Direct inhibition of bisphenols on human and rat 11ß-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis.

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11ß-Hydroxysteroid dehydrogenase 2 (11ß-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11ß-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11ß-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 µM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11ß-HSD2. Some BPs also inhibited rat renal 11ß-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 µM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11ß-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11ß-HSD2 activity and that there were species-dependent differences.

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 11ß-HSD1 and 11ß-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 11ß-HSD were evaluated. The highest inhibitory activity for 11ß-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 11ß-HSD2 (53.57% at a conc. of 10 µM). A low selectivity in the inhibition of 11ß-HSD2 was observed but, unlike 18ß-glycyrrhetinic acid, these compounds were found to inhibit the activity of 11ß-HSD2 to a greater extent than 11ß-HSD1, which makes them attractive for further research on their anti-cancer activity.

Species-specific differences in the inhibition of 11ß-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole.

11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) converts active 11ß-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11ß-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11ß-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11ß-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11ß-HSD2 was potently inhibited by all three compounds (IC50 values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC50 values compared to human), and mouse and zebrafish 11ß-HSD2 were very weakly inhibited (IC50 values above 7 µM). Sequence alignment and application of newly generated homology models for human and mouse 11ß-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11ß-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11ß-HSD2. The results provide an explanation for the observed species-specific 11ß-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11ß-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.

Comparison of flavonoids and isoflavonoids to inhibit rat and human 11ß-hydroxysteroid dehydrogenase 1 and 2.

Many flavonoids and isoflavonoids have anti-diabetic effects in animal models. However, the mechanisms that are involved are generally unclear. Since 11ß-hydroxysteroid dehydrogenases (HSD11Bs) play important roles in diabetes, we hypothesize that flavonoids and isoflavonoids may affect diabetes by targeting two isoforms of HSD11B differently. The inhibitory effects of flavonoids (apigenin and quercetin) and isoflavonoids [genistein and (±) equol] on rat and human HSD11B1 and HSD11B2 were analyzed. The potencies of inhibition on human HSD11B1 reductase was in the order of apigenin > quercetin > genistein > (±) equol, with IC50 values of 2.19, 5.36, 11.00, and over 100 µM, respectively. Genistein also inhibited rat HSD11B1 reductase with IC50 value of 24.58 µM, while other three chemicals showed no effects on the enzyme activity with IC50 values over 100 µM. However, apigenin and (±) equol did not inhibit human HSD11B2 at concentrations as high as 100 µM, while genistein and quercetin inhibited human HSD11B2 by 60% and 50% at 100 µM, respectively. The effective flavonoids and isoflavonoids are noncompetitive inhibitors of HSD11B1 when steroid substrates were used. Docking analysis showed that they bound to the steroid-binding site of the human HSD11B1. These data indicate that apigenin is a selective inhibitor of human HSD11B1 of two HSD11B isoforms, which may be useful in managing symptoms of the metabolic syndrome.

11ß-HSD2 SUMOylation Modulates Cortisol-Induced Mineralocorticoid Receptor Nuclear Translocation Independently of Effects on Transactivation.

The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) has an essential role in aldosterone target tissues, conferring aldosterone selectivity for the mineralocorticoid receptor (MR) by converting 11ß-hydroxyglucocorticoids to inactive 11-ketosteroids. Congenital deficiency of 11ß-HSD2 causes a form of salt-sensitive hypertension known as the syndrome of apparent mineralocorticoid excess. The disease phenotype, which ranges from mild to severe, correlates well with reduction in enzyme activity. Furthermore, polymorphisms in the 11ß-HSD2 coding gene (HSD11B2) have been linked to high blood pressure and salt sensitivity, major cardiovascular risk factors. 11ß-HSD2 expression is controlled by different factors such as cytokines, sex steroids, or vasopressin, but posttranslational modulation of its activity has not been explored. Analysis of 11ß-HSD2 sequence revealed a consensus site for conjugation of small ubiquitin-related modifier (SUMO) peptide, a major posttranslational regulatory event in several cellular processes. Our results demonstrate that 11ß-HSD2 is SUMOylated at lysine 266. Non-SUMOylatable mutant K266R showed slightly higher substrate affinity and decreased Vmax, but no effects on protein stability or subcellular localization. Despite mild changes in enzyme activity, mutant K266R was unable to prevent cortisol-dependent MR nuclear translocation. The same effect was achieved by coexpression of wild-type 11ß-HSD2 with sentrin-specific protease 1, a protease that catalyzes SUMO deconjugation. In the presence of 11ß-HSD2-K266R, increased nuclear MR localization did not correlate with increased response to cortisol or increased recruitment of transcriptional coregulators. Taken together, our data suggests that SUMOylation of 11ß-HSD2 at residue K266 modulates cortisol-mediated MR nuclear translocation independently of effects on transactivation.

