Memory, mood and associated neuroanatomy in individuals with steroid sulphatase deficiency (X-linked ichthyosis).

Steroid sulphatase (STS) cleaves sulphate groups from steroid hormones, and steroid (sulphate) levels correlate with mood and age-related cognitive decline. In animals, STS inhibition or deletion of the associated gene, enhances memory/neuroprotection and alters hippocampal neurochemistry. Little is known about the consequences of constitutive STS deficiency on memory-related processes in humans. We investigated self-reported memory performance (Multifactorial Memory Questionnaire), word-picture recall and recent mood (Kessler Psychological Distress Scale, K10) in adult males with STS deficiency diagnosed with the dermatological condition X-linked ichthyosis (XLI; n = 41) and in adult female carriers of XLI-associated genetic variants (n = 79); we compared results to those obtained from matched control subjects [diagnosed with ichthyosis vulgaris (IV, n = 98) or recruited from the general population (n = 250)]. Using the UK Biobank, we compared mood/memory-related neuroanatomy in carriers of genetic deletions encompassing STS (n = 28) and non-carriers (n = 34,522). We found poorer word-picture recall and lower perceived memory abilities in males with XLI and female carriers compared with control groups. XLI-associated variant carriers and individuals with IV reported more adverse mood symptoms, reduced memory contentment and greater use of memory aids, compared with general population controls. Mood and memory findings appeared largely independent. Neuroanatomical analysis only indicated a nominally-significantly larger molecular layer in the right hippocampal body of deletion carriers relative to non-carriers. In humans, constitutive STS deficiency appears associated with mood-independent impairments in memory but not with large effects on underlying brain structure; the mediating psychobiological mechanisms might be explored further in individuals with XLI and in new mammalian models lacking STS developmentally.

Genetic analysis of seven patients with inherited ichthyosis and Nagashima­type palmoplantar keratoderma.

Inherited ichthyosis comprises a series of heterogeneous dermal conditions; it mainly manifests as widespread hyperkeratosis, xerosis and scaling of the skin. At times, overlapping symptoms require differential diagnosis between ichthyosis and several other similar disorders. The present study reports seven patients with confirmed or suspected to be associated with ichthyosis by conducting a thorough clinical and genetic investigation. Genetic testing was conducted using whole­exome sequencing, with Sanger sequencing as the validation method. The MEGA7 program was used to analyze the conservation of amino acid residues affected by the detected missense variants. The enrolled patients exhibited ichthyosis­like but distinct clinical manifestations. Genetic analysis identified diagnostic variations in the FLG, STS, KRT10 and SERPINB7 genes and clarified the carrying status of each variant in the respective family members. The two residues affected by the detected missense variants remained conserved across multiple species. Of note, the two variants, namely STS: c.452C>T(p.P151L) and c.647_650del(p.L216fs) are novel. In conclusion, a clear genetic differential diagnosis was made for the enrolled ichthyosis­associated patients; the study findings also extended the mutation spectrum of ichthyosis and provided solid evidence for the counseling of the affected families.

Synthesis, biological evaluation, and stability studies of raloxifene mono- and bis-sulfamates as dual-targeting agents.

