Discovery and structure–activity relationships of a novel class of quinazoline glucokinase activators

We describe design, syntheses and structure–activity relationships of a novel class of 4,6-disubstituted quinazoline glucokinase activators. Prototype quinazoline leads (4 and 5) were designed based on the X-ray analyses of the previous 2-aminobenzamide lead classes. Modifications of the quinazoline leads led to the identification of a potent GK activator (21d).     

Relationships Between Slc1a5 and Osteoclastogenesis

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5^−/− mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5^+/+ and Slc1a5^−/− bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5^−/− bone marrow cells was reduced to 70% of that of cells from Slc1a5^+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5^+/+ or Slc1a5^−/− mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5^−/− mice was severely impaired compared with those from Slc1a5^+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5^−/− osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Osteoclasts are giant multinucleated cells of hematopoietic origin that are responsible for bone resorption. The differentiation of osteoclasts can be induced by treating bone marrow macrophages with RANKL.² After stimulation, bone marrow macrophages mature and then fuse to become multinucleated osteoclasts. The processes of osteoclastogenesis and bone resorption are known to be energy-demanding,⁸ but little is known about the amino acid requirements of osteoclasts. In this study, we investigated the role of glutamine in osteoclastogenesis. Glutamine was selected for this work because it provides an excellent example of amino acid metabolism.Although glutamine acts as an essential amino acid in some specific physiologic situations, it is classified as a nonessential amino acid.⁵ The need for the biosynthesis and metabolism of amino acids is significantly increased in cells with high rates of proliferation, such as functionally active cells and cancer cells. The activity of amino acid synthetases such as glutamine synthetase is increased in these cells. In addition, glutamine transporters on the plasma membrane are important, because they mediate glutamine uptake to meet the intracellular glutamine demand. The transporter Slc1a5, also known as ASCT2, is particularly important for glutaminolysis and mTOR signaling.^14,16Glutamine concentrations in tissue and blood are regulated by the activities of glutamine synthetase and glutaminase. ­Endogenous synthesis cannot meet the cell’s demands for glutamine in conditions including cancer, infections, and intense physical exercise. Glutamine is released into the blood from the lungs, adipocytes, and skeletal muscles and is transported into the cytoplasm via glutamine acid transporter molecules on the cell membrane. Glutamine is required for the growth of cancer cells; upregulation of the expression of the proteins involved in glutamine transport has been observed in tumor cells.⁴ Slc1a5 (ASCT2) is a small neutral amino acid exchanger that is overexpressed in many cancers and is the most well-described glutamine transporter in cancer cells.⁹ However, previous studies^1,10,22,23 have reported that silencing, deletion, and amino-acid analog substitution of Slc1a5 in cancer cells generated different results for mTORC1 signaling, proliferation, and cell migration.^{1,3,4,10,22,23} Additional work^3,4 has shown that Slc1a5 is indispensable for tumor growth and mTORC1 signaling. Slc1a5 is important in accumulating nonessential amino acids to quickly restore amino acid composition during imbalanced amino acid usage,⁴ whereas Slc38a1 (SNAT1) and Slc38a2 (SNAT2) mediate the net import of glutamine.In bone homeostasis, glutamine is a critical regulator of energy for protein and nucleic acid synthesis via the tricarboxylic acid cycle. Active glutamine metabolism stimulates the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts. The enzyme glutaminase deaminates glutamine to form glutamate. Glutaminase deficiency in osteoblasts and chondrocytes leads to reduced osteoblast formation and decreased bone mass, resulting in potentially dangerous conditions, such as osteoporosis.²⁴ In osteoclasts, glutamine is an important source of fuel for protein and nucleic acid biosynthesis. Therefore, Slc1a5 deficiency in mice may influence bone homeostasis, including osteoclastogenesis. We therefore created Slc1a5-deficient mice to investigate the contribution of Slc1a5 to the development and functional properties of osteoclasts.     

Leptin and insulin down-regulate angiopoietin-like protein 3, a plasma triglyceride-increasing factor

We reported previously that angiopoietin-like protein3 (ANGPTL3), a liver-specific secretory factor, increased plasma triglyceride (TG) via inhibition of lipoprotein lipase and free fatty acid (FFA) by activating adipose-lipolysis. The current study examined the regulation of Angptl3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angptl3 expression and plasma ANGPTL3 levels were increased in leptin-resistant C57BL/6J(db/db) and -deficient C57BL/6J(ob/ob) mice, relative to the control. Leptin supplements decreased Angptl3 gene expression and plasma ANGPTL3 in C57BL/6J(ob/ob) mice. The changes of Angptl3 were associated with alterations of plasma TG and FFA levels. Leptin treatment directly suppressed Angptl3 gene expression in hepatocytes. Angptl3 gene expression and plasma protein levels were also increased in insulin-deficient streptozotocin-treated mice. Insulin treatment of hepatocytes decreased Angptl3 gene expression and protein secretion. Our results suggest that elevated ANGPTL3 by leptin- or insulin-resistance is attributed to increased plasma TG and FFA concentrations in obesity.     

