Molecular biology of 11β-hydroxylase and 11β-hydroxysteroid dehydrogenase enzymes

There are two steroid 11β-hydroxylase isozymes encoded by the CYP11B1 and CYP11B2 genes on human chromosome 8q. The first is expressed at high levels in the normal adrenal gland, has 11β-hydroxylase activity and is regulated by ACTH. Mutations in the corresponding gene cause congenital adrenal hyperplasia due to 11β-hydroxylase deficiency; thus, this isozyme is required for cortisol biosynthesis. The second isozyme is expressed at low levels in the normal adrenal gland but at higher levels in aldosterone-secreting tumors, and has 11β-hydroxylase, 18-hydroxylase and 18-oxidase activities. The corresponding gene is regulated by angiotensin II, and mutations in this gene are found in persons who are unable to synthesize aldosterone due to corticosterone methyloxidase II deficiency. Thus, this isozyme is required for aldosterone biosynthesis.

Cortisol and aldosterone are both effective ligands of the “mineralocorticoid” receptor in vitro, but only aldosterone is a potent mineralocorticoid in vivo. This apparent specificity occurs because 11β-hydroxysteroid dehydrogenase in the kidney converts cortisol to cortisone, which is not a ligand for the receptor. This enzyme is a “short-chain” dehydrogenase which is encoded by a single gene on human chromosome 1. It is possible that mutations in this gene cause a form of childhood hypertension called apparent mineralocorticoid excess, in which the mineralocorticoid receptor is not protected from high concentrations of cortisol.     

A urine-concentrating defect in 11β-hydroxysteroid dehydrogenase type 2 null mice

In aldosterone target tissues, 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) is coexpressed with mineralocorticoid receptors (MR) and protects the receptor from activation by glucocorticoids. Null mutations in the encoding gene, HSD11B2, cause apparent mineralocorticoid excess, in which hypertension is thought to reflect volume expansion secondary to sodium retention. Hsd11b2(-/-) mice are indeed hypertensive, but impaired natriuretic capacity is associated with significant volume contraction, suggestive of a urine concentrating defect. Water turnover and the urine concentrating response to a 24-h water deprivation challenge were therefore assessed in Hsd11b2(-/-) mice and controls. Hsd11b2(-/-) mice have a severe and progressive polyuric/polydipsic phenotype. In younger mice (～2 mo of age), polyuria was associated with decreased abundance of aqp2 and aqp3 mRNA. The expression of other genes involved in water transport (aqp4, slc14a2, and slc12a2) was not changed. The kidney was structurally normal, and the concentrating response to water deprivation was intact. In older Hsd11b2(-/-) mice (>6 mo), polyuria was associated with a severe atrophy of the renal medulla and downregulation of aqp2, aqp3, aqp4, slc14a2, and slc12a2. The concentrating response to water deprivation was impaired, and the natriuretic effect of the loop diuretic bumetanide was lost. In older Hsd11b2(-/-) mice, the V2 receptor agonist desmopressin did not restore full urine concentrating capacity. We find that Hsd11b2(-/-) mice develop nephrogenic diabetes insipidus. Gross changes to renal structure are observed, but these were probably secondary to sustained polyuria, rather than of developmental origin.     

Chronic food restriction up-regulates 11-β-hydroxysteroid dehydrogenase

Corticosterone — product of 11-β-hydroxysteroid dehydrogenase type I (11βHSD1) stimulates adipocytes differentiation and activates lipogenic enzymes gene expression in white adipose tissue (WAT) of rats. The aim of the study was to examine the effect of chronic food restriction, often practised by obese individuals trying to lose body mass, on: a) 11βHSD1 gene expression, b) expression of genes associated with adipocyte differentiation (PPARg, SREBP-1, adiponectin), and c) expression of genes associated with lipogenesis in WAT of rats. Two-month old rats were divided into a control and a food restricted group obtaining 50% of food consumed by controls for 30 days. mRNA levels of studied genes in perirenal WAT were analysed by real-time PCR. 11βHSD1 and lipogenic enzymes activities were measured by radiometric conversion assay and by spectrophotometric assay respectively. Food restriction caused significant increase of 11βHSD1, PPARg, SREBP1, adiponectin and lipogenic enzymes mRNA levels in perirenal WAT. 11βHSD1 and some lipogenic enzymes activities were also increased by food restriction. The coordinated up-regulation of 11βHSD1, and genes associated with adipocyte differentiation and lipogenesis by food restriction suggests that such nutritional condition shifts WAT metabolism, that would permit this tissue to synthesize and accumulate triacylglycerols immediately after refeeding.     