All three possible sulfamate derivatives of the selective estrogen receptor modulator Raloxifene (bis-sulfamate 7 and two mono-sulfamates 8-9) were synthesized and evaluated as inhibitors of the clinical drug target steroid sulfatase (STS), both in cell-free and in cell-based assays, and also as estrogen receptor (ER) modulators. Bis-sulfamate 7 was the most potent STS inhibitor with an IC50 of 12.2 nM in a whole JEG3 cell-based assay, with the two mono-sulfamates significantly weaker. The estrogen receptor-modulating activities of 7-9 showed generally lower affinities compared to Raloxifene HCl, diethylstilbestrol and other known ligands, with mono-sulfamate 8 being the best ligand (Ki of 1.5 nM) for ERα binding, although 7 had a Ki of 13 nM and both showed desirable antagonist activity. The antiproliferative activities of the sulfamate derivatives against the T-47D breast cancer cell line showed 7 as most potent (GI50 = 7.12 µM), comparable to that of Raloxifene. Compound 7 also showed good antiproliferative potency in the NCI-60 cell line panel with a GI50 of 1.34 µM against MDA-MB-231 breast cancer cells. Stability testing of 7-9 showed that bis-sulfamate 7 hydrolyzed by desulfamoylation at a surprisingly rapid rate, initially leading selectively to 8 and finally to Raloxifene 3 without formation of 9. The mechanisms of these hydrolysis reactions could be extensively rationalized. Conversion of Raloxifene (3) into its bis-sulfamate (7) thus produced a promising drug lead with nanomolar dual activity as an STS inhibitor and ERα antagonist, as a potential candidate for treatment of estrogen-dependent breast cancer.

Unraveling the molecular mechanisms of cell migration impairment and apoptosis associated with steroid sulfatase deficiency: Implications for X-linked ichthyosis.

Steroid sulfatase (STS) deficiency is responsible for X-linked ichthyosis (XLI), a genetic disorder characterized by rough and dry skin caused by excessive keratinization. The impaired keratinization process leads to reduced cell mobility and increased apoptosis, which can cause an excessive buildup of the stratum corneum. In this study, we investigated the mechanisms underlying XLI and found that STS deficiency reduces cell mobility and increases apoptosis in human keratinocyte HaCaT cells. To explore these mechanisms further, RNA-sequencing was conducted on skin tissues from STS transgenic and knockout mice. Our RNA-seq results revealed that STS deficiency plays a critical role in regulating multiple signaling pathways associated with cell mobility and apoptosis, such as Wnt/ß signaling and the Hippo signaling pathway. Knockdown of the STS gene using shRNA in HaCaT cells led to an upregulation of E-cadherin expression and suppression of key factors involved in epithelial-mesenchymal transition (EMT), such as N-cadherin and vimentin. Inhibition of EMT involved the Hippo signaling pathway and reduction of HIF-1α. Interestingly, inhibiting STS with shRNA increased mitochondrial respiration levels, as demonstrated by the extracellular flux oxygen consumption rate. Additionally, we observed a significant increase in ROS production in partial STS knockout cells compared to control cells. Our study demonstrated that the excessive generation of ROS caused by STS deficiency induces the expression of Bax and Bak, leading to the release of cytochrome c and subsequent cell death. Consequently, STS deficiency impairs cell mobility and promotes apoptosis, offering insights into the pathophysiological processes and potential therapeutic targets for XLI.

Estrogen sulfotransferase and sulfatase in steroid homeostasis, metabolic disease, and cancer.

Sulfation and desulfation of steroids are opposing processes that regulate the activation, metabolism, excretion, and storage of steroids, which account for steroid homeostasis. Steroid sulfation and desulfation are catalyzed by cytosolic sulfotransferase and steroid sulfatase, respectively. By modifying and regulating steroids, cytosolic sulfotransferase (SULT) and steroid sulfatase (STS) are also involved in the pathophysiology of steroid-related diseases, such as hormonal dysregulation, metabolic disease, and cancer. The estrogen sulfotransferase (EST, or SULT1E1) is a typical member of the steroid SULTs. This review is aimed to summarize the roles of SULT1E1 and STS in steroid homeostasis and steroid-related diseases.

Steroid sulfatase inhibitors and sulfated C19 steroids for proteotoxicity-related diseases: a patent spotlight.

Aging and proteotoxicity go hand in hand. Inhibiting proteotoxicity has been proposed to extend lifespan. This invention describes a new strategy to limit proteotoxicity and to extend the lifespan. Loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase, elevates the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. The present invention provides a group of molecules for use in the prevention of aging-associated proteotoxicity caused by protein aggregation diseases and/or to increase the lifespan of a eukaryotic organism. These molecules are either steroid sulfatase inhibitors or sulfated C19 steroids, both of which reproduce the phenotype of sul-2 mutants. One particular representative example is STX-64. Potential applications of the claims have been demonstrated in animal models of Parkinson's disease, Huntington's disease and Alzheimer's disease.