A novel mutation in HSD11B2 causes apparent mineralocorticoid excess in an Omani kindred.

Apparent mineralocorticoid excess (AME) is a rare autosomal recessive genetic disorder causing severe hypertension in childhood due to a deficiency of 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2), which is encoded by HSD11B2. Without treatment, chronic hypertension leads to early development of end-organ damage. Approximately 40 causative mutations in HSD11B2 have been identified in ∼100 AME patients worldwide. We have studied the clinical presentation, biochemical parameters, and molecular genetics in six patients from a consanguineous Omani family with AME. DNA sequence analysis of affected members of this family revealed homozygous c.799A>G mutations within exon 4 of HSD11B2, corresponding to a p.T267A mutation of 11ßHSD2. The structural change and predicted consequences owing to the p.T267A mutation have been modeled in silico. We conclude that this novel mutation is responsible for AME in this family.

Species differences of 11beta-hydroxysteroid dehydrogenase type 2 function in human and rat term placenta determined via LC-MS/MS.

INTRODUCTION: Glucocorticoid-induced fetal programming has been associated with negative metabolic and cardiovascular sequelae in the adult. The placental enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) shields the fetus from maternal glucocorticoid excess by catalyzing the conversion of these hormones into biologically inactive derivatives. In vivo experiments addressing placental barrier function are mostly conducted in rodents. Therefore we set out to characterize species-specific differences of rat and human placental 11ß-HSD2 steroid turnover, introducing Liquid Chromatography Tandem Mass-Spectrometry (LC-MS/MS) as a tool for rat tissue analysis. MATERIALS AND METHODS: Using LC-MS/MS we determined corticotropin-releasing hormone (CRH), cortisol (F), cortisone (E), corticosterone (B) and 11-dehydrocorticosterone (A) in human and rat placenta at term and measured the enzymatic 11ß-HSD glucocorticoid conversion-rates in placental microsomes of both species. In parallel, further glucocorticoid derivatives and sex steroids were determined in the same placental samples. RESULTS: In contrast to the human placenta, we did not detect CRH in the rat placenta. While cortisol (F) and cortisone (E) were exclusively present in human term placenta (E/F-ratio >1), rat placenta showed significant levels of corticosterone (B) and 11-dehydrocorticosterone (A), with an A/B-ratio <1. In line with these species-specific findings, human placenta showed a prominent 11ß-HSD2 activity, while in rat placenta higher 11ß-HSD1 glucocorticoid turnover rates were determined. DISCUSSION: Placental steroid metabolism of human and rat shows relevant species-specific differences, especially regarding the barrier function of 11ß-HSD2 at term. The exclusive expression of CRH in the human placenta further points to relevant differences in the regulation of parturition in rats. Consideration of these findings is warranted when transferring results from rodent placental glucocorticoid metabolism into humans.

Zearalenone Inhibits Rat and Human 11ß-Hydroxysteroid Dehydrogenase Type 2.