Mono-carbonyl curcumin analogues as 11β-hydroxysteroid dehydrogenase 1 inhibitors

A series of structurally novel mono-carbonyl curcumin analogues have been synthesized and biologically evaluated to test their inhibitory potencies and the structure–activity relationship (SAR) on human and rat 11β-hydroxysteroid dehydrogenase isoform (11β-HSD1) activities. 11β-HSD1 selective inhibitors have been discovered and compound A10 is discovered as a very potent with an IC₅₀ value of 97 nM without inhibiting 11β-HSD2.     

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC₅₀ value similar to that in in vitro microsomal assay (IC₅₀ = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.     

Novel enzymological profiles of human 11β-hydroxysteroid dehydrogenase type 1

The human enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) catalyzes the reversible oxidoreduction of 11β-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11β-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11β-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11β-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11β-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Discovery of cyclicsulfonamide derivatives as 11β-hydroxysteroid dehydrogenase 1 inhibitors

A new series of cyclic sulfonamide derivatives was synthesized and evaluated for their ability to inhibit 11β-HSD1. Cyclic sulfonamides with phenylacetyl substituents at the 2-position showed nanomolar inhibitory activities. Among them, compound 4e exhibited a good in vitro inhibitory activity and selectivity toward human 11β-HSD2.     

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10⁻¹³ M cortisol, whereas 1 × 10⁻⁵ M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.     

Synthesis of sterically encumbered 11β-aminoprogesterone derivatives and evaluation as 11β-hydroxysteroid dehydrogenase inhibitors and mineralocorticoid receptor antagonists

11β-Hydroxyprogesterone is a well-known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11βHSD) types 1 and 2. It also activates the mineralocorticoid receptor (MR). Modulation of corticosteroid action by inhibition of 11βHSDs or blocking MR is currently under consideration for treatment of electrolyte disturbances, metabolic diseases and chronic inflammatory disorders. We established conditions to synthesize sterically demanding 11β-aminoprogesterone, which following subsequent nucleophilic or reductive amination, allowed extension of the amino group to prepare amino acid derivatives. Biological testing revealed that some of the 11β-aminoprogesterone derivatives selectively inhibit 11βHSD2. Moreover, two compounds that did not significantly inhibit 11βHSDs had antagonist properties on MR. The 11β-aminoprogesterone derivatives form a basis for the further development of improved modulators of corticosteroid action.     

Adamantyl carboxamides and acetamides as potent human 11β-hydroxysteroid dehydrogenase type 1 inhibitors

The modulation of 11β-HSD1 activity with selective inhibitors has beneficial effects on various metabolic disorders including insulin resistance, dyslipidemia and obesity. Here we report the discovery of a series of novel adamantyl carboxamide and acetamide derivatives as selective inhibitors of human 11β-HSD1 in HEK-293 cells transfected with the HSD11B1 gene. Optimization based on an initially identified 11β-HSD1 inhibitor (3) led to the discovery of potent inhibitors with IC₅₀ values in the 100 nM range. These compounds are also highly selective 11β-HSD1 inhibitors with no activity against 11β-HSD2 and 17β-HSD1. Compound 15 (IC₅₀ = 114 nM) with weak inhibitory activity against the key human cytochrome P450 enzymes and moderate stability in incubation with human liver microsomes is worthy of further development. Importantly, compound 41 (IC₅₀ = 280 nM) provides a new lead that incorporates an adamantyl group surrogate and should enable further series diversification.     

The inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 by perfluoroalkylated substances

11β-Hydroxysteroid dehydrogenase 2 (11β-HSD2) regulates active glucocorticoid access to glucocorticoid and mineralocorticoid receptors by metabolizing it to an inactive form. Perfluoroalkylated substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. We tested the inhibitory potencies of four PFASs including perfluorooctanoic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonate (PFHxS) and perfluorobutane sulfonate (PFBS) on human and rat 11β-HSD2. PFOS was a potent inhibitor of both human (IC(50)=48 nM) and rat (IC(50)=293 nM) 11β-HSD2 activities. The potencies for the inhibition of human and rat 11β-HSD2 activities were PFOS>PFOA>PFHxS>PFBS. PFASs showed competitive inhibition of both human and rat 11β-HSD2 activities. This observation indicates that PFOS is a potent endocrine disruptor for glucocorticoid metabolism. Article from the Special issue on Targeted Inhibitors.     

