Inhibition Of 11β-HSD2 Research Articles

7963 Analysis of the inhibition of adrenal synthesis and peripheral metabolism of corticosteroids by oteseconazole and two preclinical tetrazole antifungals

Abstract Disclosure: M. Jäger: None. V. González-Ruiz: None. F.L. Joos: None. D.V. Winter: None. J. Boccard: None. T. Degenhardt: Employee; Self; Mycovia Inc. S. Brand: Employee; Self; Mycovia Inc.. S. Rudaz: None. G.R. Thompson: None. A. Odermatt: None. The triazole antifungals posaconazole and itraconazole can cause pseudohyperaldosteronism with hypertension and hypokalemia by blocking adrenal cortisol synthesis and its peripheral inactivation [1]. Mechanisms of pseudohyperaldosteronism include inhibition of adrenal cytochrome P450 enzymes CYP11B1 and CYP17A1, as well as peripherally expressed 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2). To enhance specificity for fungal CYP51, tetrazole compounds have been developed [2]. Here, we used adrenal H295R cells and enzyme activity assays to assess the inhibition of steroidogenesis by the tetrazoles oteseconazole, VT-1129 and VT-1598. In addition, the three tetrazoles were tested for inhibition of 11β-HSD2. Steroidomic footprint analyses of H295R cell supernatants using untargeted LC-HRMS with steroid annotation indicated common features of the oteseconazole and VT-1129 patterns with that of the triazole itraconazole, and similarities between VT-1598 and the triazole isavuconazole. Targeted quantification of nine adrenal steroids by UHPLC-MS/MS in supernatants of treated H295R cells showed an overall inhibition of steroidogenesis by the tetrazoles and the triazoles itraconazole and isavuconazole. Experiments using recombinant human enzymes suggested that this effect is not caused by direct inhibition of steroidogenic enzymes because no or very weak inhibition could be observed. Furthermore, oteseconazole, VT-1129 and VT-1598 did not inhibit 11β-HSD2, suggesting that they unlikely cause pseudohyperaldosteronism. Further research needs to uncover the exact mechanism for the observed overall inhibition of steroidogenesis by the tetrazoles as well as itraconazole and isavuconazole, and whether concentrations reached in patients are high enough to cause adrenal insufficiency and hyperplasia.

Chalcones from plants cause toxicity by inhibiting human and rat 11β-hydroxysteroid dehydrogenase 2: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis

Chalcones from licorice and its related plants have many pharmacological effects. However, the effects of chalcones on the activity of human and rat 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), and associated side effects remain unclear. The inhibition of 11 chalcones on human and rat 11β-HSD2 were evaluated in microsomes and a 3D-quantitative structure-activity relationship (3D-QSAR) was analyzed. Screening revealed that bavachalcone, echinatin, isobavachalcone, isobavachromene, isoliquiritigenin, licochalcone A, and licochalcone B significantly inhibited human 11β-HSD2 with IC50 values ranging from 15.62 (licochalcone A) to 38.33 (echinatin) μM. Screening showed that the above chemicals and 4-hydroxychalcone significantly inhibited rat 11β-HSD2 with IC50 values ranging from 6.82 (isobavachalcone) to 72.26 (4-hydroxychalcone) μM. These chalcones acted as noncompetitive/mixed inhibitors for both enzymes. Comparative analysis revealed that inhibition of 11β-HSD2 depended on the species. Most chemicals bind to the NAD+ binding site or both the NAD+ and substrate binding sites. Bivariate correlation analysis showed that lipophilicity and molecular weight determine inhibitory strength. Through our 3D-QSAR models, we identified that the hydrophobic region, hydrophobic aliphatic groups, and hydrogen bond acceptors are pivotal factors in inhibiting 11β-HSD2. In conclusion, many chalcones inhibit human and rat 11β-HSD2, possibly causing side effects and there is structure-dependent and species-dependent inhibition on 11β-HSD2.

In vitro methods to assess 11β-hydroxysteroid dehydrogenase type 2 activity.

