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.     

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.     

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.     

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.     

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.     

Structural assessment and identification of 11β-hydroxysteroid dehydrogenase type 1 inhibitors

Communicated by Ramaswamy H. Sarma     

Discovery of camphor-derived pyrazolones as 11β-hydroxysteroid dehydrogenase type 1 inhibitors

Starting from screening hit, (4S,7R)-1,7,8,8-tetramethyl-2-phenyl-1,2,4,5,6,7-hexahydro-4,7-methano-indazol-3-one (7), we optimized the potency and pharmacokinetic properties. This led to the identification of compounds with good in vivo activity in a mouse pharmacodynamic model of inhibition of 11βHSD1.     

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.     

Antagonism of the estradiol-mediated repression of microsomal 3β-hydroxysteroid dehydrogenase activity in rat liver by antiestrogenic substances

5α-Dihydrotestosterone, 17-hydroxyprogesterone caproate, 2-methoxyestrone and a number of nonsteroidal antiestrogens (clomiphene citrate, nafoxidine hydrochloride, tamoxifen, MER-25) were tested for their ability to block estradiol-mediated repression of the androgen-dependent 3β-hydroxy-steroid dehydrogenase activity of male rat liver. With the exception of 5α-dihydrotestosterone, which induced activity in females, none of these substances affected 3β-hydroxy-steroid dehydrogenase activity when administered alone to otherwise untreated male and female rats. Tamoxifen (100 or 500 μg/day) was the only substance which prevented a decrease in enzyme activity when given simultaneously with estradiol (5 μg/day). The estradiol-mediated decrease in activity was not antagonized by a 100-fold higher dose of androgen (5α-dihydrotestosterone, 0.5 mg/day), demonstrating the potent antiandrogenic effect of estradiol on this hepatic androgen-dependent enzyme activity.     

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.     

Hepatic reduction of the secondary bile acid 7-oxolithocholic acid is mediated by 11β-hydroxysteroid dehydrogenase 1

The oxidized bile acid 7-oxoLCA (7-oxolithocholic acid), formed primarily by gut micro-organisms, is reduced in human liver to CDCA (chenodeoxycholic acid) and, to a lesser extent, UDCA (ursodeoxycholic acid). The enzyme(s) responsible remained unknown. Using human liver microsomes, we observed enhanced 7-oxoLCA reduction in the presence of detergent. The reaction was dependent on NADPH and stimulated by glucose 6-phosphate, suggesting localization of the enzyme in the ER (endoplasmic reticulum) and dependence on NADPH-generating H6PDH (hexose-6-phosphate dehydrogenase). Using recombinant human 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), we demonstrate efficient conversion of 7-oxoLCA into CDCA and, to a lesser extent, UDCA. Unlike the reversible metabolism of glucocorticoids, 11β-HSD1 mediated solely 7-oxo reduction of 7-oxoLCA and its taurine and glycine conjugates. Furthermore, we investigated the interference of bile acids with 11β-HSD1-dependent interconversion of glucocorticoids. 7-OxoLCA and its conjugates preferentially inhibited cortisone reduction, and CDCA and its conjugates inhibited cortisol oxidation. Three-dimensional modelling provided an explanation for the binding mode and selectivity of the bile acids studied. The results reveal that 11β-HSD1 is responsible for 7-oxoLCA reduction in humans, providing a further link between hepatic glucocorticoid activation and bile acid metabolism. These findings also suggest the need for animal and clinical studies to explore whether inhibition of 11β-HSD1 to reduce cortisol levels would also lead to an accumulation of 7-oxoLCA, thereby potentially affecting bile acid-mediated functions.     

Expression and NNK reducing activities of carbonyl reductase and 11β-hydroxysteroid dehydrogenase type 1 in human lung

The tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), which is found in high amounts in tobacco products, is believed to play an important role in lung cancer induction in smokers. NNK requires metabolic activation by cytochrome P450 mediated α-hydroxylation to exhibit its carcinogenic properties. On the other hand, NNK is inactivated by carbonyl reduction to its alcohol-equivalent 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronidation and final excretion into urine or bile. Carbonyl reduction and α-hydroxylation are the predominant pathways in man, and it has been postulated that the extent of these competing pathways determines the individual susceptibility to lung cancer. Moreover, only a minor part of all habitual smokers develop lung cancer, suggesting the existence of susceptibility genes. Microsomal 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) (EC 1.1.1.146) and cytosolic carbonyl reductase (CR) (EC 1.1.1.184) have been shown to be mainly responsible for NNAL formation in liver and lung. In the present study, we performed comparative investigations of human lung tissue samples from several patients with respect to the expression and activity of 11β-HSD 1 and carbonyl reductase. We observed varying levels in 11β-HSD 1 and carbonyl reductase expression in these patients, as revealed by RT-PCR and ELISA. Also, the tissue samples showed a different activity and inhibitor profile for both enzymes. According to our results, variations in the expression and activity of NNK carbonyl reducing enzymes may constitute a major determinant in the overall NNK detoxification capacity and thus may be linked to the great differences observed in the individual susceptibility of tobacco-smoke related lung cancer.     

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.     

Solubilization of 17β-hydroxysteroid dehydrogenase from rat liver microsomes

The conditions for the solubilization of 17β-hydroxysteroid dehydrogenase from a rat liver microsomal preparation with the non-ionic detergent Triton X-100 were studied. The recoveries of 17β-hydroxysteroid dehydrogenase activity and of proteins in the solubilized form were determined as a function of detergent concentration, of pH and temperature, of incubation time and of saline concentration. The soluble fraction obtained under the optimal conditions contained 80% of the proteins and 75% of the enzymatic activity of initial microsomes. The presence of Triton X-100 in the solubilized proteins was not essential for enzyme activity.     

A novel derivatives of thiazol-4(5H)-one and their activity in the inhibition of 11β-hydroxysteroid dehydrogenase type 1

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an enzyme that catalyzes the conversion of inactive cortisone into physiologically active cortisol. Inhibiting the activity of this enzyme plays a key role in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. Therefore, new compounds that are selective inhibitors of this enzyme are constantly being looked for.In this work we present the synthesis of 2-(allylamino)thiazol-4(5H)-one derivatives by the reaction of N-allylthiourea with appropriate α-bromoesters. In the case of using of aliphatic α-bromoesters and α-bromo-β-phenylesters, the reactions were carried out in a basic medium (sodium ethoxide) and the products were isolated with a yield of up to 68%. Derivatives containing spiro systems in which carbon C-5 of the thiazole ring is the linker atom were obtained in the presence of N,N-diisopropylethylamine.Some of the obtained compounds, at a concentration of 10 μM have activity in the inhibition of 11β-HSD1 up to 71%. IC₅₀ value for the most active compound: 2-(allylamino)-1-thia-3-azaspiro[4.5]dec-2-en-4-one is 2.5 µM. With a high degree of 11β-HSD1 inhibition and a relatively large difference in the inhibition of 11β-HSD1 and 11β-HSD2 activity, this compound appears to be promising and should be subjected to further testing.     

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.     

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.