Zearalenone is a mycotoxin produced by Fusarium spp. 11ß-Hydroxysteroid dehydrogenases, isoforms 1 (HSD11B1) and 2 (HSD11B2), have been demonstrated to be the regulators of the local level of active glucocorticoid, which has a broad range of physiological actions. In the present study, the potency of zearalenone was tested for the inhibition of HSD11B1 and HSD11B2 in rat and human tissues. Zearalenone showed potent inhibition of HSD11B2 with the half-maximal inhibitory concentration (IC50) calculated at 49.63 and 32.22 µM for the rat and human, respectively. Results showed that zearalenone competitively inhibited HSD11B2 when a steroid substrate was used. However, it served as an uncompetitive inhibitory factor when the cofactor NAD(+) was used. In contrast, the potency of zearalenone to inhibit both rat and human HSD11B1 was diminished, with the concentration of 100 µM causing almost no inhibitory effect on the isoform. In conclusion, we observed that zearalenone is a selective inhibitor of HSD11B2, implying that this agent may cause excessive glucocorticoid action in local tissues such as kidney and placentas.

Identification of novel 11ß-HSD1 inhibitors by combined ligand- and structure-based virtual screening.

11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) converts cortisone to cortisol in a NADPH dependent manner. Overexpression of 11ß-HSD1 in key metabolic tissues is related to the development of type 2 diabetes, obesity, hypertension and metabolic syndrome. Using crystal structures of human 11ß-HSD1 in complex with inhibitors as source of structural information, a combined ligand and structure-based virtual screening approach was implemented to identify novel 11ß-HSD1 inhibitors. A selected group of compounds was identified in silico and further evaluated in cell-based assays for cytotoxicity and 11ß-HSD1 mediated cortisol production inhibitory capacity. The expression of 11ß-HSD1 and 11ß-HSD2 in human LS14 adipocytes was assessed during differentiation. Biological evaluation of 39 compounds in adipocytes and steroids quantification by HPLC-MS/MS identify 4 compounds that exhibit 11ß-HSD1 mediated cortisol production inhibitory activity with potencies in the micromolar range. Two compounds showed to be selective for the 11ß-HSD1 reductase activity and over 11ß-HSD2 isoform, and thus represent novel leads for the development of more active derivatives with higher efficacies targeting intracellular cortisol levels in type 2 diabetes and metabolic syndrome.

Inhibition of 11b-HSD1 by tetracyclic triterpenoids from Euphorbia kansui.

The roots of Euphorbia kansui are considered an important traditional folk medicine. In this study the ethanol extracts of E. kansui were investigated. A new tetracyclic triterpenoid, euphane-3b,20-dihydroxy-24-ene, in addition to five known triterpenoids with euphane skeletons were isolated. Their structures were elucidated on the basis of physical and spectral techniques (1D-, 2D-NMR and MS, respectively). Furthermore, these compounds 1-6 exhibited strong inhibitory activity against human 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1), with IC(50) values of 34.86 nM, 1.115 mM, 16.08 nM, 2.815 nM, 26.47 nM, 15.99 nM, and 41.86 nM, respectively. The docking results show that the ring part of compounds can insert into the hydrophobic core of h11b-HSD1 and the alkane chain orientates toward the outside. The results presented herein provide a scientific explanation for the usage of the E. kansui in clinical treatment of diabetes.

Virtual screening as a strategy for the identification of xenobiotics disrupting corticosteroid action.