11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) converts active 11β-hydroxyglucocorticoids to their inactive 11-keto forms, fine-tuning the activation of mineralocorticoid and glucocorticoid receptors. 11β-HSD2 is expressed in mineralocorticoid target tissues such as renal distal tubules and cortical collecting ducts, and distal colon, but also in placenta where it acts as a barrier to reduce the amount of maternal glucocorticoids that reach the fetus. Disruption of 11β-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypernatremia, hypokalemia and hypertension. Secondary hypertension due to 11β-HSD2 inhibition has been observed upon consumption of excessive amounts of licorice and in patients treated with the azole fungicides posaconazole and itraconazole. Furthermore, inhibition of 11β-HSD2 during pregnancy with elevated exposure of the fetus to cortisol can cause neurological complications with a lower intelligence quotient, higher odds of attention deficit and hyperactivity disorder as well as metabolic reprogramming with an increased risk of cardio-metabolic disease in adulthood. This chapter describes in vitro methods for the determination of 11β-HSD2 activity that can be applied to identify inhibitors that may cause secondary hypertension and characterize the enzyme's activity in disease models. The included decision tree and the list of methods with their advantages and disadvantages aim to enable the reader to select and apply an in vitro method suitable for the scientific question and the equipment available in the respective laboratory.

Phthalates cause a low-renin phenotype commonly found in premature infants with idiopathic neonatal hypertension.

Since the 1970s, when the initial reports of neonatal hypertension related to renal artery thromboembolism were published, other secondary causes of neonatal hypertension have been reported. Those infants with no identifiable cause of hypertension were labeled with a variety of terms. Herein, we describe such infants as having idiopathic neonatal hypertension (INH). Most, but not all, of these hypertensive infants were noted to have bronchopulmonary dysplasia (BPD). More recently, reports described common clinical characteristics seen in INH patients, whether or not they had BPD. This phenotype includes low plasma renin activity, presentation near 40weeks postmenstrual age, and a favorable response to treatment with spironolactone. A small prospective study in INH patents showed evidence of mineralocorticoid receptor activation due to inhibition of 11β-HSD2, the enzyme that converts cortisol to the less potent mineralocorticoid-cortisone. Meanwhile, phthalate metabolites have been shown to inhibit 11β-HSD2 in human microsomes. Premature infants can come in contact with exceptionally large phthalate exposures, especially those infants with BPD. This work describes a common low-renin phenotype, commonly seen in patients categorized as having INH. Further, we review the evidence that hypertension in INH patients with the low-renin phenotype may be mediated by phthalate-associated inhibition of 11β-HSD2. Lastly, we review the implications of these findings regarding identification, treatment, and prevention of the low-renin hypertension phenotype seen in premature infants categorized as having INH.

A Novel N-Tert-Butyl Derivatives of Pseudothiohydantoin as Potential Target in Anti-Cancer Therapy.

Tumors are currently more and more common all over the world; hence, attempts are being made to explain the biochemical processes underlying their development. The search for new therapeutic pathways, with particular emphasis on enzymatic activity and its modulation regulating the level of glucocorticosteroids, may contribute to the development and implementation of new therapeutic options in the treatment process. Our research focuses on understanding the role of 11β-HSD1 and 11β-HSD2 as factors involved in the differentiation and proliferation of neoplastic cells. In this work, we obtained the 9 novel N-tert-butyl substituted 2-aminothiazol-4(5H)-one (pseudothiohydantoin) derivatives, differing in the substituents at C-5 of the thiazole ring. The inhibitory activity and selectivity of the obtained derivatives in relation to two isoforms of 11β-HSD were evaluated. The highest inhibitory activity for 11β-HSD1 showed compound 3h, containing the cyclohexane substituent at the 5-position of the thiazole ring in the spiro system (82.5% at a conc. 10 µM). On the other hand, the derivative 3f with the phenyl substituent at C-5 showed the highest inhibition of 11β-HSD2 (53.57% at a conc. of 10 µM). A low selectivity in the inhibition of 11β-HSD2 was observed but, unlike 18β-glycyrrhetinic acid, these compounds were found to inhibit the activity of 11β-HSD2 to a greater extent than 11β-HSD1, which makes them attractive for further research on their anti-cancer activity.

