Discovery of a benzimidazole series as the first highly potent and selective ACSL1 inhibitors.

Long-chain acyl-CoA synthetase-1 (ACSL1), an enzyme that catalyzes the synthesis of long-chain acyl-CoA from the corresponding fatty acids, is believed to play essential roles in lipid metabolism. Structure activity relationship studies based on HTS hit compound 1 delivered the benzimidazole series as the first selective and highly potent ACSL1 inhibitors. Representative compound 13 exhibited not only remarkable inhibitory activity against ACSL1 (IC50 = 0.042 µM) but also excellent selectivity for the other ACSL isoforms. In addition, compound 13 demonstrated an in vivo suppression effect against the production of long-chain acyl-CoAs in mouse.

Tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin as a non-invasive biomarker for non-alcoholic steatohepatitis: a novel glycobiomarker for non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD) that may lead to liver cirrhosis or hepatocellular carcinoma. Here, we examined the diagnostic utility of tri-antennary tri-sialylated mono-fucosylated glycan of alpha-1 antitrypsin (AAT-A3F), a non-invasive glycobiomarker identified in a previous study of NASH diagnosis. This study included 131 biopsy-proven Japanese patients with NAFLD. We evaluated the utility of AAT-A3F in NASH diagnosis, and conducted genetic analysis to analyse the mechanism of AAT-A3F elevation in NASH. Serum AAT-A3F concentrations were significantly higher in NASH patients than in NAFL patients, and in patients with fibrosis, lobular inflammation, and ballooning. Hepatic FUT6 gene expression was significantly higher in NASH than in NAFL. IL-6 expression levels were significantly higher in NASH than in NAFL and showed a positive correlation with FUT6 expression levels. The serum-AAT-A3F levels strongly correlated with hepatic FUT6 expression levels. AAT-A3F levels increased with fibrosis, pathological inflammation, and ballooning in patients with NAFLD and may be useful for non-invasive diagnosis of NASH from the early stages of fibrosis.

Quantifying Protein-Specific N-Glycome Profiles by Focused Protein and Immunoprecipitation Glycomics.

Serum N-glycans have been reported to be potential diagnostic and therapeutic biomarkers for many diseases and conditions, such as inflammation, fibrosis, and cancer progression. We previously described the focused protein glycomic analysis (FPG) from gel-separated serum proteins. With this methodology, we sought novel glycan biomarkers for nonalcoholic steatohepatitis (NASH) and successfully identified some N-glycans that were significantly elevated in NASH patients compared to nonalcoholic fatty liver patients. Among them, trisialylated monofucosylated triantennary glycan (A3F) of alpha-1 antitrypsin showed the most dynamic change. For rapid identification of N-glycans on the focused proteins, we constructed a simplified method called immunoprecipitation glycomics (IPG), where the target proteins were immunoprecipitated with affinity beads and subsequently subjected to glycomic analysis by MALDI-TOF MS. Focusing on alpha-1 antitrypsin and ceruloplasmin as the target proteins, we compared the values of N-glycans determined by FPG and IPG. The quantified values of each N-glycan by these two methods showed a statistically significant correlation, indicating that high throughput and quantitative N-glycomics of targeted proteins can be achieved by the simplified IPG method. Thus, an analytical strategy combining FPG and IPG can be adapted to general biomarker discovery and validation in appropriate disease areas.

Glycomics for drug discovery: metabolic perturbation in androgen-independent prostate cancer cells induced by unnatural hexosamine mimics.

Inhibited: N-acetylglucosamine (GlcNAc) derivatives with a fluorine atom at the C4 position (2-4) were synthesized, and their ability to inhibit cancer-cell growth was investigated. The administration of these 4F-GlcNAc derivatives to cells led to the unnatural sugar nucleotide 1. Furthermore, N-glycan profiles of cells were determined by using a glycoblotting-based enrichment analysis, which is suitable for high-throughput screenings for drug discovery.

Alterations of high-mannose type N-glycosylation in human and mouse osteoarthritis cartilage.

OBJECTIVE: The process of N-glycosylation is involved in the pathogenesis of various diseases. However, little is known about the contribution of changes in N-glycans in osteoarthritis (OA). The aim of this study was to identify the alterations in N-glycans in human OA cartilage, to characterize the messenger RNA (mRNA) expression of N-glycan biosynthesis enzyme genes (N-glycogenes) in mouse articular chondrocytes during cartilage degradation, and to analyze the relationship between altered N-glycan patterns and mechanisms of cartilage degradation. METHODS: Alterations in N-glycans were analyzed in human OA cartilage and degraded mouse cartilage by high-performance liquid chromatography and mass spectrometry. N-glycogene mRNA expression in mouse chondrocytes was measured using reverse transcription-polymerase chain reaction. To assess the relationship between the altered N-glycans and degradation of mouse cartilage, experiments involving either knockdown or overexpression of N-glycogenes were performed in mouse articular chondrocytes. RESULTS: Alterations in high-mannose type N-glycans were observed in both human OA cartilage and degraded mouse cartilage. The expression of ß1,2N-acetylglucosaminyltransferase I (GlcNAc-TI) mRNA, which converts high-mannose type N-glycans, was significantly increased in degraded mouse cartilage. Mouse chondrocytes with suppressed GlcNAc-TI expression had reduced levels of matrix metalloproteinase 13 (MMP-13) and ADAMTS-5 (aggrecanase 2) mRNA following stimulation with interleukin-1α (IL-1α). In contrast, mouse chondrocytes overexpressing GlcNAc-TI had increased levels of MMP-13 and ADAMTS-5 mRNA following stimulation with IL-1α. CONCLUSION: These findings indicate that alterations in high-mannose type N-glycans and N-glycogenes in chondrocytes correlate with the release of MMP-13 and ADAMTS-5 during cartilage degradation. These findings suggest that N-glycans play a crucial role in the initiation and progression of OA.