BACKGROUND: Impaired corticosteroid action caused by genetic and environmental influence, including exposure to hazardous xenobiotics, contributes to the development and progression of metabolic diseases, cardiovascular complications and immune disorders. Novel strategies are thus needed for identifying xenobiotics that interfere with corticosteroid homeostasis. 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) and mineralocorticoid receptors (MR) are major regulators of corticosteroid action. 11ß-HSD2 converts the active glucocorticoid cortisol to the inactive cortisone and protects MR from activation by glucocorticoids. 11ß-HSD2 has also an essential role in the placenta to protect the fetus from high maternal cortisol concentrations. METHODS AND PRINCIPAL FINDINGS: We employed a previously constructed 3D-structural library of chemicals with proven and suspected endocrine disrupting effects for virtual screening using a chemical feature-based 11ß-HSD pharmacophore. We tested several in silico predicted chemicals in a 11ß-HSD2 bioassay. The identified antibiotic lasalocid and the silane-coupling agent AB110873 were found to concentration-dependently inhibit 11ß-HSD2. Moreover, the silane AB110873 was shown to activate MR and stimulate mitochondrial ROS generation and the production of the proinflammatory cytokine interleukin-6 (IL-6). Finally, we constructed a MR pharmacophore, which successfully identified the silane AB110873. CONCLUSIONS: Screening of virtual chemical structure libraries can facilitate the identification of xenobiotics inhibiting 11ß-HSD2 and/or activating MR. Lasalocid and AB110873 belong to new classes of 11ß-HSD2 inhibitors. The silane AB110873 represents to the best of our knowledge the first industrial chemical shown to activate MR. Furthermore, the MR pharmacophore can now be used for future screening purposes.

Species-specific differences in the inhibition of human and zebrafish 11ß-hydroxysteroid dehydrogenase 2 by thiram and organotins.

Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11ß-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11ß-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11ß-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11ß-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11ß-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11ß-HSD2 to thiram. Mutating cysteine-264 on human 11ß-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11ß-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11ß-HSD2 for sensitivity to sulfhydryl modifying agents.

Molecular docking and structural analysis of cofactor-protein interaction between NAD⁺ and 11ß-hydroxysteroid dehydrogenase type 2.

Molecular docking and structural analysis of the cofactor-protein interaction between NAD(+) and human (h) or mouse (m) 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) were performed with the molecular operating environment (MOE). 11ßHSD1 (PDB code: 3HFG) was selected as a template for the 3D structure modeling of 11ßHSD2. The MOE docking (MOE-dock) and the alpha sphere and excluded volume-based ligand-protein docking (ASE-dock) showed that both NAD(+)-h11ßHSD2 and NAD(+)-m11ßHSD2 models have a similar binding orientation to the template cofactor-protein model. Our present study also revealed that Asp91, Phe94, Tyr232 and Thr267 could be of importance in the interaction between NAD(+) and 11ßHSD2. NADP(+) was incapable of entering into the cofactor-binding site of the 11ßHSD2 models. The present study proposes the latest models for 11ßHSD2 and its cofactor NAD(+), and to the best of our knowledge, this is the first report of a m11ßHSD2 model with NAD(+).

Ligand-receptor interaction between triterpenoids and the 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) enzyme predicts their toxic effects against tumorigenic r/m HM-SFME-1 cells.

The present study deals with in silico prediction and in vitro evaluation of the selective cytotoxic effects of triterpenoids on tumorigenic human c-Ha-ras and mouse c-myc cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells. Ligand fitting of five different triterpenoids to 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) was analyzed with a molecular modeling method, and glycyrrhetinic acid (GA) was the best-fitted triterpenoid to the ligand binding site in 11ßHSD2. Analysis of antiproliferative effects revealed that GA, oleanolic acid, and ursolic acid had selective toxicity against the tumor cells and that GA was the most potent triterpenoid in its selectivity. The toxic activity of the tested triterpenoids against the tumor cells showed good correlations with the partition coefficient (logP) and polar surface area values. Time-lapse microscopy, fluorescence staining, and confocal laser scanning microscopic observation revealed that GA induced morphologic changes typical of apoptosis such as cell shrinkage and blebbing and also disrupted the cytoskeletal proteins. Furthermore, GA exhibited a strong inhibitory effect on 11ßHSD2 activity in the tumor cells. Our current results suggest that analysis of the ligand-receptor interaction between triterpenoids and 11ßHSD2 can be utilized to predict their antitumor effects and that GA can be used as a possible chemopreventive and therapeutic antitumor agent. To the best of our knowledge, this is the first report on in silico prediction of the toxic effects of triterpenoids on tumor cells by 11ßHSD2 inhibition.

Protective effect of aescin from the seeds of Aesculus hippocastanum on liver injury induced by endotoxin in mice.