Molecular mechanisms of posaconazole- and itraconazole-induced pseudohyperaldosteronism and assessment of other systemically used azole antifungals

Recent reports described cases of severe hypertension and hypokalemia accompanied by low renin and aldosterone levels during antifungal therapy with posaconazole and itraconazole. These conditions represent characteristics of secondary endocrine hypertension caused by mineralocorticoid excess. Different mechanisms can cause mineralocorticoid excess, including inhibition of the adrenal steroidogenic enzymes CYP17A1 and CYP11B1, inhibition of the peripheral cortisol oxidizing enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) or direct activation of the mineralocorticoid receptor (MR). Compared to previous experiments revealing a threefold more potent inhibition of 11β-HSD2 by itraconazole than with posaconazole, the current study found sevenfold stronger CYP11B1 inhibition by posaconazole over itraconazole. Both compounds most potently inhibited CYP11B2. The major pharmacologically active itraconazole metabolite hydroxyitraconazole (OHI) resembled the effects of itraconazole but was considerably less active. Molecular modeling calculations assessed the binding of posaconazole, itraconazole and OHI to 11β-HSD2 and the relevant CYP enzymes, and predicted important interactions not formed by the other systemically used azole antifungals, thus providing an initial explanation for the observed inhibitory activities. Together with available clinical observations, the presented data suggest that itraconazole primarily causes pseudohyperaldosteronism through cortisol-induced MR activation due to 11β-HSD2 inhibition, and posaconazole by CYP11B1 inhibition and accumulation of the mineralocorticoids 11-deoxycorticosterone and 11-deoxycortisol because of hypothalamus-pituitary-adrenal axis (HPA) feedback activation. Therapeutic drug monitoring and introduction of upper plasma target levels may help preventing the occurrence of drug-induced hypertension and hypokalemia. Furthermore, the systemically used azole antifungals voriconazole, isavuconazole and fluconazole did not affect any of the mineralocorticoid excess targets, offering alternative therapeutic options.

Application of ELISA Technique and Human Microsomes in the Search for 11β-Hydroxysteroid Dehydrogenase Inhibitors.

The metabolic syndrome is defined by impaired carbohydrate metabolism and lipid disorders and often accompanied by hypertension, all of which will lead to obesity and insulin resistance. Glucocorticoids play a regulatory role in the metabolism of proteins, lipids, and carbohydrates. There is growing evidence for a role of glucocorticoids in the development of the metabolic syndrome. The most important factor that regulates the access of endogenous glucocorticoids to receptors after release of glucocorticoids and their diffusion into the cytoplasm of target cells is the steroid metabolism involving a microsomal enzyme, 11β-hydroxysteroid dehydrogenase (11β-HSD). The changes in intracellular glucocorticoid metabolism in the pathogenesis of obesity indicate the participation of modulation by 11β-HSD1, which may represent a new therapeutic target for the treatment of diseases such as type 2 diabetes, visceral obesity, or atherosclerosis. The aim of our study was to determine the fast and effective method to assess inhibition activity of compounds in relation with 11β-hydroxysteroid dehydrogenase. The material for this study was human liver and kidney microsomes. In this study we used ELISA technique using 96-well microplates coated with antibodies which were specific for analyzed enzymes. The method can quickly and efficiently measure the inhibition of both 11β-HSD1 and 11β-HSD2. This method can be used to search for and determine inhibitors of this enzyme. Cortisone and cortisol were used as the substrates for corresponding enzyme assays. Furthermore, 3-N-allyl-2-thiouracil derivatives were used by us for comparison purposes in developing the method, although, due to their structure, those derivatives have not previously been considered as potential inhibitors of 11β-HSD1. 3-N-Allyl-2-thiouracil derivatives are a group worth considering, because by modifying their structure (e.g., by introducing other substituents into the pyrimidine ring) it will be possible to obtain an increase in the activity of compounds in this regard. In conclusion, this study shows an efficient and fast method of determining inhibition activity of compounds in relation with 11β-hydroxysteroid dehydrogenase.

Corticosteroid Regulation of ENaC‐Mediated Na+ Transport in a Cellular Model of the Cortical Collecting Duct