Aromatase and steroid sulfatase from human placenta.

Cytochrome P450 aromatase (AROM) and steroid (estrone (E1)/dehydroepiandrosterone (DHEA)) sulfatase (STS) are the two key enzymes responsible for the biosynthesis of estrogens in human, and maintenance of the critical balance between androgens and estrogens. Human AROM, an integral membrane protein of the endoplasmic reticulum, is a member of the Fe-heme containing cytochrome P450 superfamily having a cysteine thiolate as the fifth Fe-coordinating ligand. It is the only enzyme known to catalyze the conversion of androgens with non-aromatic A-rings to estrogens characterized by the aromatic A-ring. Human STS, also an integral membrane protein of the endoplasmic reticulum, is a Ca2+-dependent enzyme that catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, the precursors of the most potent forms of estrogens and androgens, namely, 17ß-estradiol (E2), 16α,17ß-estriol (E3), testosterone (TST) and dihydrotestosterone (DHT). Expression of these steroidogenic enzymes locally within various organs and tissues of the endocrine, reproductive, and central nervous systems is the key for maintaining high levels of the reproductive steroids. Thus, the enzymes have been drug targets for the prevention and treatment of diseases associated with steroid hormone excesses, especially in breast and prostate malignancies and endometriosis. Both AROM and STS have been the subjects of vigorous research for the past six decades. In this article, we review the procedures of their extraction and purification from human term placenta are described in detail, along with the activity assays.

STS and PUDP Deletion Identified by Targeted Panel Sequencing with CNV Analysis in X-Linked Ichthyosis: A Case Report and Literature Review.

X-linked recessive ichthyosis (XLI) is clinically characterized by dark brown, widespread dryness with polygonal scales. We describe the identification of STS and PUDP deletions using targeted panel sequencing combined with copy-number variation (CNV) analysis in XLI. A 9-month-old infant was admitted for genetic counseling. Since the second day after birth, the infant's skin tended to be dry and polygonal scales had accumulated over the abdomen and upper extremities. The infant's maternal uncle and brother (who had also exhibited similar skin symptoms from birth) presented with polygonal scales on their trunks. CNV analysis revealed a hemizygous deletion spanning 719.3 Kb on chromosome Xp22 (chrX:7,108,996-7,828,312), which included a segment of the STS gene and exhibited a Z ratio of -2 in the proband. Multiplex ligation-dependent probe amplification (MLPA) confirmed this interstitial Xp22.31 deletion. Our report underscores the importance of implementing CNV screening techniques, including sequencing data analysis and gene dosage assays such as MLPA, to detect substantial deletions that encompass the STS gene region of Xq22 in individuals suspected of having XLI.

Identification of a novel partial deletion of STS associated with pre-Descemet corneal dystrophy and X-linked ichthyosis.

Purpose: Pre-Descemet corneal dystrophy (PDCD) with X-linked ichthyosis (XLI) is associated with mutations in or deletions of the steroid sulfatase gene (STS). As only three cases of genetically confirmed PDCD associated with XLI have been reported, we sought to expand our understanding of the genetic basis of PDCD by screening STS in two previously unreported families. Materials and Methods: The affected individuals underwent cutaneous and slit-lamp examinations. Saliva samples collected from each affected individual served as a source of DNA for the amplification of the 10 coding exons of STS and flanking DNA markers. Results: The slit-lamp examination of three affected men (two of whom were brothers) from two families revealed bilateral punctate posterior corneal stromal opacities anterior to the Descemet membrane. Cutaneous examination demonstrated dry, coarse, scaly ichthyotic changes characteristic of XLI in all individuals. Genetic examination of the STS locus on the X chromosome in Case 1 revealed a deletion that spanned across DNA markers DXS1130-DXS237, which includes all the coding exons (exons 1-10) of STS. Genetic screening of Cases 2 and 3 revealed a partial deletion of the STS locus involving exons 1-7 and flanking DNA marker DXS1130 on the X chromosome. Conclusions: PDCD with XLI may be associated with either partial or complete deletion of STS. Despite the identification of point mutations, partial deletion, and complete deletion of STS in different affected families reported to date, there was no apparent difference in the affected phenotype between the families, suggesting that the identified variants likely all resulted in loss of function of steroid sulfatase.

