Pyridine amides as potent and selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored.

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides.

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.

Cofactor-specific modulation of 11beta-hydroxysteroid dehydrogenase 1 inhibitor potency.

11beta-hydroxysteroid dehydrogenase 1 regulates the tissue availability of cortisol by interconverting cortisone and cortisol. It is capable of functioning as both a reductase and a dehydrogenase depending upon the surrounding milieu. In this work, we have studied the reaction mechanism of a soluble form of human 11beta-hydroxysteroid dehydrogenase 1 and its mode of inhibition by potent and selective inhibitors belonging to three different structural classes.

A general liquid chromatography/mass spectroscopy-based assay for detection and quantitation of methyltransferase activity.

Methyltransferases form a large class of enzymes, most of which use S-adenosylmethionine as the methyl donor. In fact, S-adenosylmethionine is second only to ATP in the variety of reactions for which it serves as a cofactor. Several methods to measure methyltransferase activity have been described, most of which are applicable only to specific enzymes and/or substrates.

Discovery of a potent and novel motilin agonist.

A novel series of dihydro- and tetrahydrotriazolopyridazine-1,3-dione-based amino acid derivatives were identified as very potent motilin receptor agonists. Incorporating one additional phenylethyl glycinamide subunit to 1 (EC(50) = 660 nM) was found to improve in vitro potency approximately 3000-fold, resulting in compound 10 (EC(50) = 0.22 nM).

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo.

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0.

Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis.

Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose.

Structural characterization of a new galactofuranose-containing glycolipid antigen of Paracoccidioides brasiliensis.

An acidic glycolipid (Band 1), purified from P. brasiliensis by a combination of ion exchange chromatography, HPLC, and HPTLC, was found to be reactive with sera of all patients with paracoccidioidomycosis (PCM). Monosaccharide analysis of Band 1 yielded mannose and galactose in a 2:1 ratio, while mild acid hydrolysis and mild periodate oxidation/NaB3H4 reduction indicated the presence of a terminal galactofuranose.

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 2. Characterization of E-selectin binding fractions, and structural requirements for physiological binding to E-selectin.

E-selectin binding gangliosides were isolated from myelogenous leukemia HL60 cells, and the E-selectin binding pattern was compared with that of human neutrophils as described in the preceding paper in this issue. The binding fractions were identified as monosialogangliosides having a series of unbranched polylactosamine cores. Structures of fractions 12-3, 13-1, 13-2, and 14, which showed clear binding to E-selectin under the conditions described in the preceding paper, were characterized by functional group analysis by application of monoclonal antibodies, 1H-NMR, FAB-MS, and electrospray mass spectrometry with collision-induced dissociation of permethylated fractions.

Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 1. Separation of E-selectin binding from nonbinding gangliosides, and absence of sialosyl-Le(x) having tetraosyl to octaosyl core.

Previous studies suggested that sialosyl-Le(x) (SLex) is a ligand expressed in human neutrophils and myelogenous leukemia HL60 cells which binds to E-selectin and possibly P-selectin. However, clear data on structures of carbohydrate epitopes in these cells were lacking. A systematic study was therefore initiated, employing a large quantity of HL60 cells (> or = 1200 mL packed) and human leukocytes (approximately 100 mL packed).

Myeloglycan, a series of E-selectin-binding polylactosaminolipids found in normal human leukocytes and myelocytic leukemia HL60 cells.

Sialosyl-Lex (SLex) is assumed to be the binding epitope of E- and P-selectin in normal human neutrophils and myelocytic leukemia HL60 cells. Glycosphingolipid (GSL) fractions from large quantities of normal human neutrophils and HL60 cell extract did not contain SLex GSLs having 6-10 sugar residues, as commonly found in solid tumor cells and tissues. Instead, the binding target of E-selectin was revealed to be a series of long-chain, unbranched polylactosamine GSLs with terminally sialylated, internally alpha 1-->3 polyfucosylated structure as the major component, or having SLex at the terminus and internally polyfucosylated structure as a minor component.

Human tumor-associated Le(a)-Le(x) hybrid carbohydrate antigen IV3(Gal beta 1-->3[Fuc alpha 1-->4]GlcNAc)III3FucnLc4 defined by monoclonal antibody 43-9F: enzymatic synthesis, structural characterization, and comparative reactivity with various antibodies.

Two Le(a)-cross-reacting monoclonal antibodies (mAbs) were previously established which define complex tumor-associated carbohydrate antigens. The first mAb, 43-9F, was raised against human squamous cell lung carcinoma and shows preferential reactivity with various human cancers over normal cells. Its tumor cell binding activity is best inhibited by a milk oligosaccharide characterized as Le(a)-Le(x) [Mårtensson, S.

A revised structure for the disialosyl globo-series gangliosides of human erythrocytes and chicken skeletal muscle.

Disialosyl globo-series gangliosides have previously been isolated from chicken skeletal muscle (E. L. Hogan, R.

Common tetrasaccharide epitope NeuAc alpha 2-->3Gal beta 1-->3(Neu-Ac alpha 2-->6)GalNAc, presented by different carrier glycosylceramides or O-linked peptides, is recognized by different antibodies and ligands having distinct specificities.