Na+ absorption in the distal nephron is under strict hormonal control, perhaps most notably by the mineralocorticoid aldosterone. The specificity of aldosterone action is conferred by the enzyme 11β‐hydroxysteroid dehydrogenase 2 (11βHSD2) which is thought to protect the mineralocorticoid receptor (MR) from illicit occupancy by glucocorticoids, which circulate freely at ~100 fold greater concentration. In vivo studies challenge this model e.g. mice haploinsufficient for 11βHSD2 exhibit salt‐sensitive hypertension associated with downregulated ENaC‐mediated Na+ absorption1. This downregulation can be reversed by glucocorticoid receptor (GR), but not MR, blockade, strongly indicating a role for the GR. The present study has therefore utilised a cellular model of the collecting duct to interrogate the molecular pathways enabling corticosteroids to regulate ENaC activity.ENaC‐mediated Na+ transport was quantified across monolayers of mCCDcl1 cells via electrometric measurements of amiloride‐sensitive current (Iami). SGK1 abundance and activity were monitored by Western blot analysis.Time course experiments revealed that both aldosterone (3nM) and the synthetic glucocorticoid dexamethasone (100nM) stimulated Iami within 1h. Whilst aldosterone‐induced Iami peaked at 3–6h before returning to baseline, dexamethasone‐induced Iami remained stimulated after 24h. Increasing concentrations of the endogenous glucocorticoid corticosterone (0.01–100nM) did not alter Iami, indicative of functional 11βHSD2. At concentrations ≥1μM however, Iami was stimulated indicating the capacity of the enzyme had been surpassed. Inhibition of 11βHSD2 with carbenoxolone (CBX) unmasked a concentration‐dependent stimulation of Iami by corticosterone appearing at 1nM. Changes in Iami correlated with increased abundance and activity of the serum and glucocorticoid‐induced kinase 1 (SGK1). Aldosterone also stimulated Iami, as well as SGK1 abundance and activity, in a concentration‐dependent manner but was unaffected by pre‐treatment with CBX confirming that aldosterone is not a substrate for 11βHSD2. To determine the steroid receptors involved in mediating the effects of corticosteroids, Iami was measured following pharmacological inhibition of the MR using the non‐steroidal compound PF‐03882845 or of the GR using mifepristone. Application of aldosterone (3nM), dexamethasone (100nM) or corticosterone (100nM, in the presence of CBX) for 3h, stimulated Iami by 2.8‐, 3.9‐ and 3.6‐fold, respectively. Both aldosterone‐ and corticosterone‐induced Iami were inhibited predominantly (>65%) by MR antagonism, but were also inhibited by GR antagonism, ~55% and ~20%, respectively. In contrast, dexamethasone‐induced Iami was inhibited predominantly by GR antagonism, with only a small component inhibited by MR blockade.Whilst stimulation of ENaC‐mediated Na+ transport by aldosterone and corticosterone, unlike dexamethasone, is predominantly mediated by MR, these data indicate that the GR may play a permissive role in mediating the Na+ retaining effects of endogenous corticosteroids in the distal nephron.Support or Funding InformationFunded by a Kidney Research UK Intermediate Fellowhship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Harnessing the gatekeepers of glucocorticoids for chemoprevention of non-melanoma skin cancer.

Despite effective surgical methods for non‐melanoma skin cancer (NMSC), patients suffer from tissue damage, scarring, or even disfigurement; thus, there is a need for chemopreventive approaches. Because of the complex interplay between glucocorticoids (GCs), inflammation, and cancer, we sought to determine the role of 11β‐hydroxysteroid dehydrogenase 1 and 2 (11βHSD1 and 2) in regulating GCs during skin cancer development and progression. 11βHSDs modulate the activation of GCs in a tissue‐specific manner and have been reported to play a role in development and progression of other types of cancer, but their role has not yet been reported in NMSC. Here, we found a significant upregulation of 11βHSD2 protein in skin cancer cells when compared to normal skin cells, suggesting a role for this enzyme in the multifactorial process of skin cancer development. In addition, inhibition of 11βHSD2 with siRNA resulted in significant reduction in colony formation in vitro. Finally, our in vivo study elucidated that inhibition of 11βHSD2 with pharmacological inhibitor, Glycyrrhetinic acid (GA) could significantly diminish tumorigenesis in a well‐studied in vivo mouse model of NMSC. Overall, these studies highlight for the first time a potential novel role for 11βHSD2 in NMSC development and may allow for new GC treatment approaches capable of avoiding deactivation by the enzyme. If 11βHSD2 can be inhibited as we have done here, or circumvented using modified GCs, this may lead to more efficacious outcomes for NMSC patients by preventing deactivation of the GC and minimizing resistance.

