Publications

• The discovery of 2-anilinothiazolones as 11beta-HSD1 inhibitors.

  Yuan C., St Jean D.J. Jr., Liu Q., Cai L., Li A., Han N., Moniz G., Askew B., Hungate R.W., Johansson L., Tedenborg L., Pyring D., Williams M., Hale C., Chen M., Cupples R., Zhang J., Jordan S., Bartberger M.D., Sun Y., Emery M., Wang M., Fotsch C.

  A series of 2-anilinothiazolones were prepared as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The most potent compounds contained a 2-chloro or 2-fluoro group on the aniline ring with an isopropyl substituent on the 5-position of the thiazolone ring (compounds 2 and 3, ... >> More

  A series of 2-anilinothiazolones were prepared as inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). The most potent compounds contained a 2-chloro or 2-fluoro group on the aniline ring with an isopropyl substituent on the 5-position of the thiazolone ring (compounds 2 and 3, respectively). The binding mode was determined through the X-ray co-crystal structure of the enzyme with compound 3. This compound was also approximately 70-fold selective over 11beta-HSD2 and was orally bioavailable in rat pharmacokinetic studies. However, compound 3 was >580-fold less active in the 11beta-HSD1 cell assay when tested in the presence of 3% human serum albumin. << Less

  Bioorg. Med. Chem. Lett. 17:6056-6061(2007) [PubMed] [EuropePMC]

• Lumenal orientation and post-translational modifications of the liver microsomal 11beta-hydroxysteroid dehydrogenase.

  Ozols J.

  The topology and post-translational modifications of microsomal 11 beta-hydroxysteroid dehydrogenase (11 beta-DH) was investigated using the approaches of protein structure analysis. Sequence analysis of peptides generated by chemical and enzymatic cleavages revealed that carbohydrate is attached ... >> More

  The topology and post-translational modifications of microsomal 11 beta-hydroxysteroid dehydrogenase (11 beta-DH) was investigated using the approaches of protein structure analysis. Sequence analysis of peptides generated by chemical and enzymatic cleavages revealed that carbohydrate is attached at Asn-122, -161, and -206. Enzymatic deglycosylation reactions of the protein identified the attached glycans as high mannose carbohydrates, implying that the bulk of the protein molecule is oriented on the lumenal side of the endoplasmic membrane. The carbohydrate moiety of native dehydrogenase was cleaved by endo-N-acetylglucosaminidase H without significantly affecting the 11 beta-DH activity. Chemical modification of cysteinyl residues, followed by amino acid sequence analysis, identified one disulfide bond linking Cys-77 and Cys-212. This disulfide bond was inaccessible to thiol reagents, unless the protein was denatured. Contrary to the partially purified 11 beta-DH preparations, the purified enzymatically active protein failed to bind to a 2,5'-ADP affinity column, suggesting that a conformational change has occurred in the enzyme during purification. The proposed model of the 11 beta-DH has a single trans-membrane segment at the N terminus, with the bulk of the polypeptide chain projecting into the lumen of endoplasmic reticulum. Limited proteolysis studies of 11 beta-DH concluded an absence of a flexible intradomain segment between the membranous and the lumenal domains. The lumenal localization of the 11 beta-DH requires a mechanism by which cortisol is transported to the endoplasmic reticulum of the lumen. << Less

  J. Biol. Chem. 270:2305-2312(1995) [PubMed] [EuropePMC]

• Appropriate function of 11beta-hydroxysteroid dehydrogenase type 1 in the endoplasmic reticulum lumen is dependent on its N-terminal region sharing similar topological determinants with 50-kDa esterase.

  Frick C., Atanasov A.G., Arnold P., Ozols J., Odermatt A.

  By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated t ... >> More

  By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11beta-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys(5) by Ser in 11beta-HSD1, but not of the analogous Lys(4) by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu(25)/Glu(26) in 11beta-HSD1 and Asp(25) in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys(35)/Lys(36) and Glu(25)/Glu(26) on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11beta-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11beta-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins. << Less

  J. Biol. Chem. 279:31131-31138(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Adamantane sulfone and sulfonamide 11-beta-HSD1 Inhibitors.

  Sorensen B., Winn M., Rohde J., Shuai Q., Wang J., Fung S., Monzon K., Chiou W., Stolarik D., Imade H., Pan L., Deng X., Chovan L., Longenecker K., Judge R., Qin W., Brune M., Camp H., Frevert E.U., Jacobson P., Link J.T.

  Potent and selective adamantane sulfone and sulfonamide inhibitors of 11-beta-HSD-1 have been discovered. Selected compounds from these series have robust pharmacokinetic profiles and strongly inhibit liver, fat, and brain HSD1 for extended periods after oral dosing.

  Bioorg. Med. Chem. Lett. 17:527-532(2007) [PubMed] [EuropePMC]

• At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

  d'Andrea S., Canonge M., Beopoulos A., Jolivet P., Hartmann M.A., Miquel M., Lepiniec L., Chardot T.

  In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant P ... >> More

  In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined. << Less

  Biochimie 89:222-229(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The N-terminal anchor sequences of 11beta-hydroxysteroid dehydrogenases determine their orientation in the endoplasmic reticulum membrane.

  Odermatt A., Arnold P., Stauffer A., Frey B.M., Frey F.J.

  11beta-Hydroxysteroid dehydrogenase enzymes (11beta-HSD) regulate the ratio of active endogenous glucocorticoids to their inactive keto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors. In this study, the topology and intracellular localization of 11beta-HS ... >> More

  11beta-Hydroxysteroid dehydrogenase enzymes (11beta-HSD) regulate the ratio of active endogenous glucocorticoids to their inactive keto-metabolites, thereby controlling the access of glucocorticoids to their cognate receptors. In this study, the topology and intracellular localization of 11beta-HSD1 and 11beta-HSD2 have been analyzed by immunohistochemistry and protease protection assays of in vitro transcription/translation products. 11beta-HSD constructs, tagged with the FLAG epitope, were transiently expressed in HEK-293 cells. The enzymatic characteristics of tagged and native enzymes were indistinguishable. Fluorescence microscopy demonstrated the localization of both 11beta-HSD1 and 11beta-HSD2 exclusively to the endoplasmic reticulum (ER) membrane. To examine the orientation of tagged 11beta-HSD enzymes within the ER membrane, we stained selectively permeabilized HEK-293 cells with anti-FLAG antibody. Immunohistochemistry revealed that the N terminus of 11beta-HSD1 is cytoplasmic, and the catalytic domain containing the C terminus is protruding into the ER lumen. In contrast, the N terminus of 11beta-HSD2 is lumenal, and the catalytic domain is facing the cytoplasm. Chimeric proteins where the N-terminal anchor sequences of 11beta-HSD1 and 11beta-HSD2 were exchanged adopted inverted orientation in the ER membrane. However, both chimeric proteins were not catalytically active. Furthermore, mutation of a tyrosine motif to alanine in the transmembrane segment of 11beta-HSD1 significantly reduced V(max). The subcellular localization of 11beta-HSD1 was not affected by mutations of the tyrosine motif or of a di-lysine motif in the N terminus. However, residue Lys(5), but not Lys(6), turned out to be critical for the topology of 11beta-HSD1. Mutation of Lys(5) to Ser inverted the orientation of 11beta-HSD1 in the ER membrane without loss of catalytic activity. Our results emphasize the importance of the N-terminal transmembrane segments of 11beta-HSD enzymes for their proper function and demonstrate that they are sufficient to determine their orientation in the ER membrane. << Less

  J. Biol. Chem. 274:28762-28770(1999) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.