Inhibition of Sulfotransferase E1 (SULT1E1)

Biological state

Sulfotransferases (SULTs) are phase-2 enzymes responsible for the sulfation of various endogenous and exogenous substrates using 3’-phosphoadenosine 5’- phosphosulfate (PAP-sulfate, PAPS) as a sulfate donor (Fig. 1).

Figure 1.  SULT-catalyzed reaction with PAPS as the cosubstrate (Wang and James, 2015).

SULTs are single globular proteins with a characteristic α/β motif consisting of a five-stranded parallel β -sheet surrounded by α -helices. The b-sheet constitutes the PAPS-binding site and the core of the catalytic site. While sequence conservation is rather low between SULTs, their fold and catalytic features are highly conserved. Two essential components of their actions are the PAPS binding activity (which is common among various SULTs) and the substrate binding activity (which is specific for individual SULTs) (Wang and James, 2006). SULTs are traditionally named after their preferred substrate. Nevertheless, SULTs show a broad substrates specificity, thanks to plasticity at the active site (Mueller et al., 2015).

The occupation of one or both of the two essential components of SULT (PAPS and substrate binding regions) may cause inhibition of SULT activity. Various xenobiotics including dietary and environmental chemicals, some therapeutic drugs, PAPS analogues, derivatives of substrate, some library compounds, etc were shown to inhibit one or more SULT. Some inhibitors were isoform selective, while others inhibited all forms of SULT (Wang and James, 2006).

At least 9 different forms of human SULT have been cloned and sequenced and according to their amino acid identity they are classified into distinct families (> 45% amino acid sequence identity) and subfamilies (> 60% amino acid sequence identity). The SULT1 family members are divided in four subfamilies e.g., 1A, 1B, 1C and 1E.

SULT1E1 is a key enzyme in estrogen homeostasis, the sulfation of estrogens leads to their inactivation i.e., the molecule has no effect at the estrogen receptor (UniProt, available at: https://www.uniprot.org/; Hempel et al., 2000) (Fig. 2).

Figure 2. Schematic sulfation pathway of estrogens. STS, steroid sulfatase; PAPS, 3’ -phosphoadenosine 5’ -phosphosulfate; PAP, 3’ -phosphoadenosine 5’ -phosphate (adapted from Yi et al., 2021).

The enzyme also sulfates dehydroepiandrosterone (DHEA), pregnenolone, (24S)-hydroxycholesterol and xenobiotic compounds like ethinylestradiol, equalenin, diethyl stilbesterol and 1-naphthol at significantly lower efficiency. In fact, the preferred substrate of SULT1E1 is oestradiol as reflected by the low Km and high Vmax/Km values (Figure 3) (UniProt; Hempel et al., 2000).

Figure 3. Kinetic Analysis of Wild-Type humSULT1E1 Proteins with Various Sulfotransferase Substrates 9adapted from Hempel et al., 2000).

The maximal E2 sulfation was observed at a concentration of approx. 20 nM with substrate inhibition at higher E2 concentrations (Falany et al., 1995).

Biological compartment

In the mammalian organisms there are membrane and cytosolic SULTs. The membrane bound sulfotransferases sulfonate large endogenous molecules playing a crucial role in the post-translation modification of proteins. Cytosolic sulfotransferases sulfonate endogenous steroids and neurotransmitters, this resulting in alteration of their activity (UniProt: Hempel et al., 2000). SULT1E1 enzyme belongs to the category of cytoplasmic soluble sulfotransferases, with the protein mainly localised in the cytosol (Reinen and Vermeulen 2015).

SULT1E1 gene is moderately to highly (intestine, liver, skin, vagina) expressed in various human body tissues (UniProt, Human Protein Atlas - HPA). Regarding the uterus, it was demonstrated by Falany et al., 1998, and then by Utsunomiya and colleagues in 2004, that normal cycling endometrium expresses active SULT1E1 protein in glandular cells during the secretory phase of menstrual cycle. The results of the studies above mentioned indicate that the expression of SULT1E1 in human endometrial tissues is most likely regulated by progesterone secreted from the ovaries.

Orthologs to SULT1E1 are described in various mammalian species (including rat and mouse) (bgee.org).

In rodents, data demonstrates expression of SULT1E1 in the uterus of mouse, whereas no clear information is available in rats (bgee.org). This is in line with the information reported in another study indicating that the expression of SULT1E1 is species specific; however, despite differences in the tissue distribution pathway it can be concluded that SULT1E1 is mainly expressed in male and female sex organs as well as placenta (Alnouti and Klaassen, 2006).

General role in biology

SULT1E1 is an enzyme responsible for a major pathway of E2 biotransformation (sulfation) and plays a key role in E2 bioavailability and activity in target tissues.