Posaconazole-Induced Hypertension and Hypokalemia: Mechanistic Evaluation

Background Recent studies have shown the new posaconazole delayed-release tablets have superior bioavailability compared with the liquid suspension formulation. As higher serum posaconazole concentrations have been associated with improved clinical responses, this formulation has been a welcome addition to available treatment options. However, higher serum and tissue levels are likely to reveal previously undescribed toxicity as adverse events attributed to “off-target” effects are observed.Methods We prospectively identified two patients with new onset hypertension, hypokalemia, and alkalosis after starting posaconazole tablets. Patient vital signs and laboratory values were within normal limits prior to starting posaconazole; however, following over 30 days of therapy both patients became newly hypertensive (mean systolic BP increase 59 mmHg). Serum posaconazole levels were 4.3–4.6 μg/ml. Complete suppression of renin and aldosterone, with increased 11-deoxycortisol, estradiol levels, and cortisol/cortisone ratios were observed in both patients. The TTKG in both patients was inappropriately elevated.ResultsPosaconazole-induced disruption of the steroid biosynthesis pathway in patients has not previously been described, but has been suggested by in vitro studies. Our patients’ laboratory results show clinically significant inhibition of 11β-hydroxysteroid dehydrogenase enzyme type 2 isoform (11β-HSD2) as evidenced by: the elevated 11-deoxycortisol (with subsequent suppression of renin and aldosterone), the highly elevated cortisol/cortisone ratio, and the inappropriately elevated TTKG in the setting of hypokalemia. The normal deoxycorticosterone confirms normal function of 11β-hydroxylase and the observed effects in our patients are thus downstream from this enzyme.Conclusion Our findings support in vitro predictions and highlight the clinical sequelae of posaconazole-mediated inhibition of 11β-HSD2. Additional studies are necessary to determine the frequency of posaconazole induced apparent mineralocorticoid excess syndrome and whether other azole antifungals can be associated with this phenomenon.Disclosures All authors: No reported disclosures.

Recent advances in the study of 11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2)Inhibitors.

11β-Hydroxysteroid dehydrogenase (11β-HSD), which interconverts hormonally active cortisol and inactive cortisone in multiple human tissues, has two distinct isoforms named 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) and 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). 11β-HSD2 is an NAD+-dependent oxidase which lowers cortisol by converting it to cortisone while 11β-HSD1 mainly catalyzes the reduction which converts cortisone into cortisol. Selective inhibition of 11β-HSD2 is generally detrimental to health because the accumulation of cortisol can cause metabolic symptoms such as apparent mineralocorticoid excess (AME), fetal developmental defects and lower testosterone levels in males. There has been some advances on the study of 11β-HSD2 inhibitors and we think it necessary to make a summary of the characteristics and inhibiting properties of latest 11β-HSD2 inhibitors. As another review on 11β-HSD2 inhibitors has been issued on 2011 (see review (Ma et al., 2011)), this mini-review concerns advances during the last 5 years.

Inhibition of 11β-hydroxysteroid dehydrogenase 2 by the fungicides itraconazole and posaconazole

Impaired 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2)-dependent cortisol inactivation can lead to electrolyte dysbalance, hypertension and cardiometabolic disease. Furthermore, placental 11β-HSD2 essentially protects the fetus from high maternal glucocorticoid levels, and its impaired function has been associated with altered fetal growth and a higher risk for cardio-metabolic diseases in later life. Despite its important role, 11β-HSD2 is not included in current off-target screening approaches. To identify potential 11β-HSD inhibitors among approved drugs, a pharmacophore model was used for virtual screening, followed by biological assessment of selected hits. This led to the identification of several azole fungicides as 11β-HSD inhibitors, showing a significant structure-activity relationship between azole scaffold size, 11β-HSD enzyme selectivity and inhibitory potency. A hydrophobic linker connecting the azole ring to the other, more polar end of the molecule was observed to be favorable for 11β-HSD2 inhibition and selectivity over 11β-HSD1. The most potent 11β-HSD2 inhibition, using cell lysates expressing recombinant human 11β-HSD2, was obtained for itraconazole (IC50 139±14nM), its active metabolite hydroxyitraconazole (IC50 223±31nM) and posaconazole (IC50 460±98nM). Interestingly, experiments with mouse and rat kidney homogenates showed considerably lower inhibitory activity of these compounds towards 11β-HSD2, indicating important species-specific differences. Thus, 11β-HSD2 inhibition by these compounds is likely to be overlooked in preclinical rodent studies. Inhibition of placental 11β-HSD2 by these compounds, in addition to the known inhibition of cytochrome P450 enzymes and P-glycoprotein efflux transport, might contribute to elevated local cortisol levels, thereby affecting fetal programming.

