A novel [5.2.1]bicyclic amine is a potent analgesic without µ opioid activity.

We identified (5R)-6-methyl-5-phenyl-1,3,4,5,6,7-hexahydro-2,5-methano-2,6-benzodiazonine (DS21980956: 4-(R)) as a novel [5.2.1]bicyclic basic compound. The scaffold was inspired by fentanyl or pethidine, which possess potent analgesic activities. DS21980956 had potent analgesic activity in the mouse acetic acid writhing test or tail flick test without agonistic activity at the µ opioid receptor (MOR). The mechanism of analgesic action of DS21980956 was considered to differ from a biased ligand, for example, TRV-130 (3, oliceridine).

Discovery of DS42450411 as a potent orally active hepcidin production inhibitor: Design and optimization of novel 4-aminopyrimidine derivatives.

Hepcidin has emerged as the central regulatory molecule in systemic iron homeostasis. The inhibition of hepcidin may be a favorable strategy for the treatment of anemia of chronic disease. Here, we have reported the design, synthesis, and structure-activity relationships (SAR) of a series of 4-aminopyrimidine compounds as inhibitors of hepcidin production. The optimization study of 1 led to the design of a potent and bioavailable inhibitor of hepcidin production, 34 (DS42450411), which showed serum hepcidin-lowering effects in a mouse model of interleukin-6-induced acute inflammation.

Target identification of hepcidin production inhibitors by a combination of chemical proteomics and radioactive compound binding assay.

Hepcidin is a peptide hormone and has emerged as the central molecule regulating systemic iron homeostasis. Hepcidin inhibition could be a strategy for treating anemia of chronic disease. We previously reported the discovery of DS79182026, a new inhibitor of hepcidin production, from phenotypic screening using the human hepatocyte HepG2 cell line. In this study, we utilized a combination of affinity purification-based chemical proteomics and radioactive compound binding assay, and identified several candidate proteins. Purified recombinant proteins were subjected to radioactive compound binding assays for validation, and ALK2 and ALK3 demonstrated specific binding to the compound. Since ALK2 is known to be related to hepcidin production, we focused on ALK2 and found that its knockdown decreased hepcidin expression; we also found a strong correlation (R = 0.920) between pharmacological activity and compound affinity to ALK2. These results indicate that ALK2 is the primary target protein of our new hepcidin production inhibitors.

Discovery of DS28120313 as a potent orally active hepcidin production inhibitor: Design and optimization of novel 4,6-disubstituted indazole derivatives.

Hepcidin has emerged as the central regulatory molecule in systemic iron homeostasis, and its inhibition could be a favorable strategy for treating anemia of chronic disease (ACD). Here, we report the design, synthesis and structure-activity relationships (SAR) of a series of 4,6-disubstituted indazole compounds as hepcidin production inhibitors. The optimization study of multi-kinase inhibitor 1 led to the design of a potent and bioavailable hepcidin production inhibitor, 32 (DS28120313), which showed serum hepcidin-lowering effects in an interleukin-6-induced acute inflammatory mouse model.

Discovery of DS79182026: A potent orally active hepcidin production inhibitor.

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of benzisoxazole compounds as orally active hepcidin production inhibitors. The optimization study of multi kinase inhibitor 1 led to a potent and bioavailable hepcidin production inhibitor 38 (DS79182026), which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

Synthesis and SAR studies of 3,6-disubstituted indazole derivatives as potent hepcidin production inhibitors.

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of indazole compounds as hepcidin production inhibitors. The optimization study of compound 1 led to a potent hepcidin production inhibitor 45, which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

Multipeptide precursor structure of acaloleptin A isoforms, antibacterial peptides from the Udo longicorn beetle, Acalolepta luxuriosa.

We previously purified acaloleptin A1, A2, and A3, antibacterial peptides that are produced in the larval hemolymph of Acalolepta luxuriosa (Udo longicorn beetle). In this study, we performed cDNA cloning. The cDNA sequence showed a predicted acaloleptin A precursor that consisted of five acaloleptin A isoforms. Four (isoforms 1, 2, 3 and 4) of the five isoforms of the acaloleptin A precursor had high-level sequence identities with each other, but the N-terminal region of isoform 5 differed from those of the other acaloleptin A isoforms. Northern and Western blot analyses showed that acaloleptin A isoforms were mass-produced soon after bacterial inoculation. Finally, we purified isoform 5 from hemolymph of the immunized larvae. Isoform 5, unlike acaloleptin A1, A2 and A3, showed antimicrobial activities against a Gram-positive bacterium, Micrococcus luteus and a fungus, Magnaporthe grisea. These results suggest that the multipeptide structure of the acaloleptin A precursor allows A. luxuriosa high-level production of antibacterial peptides and resistance to a wide range of microorganisms.

Mating disruption for control of Melanotus okinawensis (Coleoptera: elateridae) with synthetic sex pheromone.