New structural insights provide a different angle on steroid sulfatase action.

A central part of human sulfation pathways is the spatially and temporally controlled desulfation of biologically highly potent steroid hormones. The responsible enzyme - steroid sulfatase (STS) - is highly expressed in placenta and peripheral tissues, such as fat, colon, and the brain. The shape of this enzyme and its mechanism are probably unique in biochemistry. STS was believed to be a transmembrane protein, spanning the Golgi double-membrane by stem region formed by two extended internal alpha-helices. New crystallographic data however challenge this view. STS now is portraited as a trimeric membrane-associated complex. We discuss the impact of these results on STS function and sulfation pathways in general and we hypothesis that this new STS structural understanding suggests product inhibition to be a regulator of STS enzymatic activity.

Potent Dual Inhibitors of Steroid Sulfatase and 17ß-Hydroxysteroid Dehydrogenase Type 1 with a Suitable Pharmacokinetic Profile for In Vivo Proof-of-Principle Studies in an Endometriosis Mouse Model.

Treating estrogen-dependent diseases like endometriosis with drugs suppressing local estrogen activation may be superior to existing endocrine therapies. Steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) are key enzymes of local estrogen activation. We describe the rational design, synthesis, and biological profilation of furan-based compounds as a novel class of dual STS/17ß-HSD1 inhibitors (DSHIs). In T47D cells, compound 5 showed irreversible inhibition of STS and potent, reversible inhibition of 17ß-HSD1. It was selective over 17ß-HSD2 and displayed high metabolic stabilities in human and mouse liver S9 fractions. No effect on cell viability was detected up to 31 µM (HEK293) and 23 µM (HepG2), respectively, and there was no activation of the aryl hydrocarbon receptor (AhR) up to 3.16 µM. Single daily application to mice revealed steady-state plasma levels high enough to make this compound eligible for an in vivo proof-of-principle study in a mouse endometriosis model.

Design, structure-activity relationships, and enzyme kinetic studies of tricyclic and tetracyclic coumarin-based sulfamates as steroid sulfatase inhibitors.

Inhibition of steroid sulfatase (STS) decreases estrogen production and thus, suppresses tumor proliferation. Inspired by irosustat, the first STS inhibitor in clinical trials, we explored twenty-one tricyclic and tetra-heterocyclic coumarin-based derivatives. Their STS enzyme kinetic parameters, docking models, and cytotoxicity toward breast cancer and normal cells were evaluated. Tricyclic derivative 9e and tetracyclic derivative 10c were the most promising irreversible inhibitors developed in this study, with KI of 0.05 and 0.4 nM, and kinact/KI ratios of 28.6 and 19.1 nM-1min-1 on human placenta STS, respectively.

Structures and functions of human placental aromatase and steroid sulfatase, two key enzymes in estrogen biosynthesis.