Regulation of 11β-hydroxysteroid dehydrogenase 1 and 2 by IGF-1 in mice

Two isoforms of 11β-hydroxysteroid dehydrogenase (11β-HSD1 and 11β-HSD2) play an important role in regulation of glucocorticoid corticosterone (CORT, the active form in rodents) by the interconversion between CORT and 11-dehydrocorticosterone (11DHC, the biologically inert form). 11β-HSD1 is an NADP+/NADPH-dependent oxidoreductase which is mainly expressed in liver and kidney, while 11β-HSD2 is an NAD+-dependent oxidase which is predominantly expressed in kidney. The regulation of 11β-HSD1 and 11β-HSD2 mRNA (Hsd11b1 and Hsd11b2) levels and their activities by IGF-1 was performed in liver, kidney, and testis of IGF-1 knockout male mice. Real-time PCR showed that Hsd11b1 in liver was decreased while Hsd11b2 mRNA level was decreased in kidney of IGF-1 null mice. 11β-HSD1 and 11β-HSD2 activities fluctuated with the changes of their respective Hsd11b1 or Hsd11b2 mRNA levels. In conclusion, IGF-I tissue-specifically regulates Hsd11b1 and Hsd11b2 expression.     

Structure-dependent inhibition of human and rat 11β-hydroxysteroid dehydrogenase 2 activities by phthalates

Phthalates are diesters of phthalic acid and an alcohol moiety. Phthalates have been classified as endocrine disruptors and have a broad range of effects with unknown mechanisms. Some of the effects of phthalate are consistent with disruptions of normal glucocorticoid homeostasis, and in particular, with defective function of 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2). In the present study, we tested 12 phthalate diesters and four monoesters for the inhibition of human and rat kidney 11β-HSD2. We examined the modes of inhibition and looked for a relationship between the potency for inhibition and the chemical structures. Of the phthalate diesters we tested, dipropyl phthalate (DPrP) and di-n-butyl phthalate (DBP) significantly inhibited both human and rat 11β-HSD2 activities. The IC₅₀s were 85.59 μM for DPrP and 13.69 μM for DBP when calculated for rat 11β-HSD2. As diesters, 8 of the phthalates did not affect 11β-HSD2 enzyme activity. Compared to the diesters that were inhibitory, the 8 non-inhibitory phthalates, had either fewer carbons, that is 1 or 2 carbons in the alcohol moiety, or more carbons, 5–10, as a branched or unbranched chain in the alcohol moeity. However, phthalates could be inhibitors with six carbons in the alcohol moiety if the carbons were cyclized, as in dicyclohexyl phthalate (DCHP), which inhibited rat 11β-HSD2 with an IC₅₀ of 32.64 μM. Thus, whether a phthalate is an inhibitor may reflect the size and shape of the compound. Although the diesters are the compounds used in manufacturing and present as environmental contaminants, it is the monoester metabolites that are detected in human serum and urine. We showed that mono (2-ethylhexyl) phthalate (MEHP) significantly inhibited human (IC₅₀ = 110.8 ± 10.9) and rat (121.8 ± 8.5 μM) 11β-HSD2 activity even though its parent compound, di(2-ethylhexyl) phthalate (DEHP) did not. MEHP was a competitive inhibitor of 11β-HSD2 enzymatic activity. We conclude that phthalates of a certain size act as competitive inhibitors.     

Comparative properties of Aβ1–42, Aβ11–42, and [Pyr11]Aβ11–42 generated from O-acyl isopeptides

The use of water-soluble O-acyl isopeptides enabled us to investigate the biochemical properties of Aβ11–42 species, by preparing highly concentrated stock solutions after a pretreatment. Aβ11–42 and [Pyr¹¹]Aβ11–42 showed comparable aggregation capability and cytotoxicity, suggesting that the pyroglutamate modification at Glu¹¹ does not have a crucial role in these events. However, given that Aβ11–42 is converted to [Pyr¹¹]Aβ11–42 by a glutamyl cyclase in vivo, the potential aggregative and cytotoxic nature of [Pyr¹¹]Aβ11–42 that was observed in the present study provides valuable insights into the pathological functions of pyroglutamate-modified Aβ species in Alzheimer’s disease.     

Generation of 3,8-substituted 1,2,4-triazolopyridines as potent inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1)

A series of pyridyl amide/sulfonamide inhibitors of 11β-HSD-1 were modified to incorporate a novel 1,2,4-triazolopyridine scaffold. Optimization of substituents at the 3 and 8 position of the TZP core, with a special focus on enhancing metabolic stability, resulted in the identification of compound 38 as a potent and metabolically stable inhibitor of the enzyme.     