A mating disruption experiment to control Melanotus okinawensis Ohira (Coleoptera: Elateridae) was conducted at a sugarcane (Saccharum spp.) field and a wild Japanese pampas, Miscanthus sinensis Anderss, grassland on Minami-Daito Island (3,057 ha) from 2001 to 2007. The sugarcane field and the pampas grassland were treated with synthetic sex pheromone that evaporated from a polyethylene tube dispenser. The mean total catches obtained by monitoring traps in the sugarcane fields decreased by 96.1% in 2001 from the previous year on Minami-Daito Island. The mean total trap catches in the treated area further decreased by 74.0% from 2001 until 2007 as cumulative effects. Simultaneously, the number of adults captured by hand decreased from 4.7 per sugarcane field in 2001 to 0.5 in 2007 (89.3% reduction), whereas those captured in the untreated area (Miyagi Island) did not show such a decrease. The mating rates were significantly lower in the females captured in the treated area (14.3-71.4%) than those in the untreated area (96.9-100%). However, the amount of the decrease in the trap catches was relatively small at first (39.6% reduction) in the Japanese pampas grassland on the periphery of the Island. This was probably due to the loss of pheromone substance caused by the strong seasonal wind in the periphery. However, mean total trap catches at the periphery also decreased within several years; significant decreases were detected until 2003, 2006, and 2007. These results indicated that the mating disruption effectively reduced an isolated population of M. okinawensis.

Characterization of a novel C-type lectin, Bombyx mori multibinding protein, from the B. mori hemolymph: mechanism of wide-range microorganism recognition and role in immunity.

To investigate the system used by insects to recognize invading microorganisms, we examined proteins from the larval hemolymph of Bombyx mori that bind to the cell surface of microorganisms. Two hemolymph proteins that bound to the cell surfaces of Micrococcus luteus and Saccharomyces cerevisiae were shown to be identical. This protein bound to all 11 microorganisms examined-5 Gram-negative bacteria, 3 Gram-positive bacteria, and 3 yeasts-and was consequently designated B. mori multibinding protein (BmMBP). The sequence of the cDNA encoding BmMBP revealed that it was a C-type lectin with two dissimilar carbohydrate-recognition domains (CRD1 and CRD2) distantly related to known insect C-type lectins. CRD1 and CRD2 were prepared as recombinant proteins and their binding properties were investigated using inhibition assays. Each domain had wide, dissimilar binding spectra to sugars. These properties enable BmMBP to bind to two sites on a microorganism, facilitating high-affinity binding to many types of microorganisms. The dissociation constants of BmMBP with M. luteus cells and S. cerevisiae were 1.23 x 10(-8) and 1.00 x 10(-11) M, respectively. rBmMBP triggered the aggregation of hemocytes from B. mori larvae in vitro and microorganisms recognized by BmMBP were surrounded by aggregated hemocytes in vivo, forming a nodule, which is the typical cellular reaction in insect immune responses. These observations suggest that BmMBP functions as a trigger for the nodule reaction and that the multirecognition characteristic of BmMBP plays an important role in the early stages of infection by a variety of microorganisms.

Purification and cDNA cloning of a cecropin from the longicorn beetle, Acalolepta luxuriosa.

We have cloned and characterized a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the superfamily Cerambyocidea. This peptide is active against Micrococcus luteus and Escherichia coli, and the amino acid sequence deduced by cloning of the cDNA identifies it as a coleopteran cecropin. Sequence comparisons and phylogenetic analyses performed using Clustal X suggest that this cecropin is evolutionarily intermediate between dipteran and lepidopteran cecropins. The results of MALDI-TOF mass spectrometry indicate that the mature form of this antibacterial peptide is 35 amino acid residues in length and has an amidated C-terminal isoleucine. This report is the first description of a cecropin from a coleopteran insect.

Purification and cDNA cloning of luxuriosin, a novel antibacterial peptide with Kunitz domain from the longicorn beetle, Acalolepta luxuriosa.

We have purified a novel antibacterial peptide from the hemolymph of the coleopteran insect Acalolepta luxuriosa, of the family Cerambyocidae, and named it luxuriosin. This peptide showed growth-inhibitory activity against Micrococcus luteus and germination- and/or growth-inhibitory activity against the conidia from rice blast fungus, Magnaporthe grisea. The amino acid sequence determined by cDNA cloning identified luxuriosin as a peptide of 88 amino acids with a theoretical molecular weight of 10,368.34, containing a Kunitz domain.

Purification and cDNA cloning of a novel antibacterial peptide with a cysteine-stabilized alphabeta motif from the longicorn beetle, Acalolepta luxuriosa.

An antibacterial peptide from the hemolymph of a coleopteran insect, Acalolepta luxuriosa, in the superfamily Cerambyocidea was characterized. The mature antibacterial peptide had 27 amino acid residues with a theoretical molecular weight of 3099.29 and it showed antibacterial activity against Escherichia coli and Micrococcus luteus. The deduced amino acid sequence of the peptide showed that it had a cysteine-stabilized alphabeta motif with a C...CXXXC...C...CXC consensus sequence, like insect defensins. However, the results of a multiple sequence alignment and phylogenetic analysis with CLUSTAL X indicated that this peptide is a novel peptide with a cysteine-stabilized alphabeta motif that is distant from insect defensins.

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.