Cytochrome P450 aromatase (AROM) and steroid sulfatase (STS) are the two key enzymes for the biosynthesis of estrogens in human, and maintenance of the critical balance between androgens and estrogens. Human AROM, an integral membrane protein of the endoplasmic reticulum, is a member of the cytochrome P450 superfamily. It is the only enzyme to catalyze the conversion of androgens with non-aromatic A-rings to estrogens characterized by the aromatic A-ring. Human STS, also an integral membrane protein of the endoplasmic reticulum, is a Ca2+-dependent enzyme that catalyzes the hydrolysis of sulfate esters of estrone and dehydroepiandrosterone to the unconjugated steroids, the precursors of the most potent forms of estrogens and androgens, namely, 17ß-estradiol, 16α,17ß-estriol, testosterone and dihydrotestosterone. Expression of these steroidogenic enzymes locally within organs and tissues of the endocrine, reproductive, and central nervous systems is the key for maintaining high levels of the reproductive steroids. The enzymes have been drug targets for the prevention and treatment of diseases associated with steroid hormone excesses, especially in breast, endometrial and prostate malignancies. Both enzymes have been the subjects of vigorous research for the past six decades. In this article, we review the important findings on their structure-function relationships, specifically, the work that began with unravelling of the closely guarded secrets, namely, the 3-D structures, active sites, mechanisms of action, origins of substrate specificity and the basis of membrane integration. Remarkably, these studies were conducted on the enzymes purified in their pristine forms from human placenta, the discarded and their most abundant source. The purification, assay, crystallization, and structure determination methodologies are described. Also reviewed are their functional quaternary organizations, post-translational modifications and the advancements made in the structure-guided inhibitor design efforts. Outstanding questions that still remain open are summarized in closing.

Genetic Heterogeneity of X-Linked Ichthyosis in the Republic of North Ossetia-Alania, Case Series Report.

North Caucasus has always been a residence of a lot of different authentic ethnic groups speaking different languages and still living their traditional lifestyle. The diversity appeared to be reflected in the accumulation of different mutations causing common inherited disorders. X-linked ichthyosis represents the second most common form of genodermatoses after ichthyosis vulgaris. Eight patients from three unrelated families of different ethnic origin, Kumyk, Turkish Meskhetians, and Ossetian, with X-linked ichthyosis from the North Caucasian Republic of North Ossetia-Alania were examined. NGS technology was implied for searching for disease-causing variants in one of the index patients. Known pathogenic hemizygous deletion in the short arm of chromosome X encompassing the STS gene was defined in the Kumyk family. A further analysis allowed us to establish that likely the same deletion was a cause of ichthyosis in a family belonging to the Turkish Meskhetians ethnic group. In the Ossetian family, a likely pathogenic nucleotide substitution in the STS gene was defined; it segregated with the disease in the family. We molecularly confirmed XLI in eight patients from three examined families. Though in two families, Kumyk and Turkish Meskhetian, we revealed similar hemizygous deletions in the short arm of chromosome X, but their common origin was not likely. Forensic STR markers of the alleles carrying the deletion were defined to be different. However, here, common alleles haplotype is hard to track for a high local recombination rate. We supposed the deletion could arise as a de novo event in a recombination hot spot in the described and in other populations with a recurrent character. Defined here are the different molecular genetic causes of X-linked ichthyosis in families of different ethnic origins sharing the same residence place in the Republic of North Ossetia-Alania which could point to the existing reproductive barriers even inside close neighborhoods.

Dual aromatase-steroid sulfatase inhibitors (DASI's) for the treatment of breast cancer: a structure guided ligand based designing approach.

Dual aromatase-steroid sulfatase inhibitors (DASIs) lead to significant deprivation of estrogen levels as compared to a single target inhibition and thereby exhibited an additive or synergistic effect in the treatment of hormone-dependent breast cancer (HDBC). Triazole-bearing DASI's having structural features of clinically available aromatase inhibitors are identified as lead structures for optimization as DASI's. To identify the spatial fingerprints of target-specific triazole as DASI's, we have performed molecular docking assisted Gaussian field-based comparative 3D-QSAR studies on a dataset with dual aromatase-STS inhibitory activities. Separate contours were generated for both aromatase and steroid sulphates showing respective pharmacophoric structural requirements for optimal activity. These developed 3D-QSAR models also showed good statistical measures with the excellent predictive ability with PLS-generated validation constraints. Comparative steric, electrostatic, hydrophobic, HBA, and HBD features were elucidated using respective contour maps for selective target-specific favourable activity. Furthermore, the molecular docking was used for elucidating the mode of binding as DASI's along with the MD simulation of 100 ns revealed that all the protease-ligand docked complexes are overall stable as compared to reference ligand (inhibitor ASD or Irosustat) complex. Further, the MM-GBSA study revealed that compound 24 binds to aromatase as well as STS active site with relatively lower binding energy than reference complex, respectively. A comparative study of these developed multitargeted QSAR models along with molecular docking and dynamics study can be employed for the optimization of drug candidates as DASI's.Communicated by Ramaswamy H. Sarma.