To investigate the effect and underlying mechanism of aescin on acute liver injury induced by endotoxin, liver injury was established by injecting lipopolysaccharide (LPS) in mice. Animals were assigned to seven groups: the control group and groups treated with LPS (40 mg/kg), aescin (3.6 mg/kg), LPS plus dexamethasone (4 mg/kg) and LPS plus aescin (0.9, 1.8 or 3.6 mg/kg). Hepatic histopathological changes were examined under a light microscope. Activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were determined. Levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), nitric oxide (NO) and antioxidative parameters in liver homogenate were measured. Glucocorticoid receptor (GR), 11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) expressions in liver were determined by western blotting. Treatment with escin could inhibit immigration of inflammatory cells, alleviate the degree of necrosis, and decrease serum ALT and AST activities. Aescin also down-regulated levels of inflammation mediators (TNF-α, IL-1ß and NO) and 11ß-HSD2 expression in liver, up-regulated GR expression, enhanced endogenous antioxidative capacity, but have no obvious effect on 11ß-HSD1 expression in liver. The findings suggest aescin has protective effects on endotoxin-induced liver injury, and the underlying mechanisms were associated with its anti-inflammatory effects, up-regulating GR expression, down-regulating 11ß-HSD2 experssion, and antixoidation.

Selective toxicity of glycyrrhetinic acid against tumorigenic r/m HM-SFME-1 cells is potentially attributed to downregulation of glutathione.

Natural products from plants are expected to play significant roles in creating new, safe and improved chemopreventive and therapeutic antitumor agents. Selectivity is also an important issue in cancer prevention and therapy. The present study was designed to extend our previous study on the c-Ha-ras and c-myc-induced tumor cell-selective antiproliferative effects of a licorice component, glycyrrhetinic acid (GA). An in silico ligand-receptor docking simulation revealed that GA acts as an 11ß-hydroxysteroid dehydrogenase type 2 inhibitor. GA disrupted the redox balance in tumor cells through upregulation of reactive oxygen species and downregulation of glutathione (GSH). The GA-induced GSH reduction and cytotoxicity were enhanced by an inhibitor of GSH, l-buthionine-[S,R]-sulfoximine. N-acetyl-l-cysteine, an antioxidant and precursor of GSH, restored the GA-induced GSH reduction and cytotoxicity in tumor cells. Taken together, these data highlighting the downregulation of GSH by GA and the efficacy of GSH in ameliorating GA-mediated cytotoxicity support the notion that GSH is involved in the selective toxicity of GA toward tumor cells.

Homology modeling and structural analysis of 11ß-hydroxysteroid dehydrogenase type 2.

11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) was homology-modeled by a Boltzmann-weighted randomized modeling procedure, using the X-ray crystal structure of 11ßHSD1 (PDB code: 3HFG) as a template. The model exhibited significant 3D similarities to 11ßHSD1. The contact energy profiles of the 11ßHSD2 model were in good agreement with that of the X-ray structure of 11ßHSD1. The secondary structure of the 11ßHSD2 model exhibited a central 6-stranded all-parallel ß-sheet sandwich-like structure, flanked on both sides by 3-helices. Ramachandran plots revealed that only 1.9% of the amino acid residues were in the disfavored region for 11ßHSD2. Furthermore, the ligand-binding site (LBS) volume was calculated to be 845 Å(3), which suggests that the LBS of 11ßHSD2 is sufficiently large to contain cofactors and substrates (ligands), such as NAD(+) and cortisol. The electrostatic analysis revealed that the 11ßHSD2 model had a positive potential at the LBS, which indicates that 11ßHSD2 possibly attracts negatively charged ligands at the LBS. These results indicate that the model was successfully evaluated and analyzed. Consequently, it is proposed that the 11ßHSD2 model in the present study will be suitable for further in silico structure-based de novo antitumor drug designing. To the best of our knowledge, this is the latest report of an accurate 11ßHSD2 model to target 11ßHSD2 for the development of anticancer drugs.

In silico structure-function analysis of pathological variation in the HSD11B2 gene sequence.