The sulfation of E2 in the liver makes the compound more soluble and favours its renal excretion, but also creates plasmatic stores of sulphated E2 increased stability and longer half-life than unconjugated compounds (e.g., 10– 12 h vs. 20–30 min for and a longer half-life than E2) that serve as a reservoir for the formation of biologically active steroids (Reed et al., 2005, Shevtsov et al., 2003). The sulfation of E2 in estrogen sensitive tissues by locally expressed SULT1E1, contributes to determining the ratio of the active free estrogen to the inactive estrogen sulphate in an interplay with desulphation by the enzyme sulfatase (reviewed by Alnouti and Klaassen, 2006). Local SULT1E1 is suggested to play an important role in protecting estrogen sensitive tissues from extreme estrogenic effects (Secky et al. 2013).

Inhibition of SULT1E1 activity by Endocrine Disruptive Chemicals (EDCs) or lower SULT1E1 expression levels may lead to a higher availability of biologically active E2 in the endometrium, which can result in a higher cell proliferation or estrogen stimulated DNA synthesis (Reinen and Vermeulen 2015). The expression of SULT1E1 was significantly downregulated (p = 0.0392) in endometrial cancer tissue from premenopausal women, with significantly lower levels seen in cancer and adjacent control tissue from postmenopausal women as compared to premenopausal women (Sinhrein et al., 2017).

While inhibition of SULT1E1 can occur in various tissues, in the context of AOP504 the uterine compartment is considered.

Both SULT1E1 expression and function can be measured in tissues and in recombinant systems, such as e.g., Salmonella typhimurium (Kester et al., 2002), Escherichia coli (Parker et al., 2018), V79-1E1 Chinese hamster cells line (Hamers et al., 2006) and in HepG2 cell (Maiti et al., 2007).

Expression (gene expression and proteins level)

• qRT-PCRn, Northern blotting: SULT1E1 expression (Konings et al., 2018)
• Western blotting: SULT1E1 protein levels (Konings et al., 2018)
• Immunohistochemistry: protein levels (Konings et al., 2018)

Activity (formation of E2 sulfate)

• Sulfotransferase kinetic assay: radiometric assays (Hempel et al., 2000; Falany et al., 1995)

In these assays, different types of inhibition of SULT activity, such as competitive, non-competitive and mixed type can be also determined by Lineweaver-Burk analysis (Kester et al., 2002; Hamers et al., 2006).

• Levels of sulphated estrogens and/or ratio of conjugated E2/non conjugated E2 (in tissue or in plasma):
  • liquid chromatography/mass spectrometry (LC/MS); LC-MS/MS after chemical or enzymatic hydrolysis and extraction (Borghoff et al., 2016, Wudy et al., 2018).
  • Radioimmuno Assay (RIA)
• Computational models for SULTE1 activation and such as QSAR model for E2-SULT inhibition are also used to demonstrate the criteria for potent inhibition of estrogen sulfotransferase (Harju et al., 2007; Gosavi et al., 2013). 

SULT1E1 is the high-affinity enzyme responsible for E2 sulfation at nanomolar concentrations and it is expressed in various tissues. SULT1E1 in the liver plays a role in the sulfation of circulating E2, and intestine-liver during hepatic-mediated first-pass metabolism; SULT1E1 in estrogen sensitive tissues has a role in the local modulation of E2 sulfation. Estrogenic signalling is not elicited by sulphated E2 since it does not bind to ERs. Inhibition of SULT1E1 could decrease E2 sulfation, and consequently increase the levels of non-sulphated (active) E2 available to estrogenic activation pathways.

Sex: Both sexes.

Taxonomic Applicability: in the context of AOP504 the taxonomic applicability is restricted to mammals; however, SULT1E1 is also present in other species invertebrate species and fish/avian/amphibian. SULT1E1 was identified and cloned in human and various mammalian species (Fig. 4). Overall, it has been reported that protein sequence homology between human and some mammalian species is above 71% (UniProt, Yi et al., 2021).

Table 1. SULT1E1 expression in other mammalian species (adapted from Yi et al., 2021).

The first SULT1E1 crystal structure to be reported in mammals was the mouse estrogen sulfotransferase (mouSULT1E1); mouSULT1E1 and humSULT1E1 share the 77% amino acid identify and conserve the main active sites (Fig. 5), the Km for E2 is in the nanomolar range for both the species.

Figure 5. Schematic diagram of the humSULT1E1 substrate binding (Hempel et al., 2000).

In rodents, data demonstrates expression of SULT1E1 in uterus of mouse, whereas no clear information is available in rats (bgee.org).

Studies in marine and freshwater fish have shown that steroid catabolism pathways are broadly similar to those identified in mammals, including the participation of SULT enzymes; however, it is noted that none of the enzyme studies exhibit Km values at nanomolar levels, as reported for human SULT1E1; further research in this area is needed (James, 2011).

Invertebrates are not investigated as part of AOP504.

Life Stages: SULT1E1 is expressed in the human embryo and is also highly expressed in a wide range of foetal tissues, such as the liver, lung, kidney, and hormone-dependent tissues—such as the testis or endometrium; its expression in adults is lower than in the foetus and placenta (Yi et al., 2021). However, AOP504 is focused on adulthood.

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