The inhibitory effects of perfluoroalkyl substances on human and rat 11β-hydroxysteroid dehydrogenase 1

Perfluoroalkyl substances (PFASs) are man-made polyfluorinated compounds that are widely used and persistent in the environment. PFASs have potential effects on many biological systems including the development of lung. Glucocorticoids have been reported to promote fetal and neonatal lung development at the late stage, and 11β-hydroxysteroid dehydrogenase 1(11βHSD1) in the lung is critical for the generation of local active glucocorticoid cortisol (human) or corticosterone (rodents) from biologically inert 11keto-steroids. The purpose of the present study is to study the direct inhibitory effects of PFASs on 11βHSD1 activities and action modes. Microsomal 11βHSD1 was subjected to the exposure to various PFASs, including perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), potassium perfluorohexanesulfonate (PFHxS) and potassium perfluorobutane sulfonate (PFBS). PFOS and PFOA inhibited neonatal rat lung 11βHSD1 activity with IC(50)s of 3.45μM (95% Confidence Intervals, CI(95): 1.97-6.37μM) and 45.31μM (CI(95): 27.64-74.26μM), respectively, while PFHxS and PFBS did not inhibit the enzyme activity at 250μM. PFOS and PFOA inhibited human 11βHSD1 activity with IC(50)s of 7.56μM (CI(95): 2.86-19.97μM) and 37.61μM (CI(95): 24.49-57.75μM), respectively, while PFHxS and PFBS did not inhibit the enzyme activity at 250μM. PFASs showed competitive inhibition on both human and rat 11βHSD1. In conclusion, the present study shows that PFOS and PFOA are the inhibitors of 11βHSD1.     

Effects of antisense-mediated inhibition of 11β-hydroxysteroid dehydrogenase type 1 on hepatic lipid metabolism

11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) converts inactive 11-keto derivatives to active glucocorticoids within tissues and may play a role in the metabolic syndrome (MS). We used an antisense oligonucleotide (ASO) to knock down 11β-HSD1 in livers of C57BL/6J mice consuming a Western-type diet (WTD). 11β-HSD1 ASO-treated mice consumed less food, so we compared them to ad libitum-fed mice and to food-matched mice receiving control ASO. Knockdown of 11β-HSD1 directly protected mice from WTD-induced steatosis and dyslipidemia by reducing synthesis and secretion of triglyceride (TG) and increasing hepatic fatty acid oxidation. These changes in hepatic and plasma lipids were not associated with reductions in genes involved in de novo lipogenesis. However, protein levels of both sterol regulatory element-binding protein (SREBP) 1 and fatty acid synthase were significantly reduced in mice treated with 11β-HSD1 ASO. There was no change in hepatic secretion of apolipoprotein (apo)B, indicating assembly and secretion of smaller apoB-containing lipoproteins by the liver in the 11β-HSD1-treated mice. Our results indicate that inhibition of 11β-HSD1 by ASO treatment of WTD-fed mice resulted in improved plasma and hepatic lipid levels, reduced lipogenesis by posttranslational regulation, and secretion of similar numbers of apoB-containing lipoproteins containing less TG per particle.     

Upregulation of 11β-hydroxysteroid dehydrogenase 1 in lymphoid organs during inflammation in the rat

Glucocorticoids exert anti-inflammatory and immunomodulatory effects that may be regulated in part by the activities of the glucocorticoid-activating and -inactivating enzymes, 11β-hydroxysteroid dehydrogenase type 1 (11HSD1) and type 2 (11HSD2), respectively. Previous studies have demonstrated that inflammatory bowel diseases in humans and experimental animals upregulate 11HSD1 and downregulate 11HSD2. We investigated whether proinflammatory cytokines modulate colonic 11HSDs as well as whether lymphoid organs exhibit any 11HSD response to inflammation. Colon tissue explants exposed to tumor necrosis factor α exhibited an upregulation of 11HSD1 mRNA whereas interleukin 1β downregulated 11HSD2 mRNA. Experimental colitis induced by the intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid stimulated 11HSD1 activity not only in the colon but also in mesenteric lymph nodes and the spleen. Analysis of mRNA for 11HSD1 in colon-draining lymph nodes and the spleen showed that inflammation upregulates the expression of this enzyme in mobile lymphoid cells similar to the intraepithelial and lamina propria leukocytes isolated from the colon. It is inferred that inflammation stimulates the reactivation of glucocorticoids in lymphoid organs and in gut-associated lymphoid tissue.     

Structure-based design of 7-azaindole-pyrrolidine amides as inhibitors of 11β-hydroxysteroid dehydrogenase type I

Indole-pyrrolidines were identified as inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) by high-throughput screening. Optimisation of the initial hit through structure-based design led to 7-azaindole-derivatives, with the best analogues displaying single digit nanomolar IC(50) potency. The modeling hypotheses were confirmed by solving the X-ray co-crystal structure of one of the lead compounds. These compounds were selective against 11β-hydroxysteroid dehydrogenase type 2 (selectivity ratio >200) and exhibited good inhibition of 11β-HSD1 (IC(50)<1μM) in a cellular model (3T3L1 adipocytes).