Decreased stimulation of CD4+ T cell proliferation and IL-2 production by highly enriched populations of HIV-infected dendritic cells

APC infection and dysfunction may contribute to the immunopathogenesis of HIV disease. In this study, we examined immunologic function of highly enriched populations of HIV-infected monocyte-derived dendritic cells (DC). Compared with uninfected DC, HIV-infected DC markedly down-regulated surface expression of CD4. HIV p24(+) DC were then enriched by negative selection of CD4(+)HIV p24(-) DC and assessed for cytokine secretion and immunologic function. Although enriched populations of HIV-infected DC secreted increased IL-12p70 and decreased IL-10, these cells were poor stimulators of allogeneic CD4(+) T cell proliferation and IL-2 production. Interestingly, HIV-infected DC secreted HIV gp120 and the addition of soluble (s) CD4 (a known ligand for HIV gp120) to DC-CD4(+) T cell cocultures restored T cell proliferation in a dose-dependent manner. By contrast, addition of antiretroviral drugs did not affect CD4(+) T cell proliferation. Furthermore, recombinant HIV gp120 inhibited proliferation in uninfected cocultures of allogeneic DC and CD4(+) T cells, an effect that was also reversed by addition of sCD4. In summary, we show that HIV gp120 produced by DC infected by HIV in vitro impairs normal CD4(+) T cell function and that sCD4 completely reverses HIV gp120-mediated immunosuppression. We hypothesize that HIV-infected DC may contribute to impaired CD4(+) T cell-mediated immune responses in vivo and that agents that block this particular immunosuppression may be potential immune adjuvants in HIV-infected individuals.     

Synthesis and structure-activity relationships of 5-phenylthiophenecarboxylic acid derivatives as antirheumatic agents

5-(Phenylthiophene)-3-carboxylic acid (2a), a metabolite of esonarimod (1), which was developed as a new antirheumatic drug, was considered as a lead compound for new antirheumatic drugs. A new series of 2a derivatives were synthesized and their characteristic pharmacological effects, that is their antagonistic effect toward interleukin (IL)-1 in mice and the suppressive effect against adjuvant-induced arthritis (AIA) in rats, were evaluated and compared with those of 1. The structure-activity relationships indicated that [5-(4-bromophenyl)-thiophen-3-yl]acetic acid (5d), methyl [5-(4-chlorophenyl)-thiophen-3-yl]acetate (5h), and methyl [5-(4-bromophenyl)-thiophen-3-yl]acetate (5i) suppressed AIA more potently than 1 and all of the other synthesized compounds.     

Role of adiponectin in preventing vascular stenosis. The missing link of adipo-vascular axis

Obesity is more linked to vascular disease, including atherosclerosis and restenotic change, after balloon angioplasty. The precise mechanism linking obesity and vascular disease is still unclear. Previously we have demonstrated that the plasma levels of adiponectin, an adipose-derived hormone, decreases in obese subjects, and that hypoadiponectinemia is associated to ischemic heart disease. In current the study, we investigated the in vivo role of adiponectin on the neointimal thickening after artery injury using adiponectin-deficient mice and adiponectin-producing adenovirus. Adiponectin-deficient mice showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries. Adenovirus-mediated supplement of adiponectin attenuated neointimal proliferation. In cultured smooth muscle cells, adiponectin attenuated DNA synthesis induced by growth factors including platelet-derived growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), basic fibroblast growth factor, and EGF and cell proliferation and migration induced by HB-EGF. In cultured endothelial cells, adiponectin attenuated HB-EGF expression stimulated by tumor necrosis factor alpha. The current study suggests an adipo-vascular axis, a direct link between fat and artery. A therapeutic strategy to increase plasma adiponectin should be useful in preventing vascular restenosis after angioplasty.     

Intergranular bridges in the anterior pituitary cell and their possible involvement in Ca2+-induced granule-granule fusion

Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca²⁺-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca²⁺. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca²⁺ with Clostridium perfringens enterotoxin which induces Ca²⁺ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca²⁺-induced granule-granule fusion in anterior pituitary cells.     

Phylogeny of Bipolaris inferred from nucleotide sequences of Brn1, a reductase gene involved in melanin biosynthesis

The Brn1 reductase melanin biosynthesis gene in the fungal genus Bipolaris was sequenced in 74 strains of 22 species. The Brn1 region was highly conserved among the species examined at the nucleotide and the amino acid levels. To elucidate the phylogenetic relationships among Bipolaris species, trees were inferred from nucleotide sequences of this region. Species in these trees formed exclusive clusters clearly separated from one another, except for B. panici-miliacei and B. setariae, and B. victoriae and B. zeicola. When unidentified strains were added to this tree, they fell within known species or formed independent clusters. These data indicated that the Brn1 gene region was suitable for species-level systematics within the genus. The results also suggest that Bipolaris consists of two or more clades that may reflect teleomorphic connections.