Structure of human placental steroid sulfatase at 2.0 angstrom resolution: Catalysis, quaternary association, and a secondary ligand site.

Human placental estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (human placental steroid sulfatase; hSTS) is an integral membrane protein of the endoplasmic reticulum. This Ca2+-dependent enzyme catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, which then act as precursors for the biosynthesis of 17ß-estradiol (E2) and dihydrotestosterone (DHT), respectively, the most potent forms of estrogens and androgens. hSTS is a key enzyme for the local production of E2 and DHT in the breast and the prostate. The enzyme is known to be responsible for maintaining high levels of estrogens in the breast tumor cells. The crystal structure of hSTS purified from human placenta has previously been reported at 2.6 Å resolution. Here we present the structure of hSTS determined at the superior 2.0 Å resolution bringing new clarity to the atomic architecture of the active site. The molecular basis of catalysis and steroid-protein interaction are revisited in light of the new data. We also reexamine the enzyme's quaternary association and its implication on the membrane integration. A secondary ligand binding pocket at the intermolecular interface and adjacent to the active site access channel, buried into the gill of the mushroom-shaped molecule, has been identified. Its role as well as that of a phosphate ion bound to an exposed histidine side chain are examined from the structure-function perspective. Higher resolution data also aids in the tracing of an important loop missing in the previous structure.

The phytochemical curcumin inhibits steroid sulfatase activity in rat liver tissue and NIH-3T3 mouse fibroblast cells.

Curcumin is a phytochemical derived from the spice turmeric that is reported to have therapeutic effects. We are studying the enzyme steroid sulfatase (STS), which removes the sulfate group from inactive steroid hormones in peripheral tissues and we were interested in the effect of curcumin on STS activity due to its structural composition (polyphenolic). We sought to determine if curcumin affects STS activity in two model systems, rat liver and NIH-3T3 mouse fibroblast cells. STS assays were performed on tissue extracts of rat liver, and on NIH-3T3 microsomes and cells, with and without curcumin. Male and female rat liver extracts contained substantial amounts of STS activity, with males averaging higher (4-11 %) levels. Estradiol inhibited STS activity in livers of both sexes at 20 and 10 µM. Curcumin acted as a competitive inhibitor of STS activity in rat liver extracts, with a Ki of 19.8 µM in males and 9.3 µM in females. Curcumin also inhibited STS activity in NIH-3T3 microsomes at both 20 µM and 10 µM, and in whole NIH-3T3 cells at 20 µM. These data are the first to demonstrate STS inhibition by curcumin. Inhibition of STS results in lower active steroid hormone (estrogens and androgens) levels in tissues, possibly altering modulation of immune responses by these steroids.

Is intracrinology of endometriosis relevant in clinical practice? A systematic review on estrogen metabolism.

