven ratios between the detected metabolites were applied to distinguish the three selected disorders from control samples. Our results show the potential of the direct detection of steroid conjugates in the diagnosis of pathologies related with steroid biosynthesis.
Carla A Piccinato, Helena Malvezzi, Douglas A Gibson and Philippa T K Saunders
Endometriosis is an incurable hormone-dependent inflammatory disease that causes chronic pelvic pain and infertility characterized by implantation and growth of endometrial tissue outside the uterine cavity. Symptoms have a major impact on the quality of life of patients resulting in socioeconomic, physical and psychological burdens. Although the immune system and environmental factors may play a role in the aetiology of endometriosis, oestrogen dependency is still considered a hallmark of the disorder. The impact of oestrogens such as oestrone and particularly, oestradiol, on the endometrium or endometriotic lesions may be mediated by steroids originating from ovarian steroidogenesis or local intra-tissue production (intracrinology) dependent upon the expression and activity of enzymes that regulate oestrogen biosynthesis and metabolism. Two key pathways have been implicated: while there is contradictory data on the participation of the aromatase enzyme (encoded by CYP19A1), there is increasing evidence that the steroid sulphatase pathway plays a role in both the aetiology and pathology of endometriosis. In this review, we consider the evidence related to the pathways leading to oestrogen accumulation in endometriotic lesions and how this might inform the development of new therapeutic strategies to treat endometriosis without causing the undesirable side effects of current regimes that suppress ovarian hormone production.
B Zimmer, L Tenbusch, M C Klymiuk, Y Dezhkam and G Schuler
In the porcine testis, in addition to estrogen sulfates, the formation of numerous sulfonated neutral hydroxysteroids has been observed. However, their functions and the underlying synthetic pathways are still widely unclear. To obtain further information on their formation in postpubertal boars, the expression of sulfotransferases considered relevant for neutral hydroxysteroids (SULT2A1, SULT2B1) was investigated in the testis and defined segments of the epididymis applying real-time RT-qPCR, Western blot and immunohistochemistry (IHC). Sulfotransferase activities were assessed in tissue homogenates or cytosolic preparations applying dehydroepiandrosterone and pregnenolone as substrates. A high SULT2A1 expression was confirmed in the testis and localized in Leydig cells by IHC. In the epididymis, SULT2A1 expression was virtually confined to the body. SULT2B1 expression was absent or low in the testis but increased significantly along the epididymis. Immunohistochemical observations indicate that both enzymes are secreted into the ductal lumen via an apocrine mechanism. The results from the characterization of expression patterns and activity measurements suggest that SULT2A1 is the prevailing enzyme for the sulfonation of hydroxysteroids in the testis, whereas SULT2B1 may catalyze the formation of sterol sulfates in the epididymis. In order to obtain information on the overall steroidogenic capacity of the porcine epididymis, the expression of important steroidogenic enzymes (CYP11A1, CYP17A1, CYP19, HSD3B1, HSD17B3, SRD5A2) was monitored in the defined epididymal segments applying real-time RT-qPCR. Surprisingly, in addition to a high expression of SRD5A2 in the epididymal head, a substantial expression of HSD3B1 was detected, which increased along the organ.
G Schuler, Y Dezhkam, L Tenbusch, MC Klymiuk, B Zimmer and B Hoffmann
Boars exhibit high concentrations of sulfonated estrogens (SE) mainly originating from the testicular-epididymal compartment. Intriguingly, in porcine Leydig cells, sulfonation of estrogens is colocalized with aromatase and steroid sulfatase (STS), indicating that de novo synthesis of unconjugated estrogens (UE), their sulfonation and hydrolysis of SE occur within the same cell type. So far in boars no plausible concept concerning the role of SE has been put forward. To obtain new information on SE formation and hydrolysis, the porcine testicular-epididymal compartment was screened for the expression of the estrogen-specific sulfotransferase SULT1E1 and STS applying real-time RT-qPCR, Western blot and immunohistochemistry. The epididymal head was identified as the major site of SULT1E1 expression, whereas in the testis, it was virtually undetectable. However, SE tissue concentrations are clearly consistent with the testis as the predominant site of estrogen sulfonation. Results from measurements of estrogen sulfotransferase activity indicate that in the epididymis, SULT1E1 is the relevant enzyme, whereas in the testis, estrogens are sulfonated by a different sulfotransferase with a considerably lower affinity. STS expression and activity was high in the testis (Leydig cells, rete testis epithelium) but also present throughout the epididymis. In the epididymis, SULT1E1 and STS were colocalized in the ductal epithelium, and there was evidence for their apocrine secretion into the ductal lumen. The results suggest that in porcine Leydig cells, SE may be produced as a reservoir to support the levels of bioactive UE via the sulfatase pathway during periods of low activity of the pulsatile testicular steroidogenesis.