A novel globo-series disialoganglioside, disialosyl galactosyl globoside (Structure 1 below), defined by new monoclonal antibody (mAb) RM2, was isolated and characterized as having terminal structure identical to that of ganglio-series ganglioside GD1 alpha (Structure 2) and a common mucin-type epitope (Structure 3) widely distributed in glycoproteins such as glycophorin A. While these three structures share a common nonreducing tetrasaccharide terminus, mAb RM2 showed strong specific reactivity only with Structure 1, not with Structures 2 or 3. Another mAb, QSH2, reacted strongly with Structure 3 but did not cross-react with Structures 1 or 2.

Stage-specific glycosphingolipids from amastigote forms of Leishmania (L.) amazonensis. Immunogenicity and role in parasite binding and invasion of macrophages.

Neutral glycosphingolipids (GSLs) from amastigote forms of Leishmania (L.) amazonensis were isolated, and their structures and biological properties were characterized. Based on various immunochemical methods, these GSLs were shown to be expressed at certain stages of amastigote development.

Structural characterization of x2 glycosphingolipid, its extended form, and its sialosyl derivatives: accumulation associated with the rare blood group p phenotype.

It has been suggested that the x2 glycosphingolipid (GSL) could offer a structural basis for a P-like antigen activity found in blood group p individuals [Kannagi R., Fukuda, M.N.

Characterization of a series of novel fucose-containing glycosphingolipid immunogens from eggs of Schistosoma mansoni.

Lipid extracts of eggs, worms, and cercariae of the parasitic trematode Schistosoma mansoni have been shown to contain a large number of highly immunogenic glycolipids (Weiss, J. B., Magnani, J.

Extended type-1 chain glycosphingolipid antigens. Isolation and characterization of trifucosyl-Leb antigen (III4V4VI2Fuc3Lc6).

A Lewis-b-active glycosphingolipid containing a repetitive type-1 chain carbohydrate core was isolated from human colonic adenocarcinoma cell line Colo205. This glycosphingolipid was purified by HPLC and preparative high-performance thin-layer chromatography and its structure elucidated by positive-ion fast-atom-bombardment mass spectrometry with collision-induced disassociation, 1H-NMR spectroscopy and methylation analysis. The glycosphingolipid was found to be a trifucosylated derivative of this novel carbohydrate core, having the following structure: [formula; see text].

Differentiation of type 1 and type 2 chain linkages of native glycosphingolipids by positive-ion fast-atom bombardment mass spectrometry with collision-induced dissociation and linked scanning.

Two underivatized glycosphingolipids, Le(b) and Le(y), isomeric in carbohydrate structure (Fuc alpha 1-->2Gal beta 1--> 3[Fuc alpha 1-->4]GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer and Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3Gal beta 1--> 4Glc beta 1-->1Cer, respectively), were analyzed by positive-ion fast-atom bombardment (FAB) mass spectrometry with high energy collision-induced dissociation (CID) and linked scanning. The two isomers were distinguishable by the abundance of product ions derived from the non-reducing terminal tetrasaccharide fragment via sequential beta-eliminations of vicinally linked saccharide residues. Following earlier studies from other laboratories, which have dealt primarily with positive-ion FAB-CID mass spectrometry of simple model oligosaccharides, these results exemplify the practical application of two-sector methodology to underivatized complex glycoconjugates commonly encountered in the biomedical field.

Extended type 1 chain glycosphingolipids: dimeric Lea (III4V4Fuc2Lc6) as human tumor-associated antigen.

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis.

Strategies for characterization of ganglioside inner esters. I--Fast atom bombardment mass spectrometry.

A comparison of old and new fast atom bombardment (FAB) mass spectrometric strategies for characterization of ganglioside inner esters (lactones) is presented. Data obtained for lactones of GD3 (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) using negative ion FAB mass spectrometry of underivatized materials, negative ion FAB mass spectrometry following ammonolysis, and positive ion FAB mass spectrometry following ammonolysis and permethylation are presented and discussed. The latter method uses well-known reactions to produce a novel type of ganglioside derivative, highly amenable to analysis by positive ion FAB mass spectrometry, which is introduced to simplify unambiguous location of NeuAc residues involved in ester linkages to other sugars.

Novel tri-and tetrasialosylpoly-N-acetyllactosaminyl gangliosides of human placenta: structure determination of pentadeca- and eicosaglycosylceramides by methylation analysis, fast atom bombardment mass spectrometry, and 1H NMR spectroscopy.

A series of highly polar neolacto series (poly-N-acetyllactosaminyl) gangliosides were isolated from human placenta tissue and purified by HPLC and preparative HPTLC. Two of these ganglioside fractions (G-12 and G-13) were analyzed by 500-MHz 1H NMR spectroscopy, GC-EIMS, +FAB-MS, and sequential exoglycosidase treatments. Their structures have been identified as being of the repeating N-acetyllactosamine type, multiply branched through GlcNAc beta 1----6/3 linkages, with every nonreducing Gal terminal alpha 2----3-sialosylated, as shown below.

A novel strategy for unambiguous determination of inner esterification sites of ganglioside lactones.

A method is described which is suitable for protection of all free hydroxyl groups of a glycosphingolipid under conditions which will not cleave ester linkages, including inner ester linkages characteristic of ganglioside lactones. The protecting methoxyethoxymethyl group is stable in alkaline media, surviving permethylation procedures which introduce a methyl ether at all sites previously acylated. Hydrolysis, reduction, and acetylation then yield alditol acetate derivatives which can be analyzed by conventional GC-MS to locate the methyl ether groups.

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure.

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S.