Endometriosis is a chronic, multifactorial, estrogen-dependent disease. The abnormal endocrine microenvironment of endometriosis lesions is considered a main feature and multiple enzymatic pathways leading to local increased synthesis of estrogens have been identified. However, the relevance of intracrinology in clinical practice is still lacking. Medline, Embase, Scopus database were systematically searched for studies reporting on local estrogens metabolism of endometriotic lesions. The main enzymatic pathways involved in the intracrinology of endometriosis such as aromatase (CYP19A1), 17ß-hydroxysteroid dehydrogenase (HSD17B) type 1, type 2 and type 5, steroid sulfatase (STS), estrogen sulfotransferase (SULT1E1) were assessed with a critical perspective on their role in disease endocrine phenotyping, drug resistance and as therapeutic targets. Overall, studies heterogeneity and missing clinical data affect the interpretation of the clinical role of these enzymes. Although the use of some drugs such as aromatase inhibitors has been proposed in clinical practice for two decades, their potential clinical value is still under investigation as well as their modality of administration. A closer look at new, more realistic drug targets is provided and discussed. Altered expression of these key enzymes in the lesions have far reaching implication in the development of new drugs aimed at decreasing local estrogenic activity with a minimal effect on gonadal function; however, given the complexity of the evaluation of the expression of the enzymes, multiple aspects still remains to be clarified. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022311329, identifier CRD42022311329.

The Design, Structure-Activity, and kinetic studies of 3-Benzyl-5-oxa-1,2,3,4-Tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates as Steroid sulfatase inhibitors.

Steroid sulfatase inhibitors block the local production of estrogenic steroids and are attractive agents for the treatment of estrogen-dependent cancers. Inspiration of coumarin-based inhibitors, we synthesized thirty-two 5-oxa-1,2,3,4-tetrahydro-2H-chromeno-(3,4-c)pyridin-8-yl sulfamates, focusing on the substitution derivatives on the adjacent phenyl ring and evaluated their abilities to block STS from human placenta and MCF-7 cells. SAR analysis revealed that the incorporation of chlorine at either meta and/or para position of the adjacent phenyl ring of the tricyclic skeleton enhanced STS inhibition. Di-substitutions at the adjacent phenyl ring were superior to mono and tri-substitutions. Further kinetic analysis of these compounds revealed that chloride-bearing compounds, such as 19m, 19v, and 19w, had KI of 0.02 to 0.11 nM and kinact/KI ratios of 8.8-17.5 nM-1min-1, a parameter indicated for the efficiency of irreversible inhibition. We also used the docking model to illustrate the difference in STS inhibitory potency of compounds. Finally, the safety and anti-cancer activity of selected compounds 19m, 19v, and 19w were also studied, showing the results of low cytotoxicity on NHDF cell line and being more potent than irosustat on ZR-75-1 cell, which was a hormone-dependent cancer cell line with high STS expression.

2-Difluoromethoxy-Substituted Estratriene Sulfamates: Synthesis, Antiproliferative SAR, Antitubulin Activity, and Steroid Sulfatase Inhibition.

2-Difluoromethoxyestratriene derivatives were designed to improve potency and in vivo stability of the drug candidate 2-methoxyestradiol (2ME2). Compound evaluation in vitro against the proliferation of MCF-7 and MDA MB-231 breast cancer cells, as inhibitors of tubulin polymerisation and also steroid sulfatase (STS) both in cell lysates and in whole cells, showed promising activities. In antiproliferative assays 2-difluoromethoxyestradiol was less potent than 2ME2, but its sulfamates were often more potent than their corresponding non-fluorinated analogues. The fluorinated bis-sulfamate is a promising antiproliferative agent in MCF-7 cells (GI50 0.28 µM) vs the known 2-methoxyestradiol-3,17-O,O-bissulfamate (STX140, GI50 0.52 µM), confirming the utility of our approach. Compounds were also evaluated in the NCI 60-cell line panel and the fluorinated bis-sulfamate derivative displayed very good overall activities with a sub-micromolar average GI50 . It was a very potent STS inhibitor in whole JEG-3 cells (IC50 3.7 nM) similar to STX140 (4.2 nM) and additionally interferes with tubulin assembly in vitro and colchicine binding to tubulin. An X-ray study of 2-difluoromethoxy-3-benzyloxyestra-1,3,5(10)-trien-17-one examined conformational aspects of the fluorinated substituent. The known related derivative 2-difluoromethyl-3-sulfamoyloxyestrone was evaluated for STS inhibition in whole JEG-3 cells and showed an excellent IC50 of 55 pM.