Paul A Foster and Jonathan Wolf Mueller
Sulfation and desulfation pathways represent highly dynamic ways of shuttling, repressing and re-activating steroid hormones, thus controlling their immense biological potency at the very heart of endocrinology. This theme currently experiences growing research interest from various sides, including, but not limited to, novel insights about phospho-adenosine-5′-phosphosulfate synthase and sulfotransferase function and regulation, novel analytics for steroid conjugate detection and quantification. Within this review, we will also define how sulfation pathways are ripe for drug development strategies, which have translational potential to treat a number of conditions, including chronic inflammatory diseases and steroid-dependent cancers.
Marta Correia-da-Silva, Verónica Rocha, Cláudia Marques, Cláudia M Deus, Adriana Marques-Carvalho, Paulo J Oliveira, Andreia Palmeira, Madalena Pinto, Emília Sousa, José Manuel Sousa Lobo and Isabel Filipa Almeida
Resveratrol (RSV) is a polyphenolic compound with antioxidant, anti-inflammatory and anti-aging properties partly associated with sirtuin 1 (SIRT1)-activation in the skin. However, poor water solubility may limit RSV efficacy. This work aimed to clarify the interest of a new synthetic water-soluble RSV derivative (resveratrol glucoside sulfate, RSV-GS) for topical application. Resveratrol glucoside sulfate was synthesized using microwave-assisted sulfation. Cytotoxicity assays were performed with the keratinocyte HaCaT cell line, using MTT reduction, neutral red uptake, Alamar Blue/resazurin reduction, trypan blue exclusion and measurement of ATP concentration. Western blotting was used to evaluate SIRT1 protein content. Regarding SIRT1 binding, an in silico docking study was performed, using AutoDock Vina. Our results showed that the synthetic derivative RSV-GS was 1000 times more soluble in water than RSV and its non-sulfated glucoside. No relevant decrease in HaCaT cell viability was observed for concentrations up to 5 mM for RSV-GS, and up to 500 μM for resveratrol glucoside, while a significant decrease in HaCaT viability occurred from 100 μM for RSV. RSV-GS and RSV showed a similar behavior regarding protective effect against oxidative stress-induced cytotoxicity. SIRT1 protein content increased after treatment with 500 μM of RSV-GS and 100 μM of RSV. Moreover, in silico studies predicted that RSV-GS binds more stably to SIRT1 with a lower binding free energy than RSV. Although these results support the possible use of RSV-GS in topical formulations, in vivo safety and efficacy studies are needed before considering the use of RSV-GS in commercial products.
Francisca Carvalhal, Marta Correia-da-Silva, Emília Sousa, Madalena Pinto and Anake Kijjoa
Marine environment is rich in structurally unique molecules and can be an inspiring source of novel drugs. Currently, six marine-derived drugs are in the market with FDA approval and several more are in the clinical pipeline. Structurally diverse and complex secondary metabolites have been isolated from the marine world and these include sulfated steroids. Biological activities of nearly 150 marine sulfated steroids reported from 1978 to 2017 are compiled and described, namely antimicrobial, antitumor, cardiovascular and antifouling activities. Structure–activity relationship for each activity is discussed.
Barry V L Potter
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.