Inhibition of 11β-HSD2 expression by triclosan via induction of apoptosis in human placental syncytiotrophoblasts.

Triclosan is widely used in personal care products for its broad spectrum of antimicrobial effects, but triclosan is a potential endocrine disruptor. 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is a cortisol-inactivating enzyme that is highly expressed in human placental syncytiotrophoblasts to ensure normal fetal development in the presence of high levels of maternal cortisol in pregnancy. We investigated the effects of triclosan on 11β-HSD2 and apoptosis and the relationship between these two events in human placental syncytiotrophoblasts. Primary human placental cytotrophoblasts were isolated from term placenta. After syncytialization, the levels of 11β-HSD2 and apoptosis-related proteins including caspase3, Bcl-2, and Bax were examined after treatment with triclosan from 0.001 μM to 10 μM or triclosan (0.1 μM) in the presence and absence of apoptosis inhibitor Z-VAD-FMK (30 μM) for 24 h. Triclosan inhibited 11β-HSD2 mRNA, protein and activity levels in a concentration-dependent manner from 0.001 to 10 μM with a significant inhibition at 0.01 μM and above. Concurrently, triclosan induced apoptosis of human placental syncytiotrophoblasts as demonstrated by observations of increased nuclear condensation, DNA fragmentation and pro-apoptosis proteins such as Bax and cleaved-caspase3, decreased pro-caspase-3 and anti-apoptosis protein such as Bcl-2. Blocking apoptosis with Z-VAD-FMK attenuated the inhibition of 11β-HSD2 by triclosan significantly. Triclosan may attenuate the expression of placental 11β-HSD2 via the induction of apoptosis of placental syncytiotrophoblasts. This is likely to disrupt the placental glucocorticoid barrier and impair fetal development.

Epithelial-Specific Deletion of 11β-HSD2 Hinders Apcmin/+ Mouse Tumorigenesis

Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) promotes colorectal tumorigenesis. Glucocorticoids are endogenous and potent COX-2 inhibitors, and their local actions are downregulated by 11β-hydroxysteroid dehydrogenase type II (11β-HSD2)-mediated metabolism. Previously, it was reported that 11β-HSD2 is increased in human colonic and Apc(min/+) mouse intestinal adenomas and correlated with increased COX-2, and 11β-HSD2 inhibition suppressed the COX-2 pathway and decreased tumorigenesis. Because 11β-HSD2 is expressed in Apc(min/+) mouse intestinal adenoma stromal and epithelial cells, Apc(min/+) mice were generated with selective deletion of 11β-HSD2 in intestinal epithelial cells (Vil-Cre-HSD2(-/-) Apc(min/+)). Deletion of 11β-HSD2 in intestinal epithelia led to marked inhibition of Apc(min/+) mouse intestinal tumorigenesis. Immunostaining indicated decreased 11β-HSD2 and COX-2 expression in adenoma epithelia, whereas stromal COX-2 expression was intact in Vil-Cre-HSD2(-/-) Apc(min/+) mice. In Vil-Cre-HSD2(-/-) Apc(min/+) mouse intestinal adenomas, both p53 and p21 mRNA and protein were increased, with a concomitant decrease in pRb, indicating glucocorticoid-mediated G1-arrest. Further study revealed that REDD1 (regulated in development and DNA damage responses 1), a novel stress-induced gene that inhibits mTOR signaling, was increased, whereas the mTOR signaling pathway was inhibited. Therefore, in Vil-Cre-HSD2(-/-) Apc(min/+) mice, epithelial cell 11β-HSD2 deficiency leads to inhibition of adenoma initiation and growth by attenuation of COX-2 expression, increased cell-cycle arrest, and inhibition of mTOR signaling as a result of increased tumor intracellular active glucocorticoids. Inhibition of 11β-HSD2 may represent a novel approach for colorectal cancer chemoprevention by increasing tumor glucocorticoid activity, which in turn inhibits tumor growth by multiple pathways.