Chromatographic deuterium isotope effects of derivatized N-glycans and N-glycopeptides in a zwitterionic type of hydrophilic interaction chromatography.

HPLC/MS is now an essential method for the analysis of glycans and glycopeptides. A technique using ICATs is also becoming popular for their comparative/quantitative analysis based on MS signal intensities. However, the RP HPLC most often used causes "doublet" peaks of deuterium-labeled and nonlabeled peptides, which are well known for causing the chromatographic deuterium isotope effect. Such doublet peaks are undesirable for precise comparison of MS signal intensities. This report shows that, in the zwitterionic type of hydrophilic interaction chromatography, the chromatographic deuterium isotope effect is negligibly small for N-glycans derivatized with deuterium-labeled 2-aminopyridine and N-glycopeptides derivatized with deuterium-labeled succinic anhydride.

BlotGlycoABCTM, an integrated glycoblotting technique for rapid and large scale clinical glycomics.

Recent progress in mass spectrometry has led to new challenges in glycomics, including the development of rapid glycan enrichment techniques. A facile technique for exploration of a carbohydrate-related biomarker is important because proteomics research targets glycosylation, a posttranslational modification. Here we report an "all-in-one" protocol for high throughput clinical glycomics. This new technique integrates glycoblotting-based glycan enrichment onto the BlotGlycoABC bead, on-bead stabilization of sialic acids, and fluorescent labeling of oligosaccharides in a single workflow on a multiwell filter plate. The advantage of this protocol and MALDI-TOF MS was demonstrated through differentiation of serum N-glycan profiles of subjects with congenital disorders of glycosylation and hepatocellular carcinoma and healthy donors. The method also permitted total cellular glycomics analysis of human prostate cancer cells and normal human prostate epithelial cells. These results demonstrate the potentials of glycan enrichment/processing for biomarker discovery.

Detection of altered N-glycan profiles in whole serum from rheumatoid arthritis patients.

Altered N-glycosylation occurs in many diseases. In rheumatoid arthritis (RA), for example, reduction in galactose residues in IgG and an increase in fucose residues in alpha1-acid glycoprotein have been observed. To further analyse N-glycans in disease, we show N-glycan profiling from whole serum employing reversed phase high performance liquid chromatography/negative-ion mode by sonic spray ionization ion trap mass spectrometry with pyridylamination. Profiles from female 15 RA patients and 18 aged-matched healthy women were compared. The most significant change seen in RA was decreased levels of mono-galactosyl bi-antennary N-glycans, in agreement with the previous reports regarding IgG. We also show previously unreported differences between isomers and increased tri-antennary oligosaccharides. These results indicate that LC-MS analysis of whole serum N-glycans can identify N-glycan alterations in RA and that this is a promising method both for studies of RA mechanisms and diagnosis.

Unusual N-glycan structures in alpha-mannosidase II/IIx double null embryos identified by a systematic glycomics approach based on two-dimensional LC mapping and matrix-dependent selective fragmentation method in MALDI-TOF/TOF mass spectrometry.

alpha-Mannosidase IIx (MX) is an enzyme closely related to alpha-mannosidase II (MII), a key enzyme in N-glycan biosynthesis that catalyzes the first step in conversion of hybrid- to complex-type N-glycans in Golgi apparatus. Recently we generated MII/MX double knock-out mice and found that double nulls completely lack the complex-type N-glycans (Akama, T. O., Nakagawa, H., Wong, N. K., Sutton-Smith, M., Dell, A., Morris, H. R., Nakayama, J., Nishimura, S.-I., Pai, A., Moremen, K. W., Marth, J. D., and Fukuda, M. N. (2006) Essential and mutually compensatory roles of alpha-mannosidase II and alpha-mannosidase IIx in N-glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 8983-8988). In the present study, we determined minor but unusual N-glycan structures found in MII/MX double knock-out mice. We identified such N-glycans by a systematic glycomics approach applying a two-dimensional LC mapping database and matrix-dependent selective fragmentation technique in MALDI-TOF/TOF MS, a highly sensitive and reliable technique that provides specific fragmentations enabling the determination of precise oligosaccharide structures including regioisomers (Kurogochi, M., and Nishimura, S.-I. (2004) Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry. Anal. Chem. 76, 6097-6101). Quantitative profiling of all N-glycan structures including minor components from MII/MX nulls, MII nulls, MX nulls, and wild-type mice at embryonic day 15.5 yielded a total of 37 species when structural heterogeneity was reduced by the removal of the sialic acids. Among six unusual N-glycan structures, two glycoforms were novel and were found only in MII/MX double nulls. We characterize such structure as pseudocomplex-type N-glycans. The present study demonstrated that use of the versatile matrix-dependent selective fragmentation method in MALDI-TOF/TOF MS greatly accelerates detailed structural analysis of a trace amount of N-glycans.