11beta-Hydroxysteroid dehydrogenase type 2 (11betaHSD2) is a short-chain dehydrogenase/reductase (SDR) responsible for inactivating cortisol and preventing its binding to the mineralocorticoid receptor (MR). Nonfunctional mutations in HSD11B2, the gene encoding 11betaHSD2, cause the hypertensive syndrome of apparent mineralocorticoid excess (AME). Like other such Mendelian disorders, AME is rare but has nevertheless helped to illuminate principles fundamental to the regulation of blood pressure. Furthermore, polymorphisms in HSD11B2 have been associated with salt sensitivity, a major risk factor for cardiovascular mortality. It is therefore highly likely that sequence variation in HSD11B2, having subtle functional ramifications, will affect blood pressure in the wider population. In this study, a three-dimensional homology model of 11betaHSD2 was created and used to hypothesize the functional consequences in terms of protein structure of published mutations in HSD11B2. This approach underscored the strong genotype-phenotype correlation of AME: severe forms of the disease, associated with little in vivo enzyme activity, arise from mutations occurring in invariant alignment positions. These were predicted to exert gross structural changes in the protein. In contrast, those mutations causing a mild clinical phenotype were in less conserved regions of the protein that were predicted to be relatively more tolerant to substitution. Finally, a number of pathogenic mutations are shown to be associated with regions predicted to participate in dimer formation, and in protein stabilization, which may therefore suggest molecular mechanisms of disease.

Impaired protein stability of 11beta-hydroxysteroid dehydrogenase type 2: a novel mechanism of apparent mineralocorticoid excess.

Apparent mineralocorticoid excess (AME) is a severe form of hypertension that is caused by impaired activity of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), which converts biologically active cortisol into inactive cortisone. Mutations in HSD11B2 result in cortisol-induced activation of mineralocorticoid receptors and cause hypertension with hypokalemia, metabolic alkalosis, and suppressed circulating renin and aldosterone concentrations. This study uncovered the first patient with AME who was described in the literature, identified the genetic defect in HSD11B2, and provided evidence for a novel mechanism of reduced 11beta-HSD2 activity. This study identified a cluster of amino acids (335 to 339) in the C-terminus of 11beta-HSD2 that are essential for protein stability. The cluster includes Tyr(338), which is mutated in the index patient, and Arg(335) and Arg(337), previously reported to be mutated in hypertensive patients. It was found that wild-type 11beta-HSD2 is a relatively stable enzyme with a half-life of 21 h, whereas that of Tyr(338)His and Arg(337)His was 3 and 4 h, respectively. Enzymatic activity of Tyr(338)His was partially retained at 26 degrees C or in the presence of the chemical chaperones glycerol and dexamethasone, indicating thermodynamic instability and misfolding. The results provide evidence that the degradation of both misfolded mutant Tyr(338)His and wild-type 11beta-HSD2 occurs through the proteasome pathway. Therefore, impaired 11beta-HSD2 protein stability rather than reduced gene expression or loss of catalytic activity seems to be responsible for the development of hypertension in some individuals with AME.

Comparative genomic and phylogenetic analysis of short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity.

Human short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity (RODH-like enzymes) are thought to contribute to the oxidation of retinol for retinoic acid biosynthesis and to the metabolism of androgenic and neuroactive 3alpha-hydroxysteroids. Here, we investigated the phylogeny and orthology of these proteins to understand better their origins and physiological roles. Phylogenetic and genomic analysis showed that two proteins (11-cis-RDH and RDHL) are highly conserved, and their orthologs can be identified in the lower taxa, such as amphibians and fish. Two other proteins (RODH-4 and 3alpha-HSD) are significantly less conserved. Orthologs for 3alpha-HSD are present in all mammals analyzed, whereas orthologs for RODH-4 can be identified in some mammalian species but not in others due to species-specific gene duplications. Understanding the evolution and divergence of RODH-like enzymes in various vertebrate species should facilitate further investigation of their in vivo functions using animal models.