Down-Regulation of 11β-Hydroxysteroid Dehydrogenase Type 2 by Bortezomib Sensitizes Jurkat Leukemia T Cells against Glucocorticoid-Induced Apoptosis

11β-hydroxysteroid dehydrogenases type 2 (11β-HSD2), a key regulator for pre-receptor metabolism of glucocorticoids (GCs) by converting active GC, cortisol, to inactive cortisone, has been shown to be present in a variety of tumors. But its expression and roles have rarely been discussed in hematological malignancies. Proteasome inhibitor bortezomib has been shown to not only possess antitumor effects but also potentiate the activity of other chemotherapeutics. In this study, we demonstrated that 11β-HSD2 was highly expressed in two GC-resistant T-cell leukemic cell lines Jurkat and Molt4. In contrast, no 11β-HSD2 expression was found in two GC-sensitive non-hodgkin lymphoma cell lines Daudi and Raji as well as normal peripheral blood T cells. Inhibition of 11β-HSD2 by 11β-HSD inhibitor 18β-glycyrrhetinic acid or 11β-HSD2 shRNA significantly increased cortisol-induced apoptosis in Jurkat cells. Additionally, pretreatment of Jurkat cells with low-dose bortezomib resulted in increased cellular sensitivity to GC as shown by elevated induction of apoptosis, more cells arrested at G1 stage and up-regulation of GC-induced leucine zipper which is an important mediator of GC action. Furthermore, we clarified that bortezomib could dose-dependently inhibit 11β-HSD2 messenger RNA and protein levels as well as activity (cortisol-cortisone conversion) through p38 mitogen-activated protein kinase signaling pathway. Therefore, we suggest 11β-HSD2 is, at least partially if not all, responsible for impaired GC suppression in Jurkat cells and also indicate a novel mechanism by which proteasome inhibitor bortezomib may influence GC action.

3-Monoglucuronyl-Glycyrrhretinic Acid Is a Substrate of Organic Anion Transporters Expressed in Tubular Epithelial Cells and Plays Important Roles in Licorice-Induced Pseudoaldosteronism by Inhibiting 11β-Hydroxysteroid Dehydrogenase 2

Licorice (glycyrrhiza root) has been used as a herbal medicine worldwide with its main active constituent being glycyrrhizin (GL). Licorice sometimes causes adverse effects such as inducing pseudoaldosteronism by inhibiting type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) caused by glycyrrhetinic acid (GA), a major metabolite of GL. In this study we compared the inhibitory effects of GA, GL, and 3-monoglucuronyl-glycyrrhetinic acid (3MGA), another metabolite of GL, on 11β-HSD2 activity by using microsomes and rat kidney tissue slices. GA, 3MGA, and GL inhibited 11β-HSD2 in rat kidney microsomes, with IC(50) values of 0.32, 0.26, and 2.2 μM, respectively. However, the inhibitory activity of these compounds was reduced markedly, in the slices, in a medium containing 5% bovine serum albumin. Assays using human embryonic kidney 293 cells with transient transformation in transporter genes showed that 3MGA is a substrate of human organic anion transporter (OAT) 1, human OAT3, and human organic anion-transporting peptide 4C1, whereas GA is not. When GA (100 mg/kg/day) was administered orally for 16 days to Eisai hyperbilirubinemic rats, plasma concentrations and urinary excretion of 3MGA were significantly higher, whereas the activity of 11β-HSD2 in kidney microsomes was significantly lower compared with Sprague Dawley rats. These results suggest that 3MGA is actively transported into tubules through OATs, resulting in the inhibition of 11β-HSD2. Because the plasma level of 3MGA depends on the function of hepatic transporters, monitoring 3MGA levels in plasma or urine may be useful for preventing pseudoaldosteronism when licorice or GL is prescribed to patients.

The Anabolic Androgenic Steroid Fluoxymesterone Inhibits 11 -Hydroxysteroid Dehydrogenase 2-Dependent Glucocorticoid Inactivation

