Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae.

BACKGROUND: Production of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galß1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcß1-3GalNAcα1-O-Ser/Thr) has not been constructed. METHODS: To generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC. RESULTS: Introduction of a codon-optimized UDP-GlcNAc:ßGal ß-1,3-N-acetylglucosaminyltransferase 6 (ß3Gn-T6) gene yielded increases in ß3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of ß3Gn-T6 was observed at Mn(2+) concentrations of 10mM and above. Supplementation of MnCl2 to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl2 to the medium. CONCLUSIONS: Physiological Mn(2+) concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn(2+) supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production. GENERAL SIGNIFICANCE: This study reveals that control of Mn(2+) concentration is important for production of specific mammalian-type glycans in S. cerevisiae.

Discovery of novel phenylpyridone derivatives as potent and selective MCH1R antagonists.

The design, synthesis and structure-activity relationships of a novel class of N-phenylpyridone MCH1R antagonists are described. The core part of the N-phenylpyridone structure was newly designed and the side chain moieties that were attached to the core part were extensively explored. As a result of optimization of the N-phenylpyridone leads, we successfully developed the orally available, and brain-penetrable MCH1R selective antagonist 7c, exhibiting excellent anti-obese effect in diet-induced obese (DIO) mice.

A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca(2+) influx.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca(2+) release from intracellular stores, but to extracellular Ca(2+) influx. The response was inhibited by the presence of either L-type Ca(2+) channel blockers or K(+) ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Galphas- nor Galphaq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca(2+) influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.

Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists.

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.