Anabolic androgenic steroids (AAS) are testosterone derivatives used either clinically, in elite sports, or for body shaping with the goal to increase muscle size and strength. Clinically developed compounds and nonclinically tested designer steroids often marketed as food supplements are widely used. Despite the considerable evidence for various adverse effects of AAS use, the underlying molecular mechanisms are insufficiently understood. Here, we investigated whether some AAS, as a result of a lack of target selectivity, might inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2)-dependent inactivation of glucocorticoids. Using recombinant human 11β-HSD2, we observed inhibitory effects for several AAS. Whereas oxymetholone, oxymesterone, danazol, and testosterone showed medium inhibitory potential, fluoxymesterone was a potent inhibitor of human 11β-HSD2 (half-maximal inhibitory concentration [IC(50)] of 60-100nM in cell lysates; IC(50) of 160nM in intact SW-620, and 530nM in MCF-7 cells). Measurements with rat kidney microsomes and lysates of cells expressing recombinant mouse 11β-HSD2 revealed much weaker inhibition by the AAS tested, indicating that the adverse effects of AAS-dependent 11β-HSD2 inhibition cannot be investigated in rats and mice. Furthermore, we provide evidence that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11β-HSD2. Structural modeling revealed similar binding modes for fluoxymesterone and cortisol, supporting a competitive mode of inhibition of 11β-HSD2-dependent cortisol oxidation by this AAS. No direct modulation of mineralocorticoid receptor (MR) function was observed. Thus, 11β-HSD2 inhibition by fluoxymesterone may cause cortisol-induced MR activation, thereby leading to electrolyte disturbances and contributing to the development of hypertension and cardiovascular disease.

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11β-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.

Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2

Modulation of intracellular glucocorticoid availability is considered as a promising strategy to treat glucocorticoid-dependent diseases. 18β-Glycyrrhetinic acid (GA), the biologically active triterpenoid metabolite of glycyrrhizin, which is contained in the roots and rhizomes of licorice ( Glycyrrhiza spp.), represents a well-known but non-selective inhibitor of 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, to assess the physiological functions of the respective enzymes and for potential therapeutic applications selective inhibitors are needed. In the present study, we applied bioassays and 3D-structure modeling to characterize nine 11β-HSD1 and fifteen 11β-HSD2 inhibiting GA derivatives. Comparison of the GA derivatives in assays using cell lysates revealed that modifications at the 3-hydroxyl and/or the carboxyl led to highly selective and potent 11β-HSD2 inhibitors. The data generated significantly extends our knowledge on structure–activity relationship of GA derivatives as 11β-HSD inhibitors. Using recombinant enzymes we found also potent inhibition of mouse 11β-HSD2, despite significant species-specific differences. The selected GA derivatives potently inhibited 11β-HSD2 in intact SW-620 colon cancer cells, although the rank order of inhibitory potential differed from that obtained in cell lysates. The biological activity of compounds was further demonstrated in glucocorticoid receptor (GR) transactivation assays in cells coexpressing GR and 11β-HSD1 or 11β-HSD2. 3D-structure modeling provides an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11β-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Article from the Special issue on Targeted Inhibitors.

In silico access for the discovery of 11β-HSD1 inhibiting triterpenes from Eriobotrya japonica

An elevated activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) has been associated with metabolic disorders including obesity and type 2 diabetes, with recent findings providing evidence for beneficial effects of 11β-HSD1 inhibitors, making this enzyme a promising therapeutic target [1]. Recently we investigated different extracts of traditionally used anti-diabetic medicinal plants for potential anti-glucocorticoid effects. Among them, the extracts of E. japonica (Thunb.) Lindl. showed a dose-dependent inhibition of 11β-HSD1, with a preferential inhibition of 11β-HSD1 versus 11β-HSD2 measured in cell lysates. These results were confirmed in intact stably transfected HEK-293 cells [2]. In this study the loquat leaves were phytochemically investigated following predictions of a pharmacophore-based virtual screening. Determination of the 11β-HSD1 and 11β-HSD2 inhibitory activities in cell lysates of virtually predicted hits in combination with a bioactivity-guided approach revealed triterpenes from the ursane type as selective, low µmolar inhibitors of 11β-HSD1: corosolic acid (IC50 0.81µM), 3-epicorosolic acid methyl ester (IC50 5.2µM), 2-α hydroxy-3-oxo urs-12-en-28-oic acid (IC50 17µM), tormentic acid methyl ester (IC50 9.4µM), and ursolic acid (IC50 1.9µM). Intriguingly, a mixture of loquat constituents with moderate activities displayed a pronounced additive effect. By means of molecular modeling studies and the identification of the 11β-HSD1 inhibiting 11-keto-ursolic acid (IC50 2.1µM) and 3-acetyl-11-keto-ursolic acid (IC50 1.3µM) a structure-activity relationship was deduced for this group of pentacyclic triterpenes [3]. The mechanism elucidated in this study together with previously determined pharmacological activities provides this class of compounds from loquat with an astonishing multi-targeted anti-diabetic profile.