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A novel series of naphthalene derivatives were designed and synthesized based on the strategy focusing on the restriction of the flexible bond rotation of OX2R selective agonist YNT-185 (1) and their agonist activities against orexin receptors were evaluated. The 1,7-naphthalene derivatives showed superior agonist activity than 2,7-naphthalene derivatives, suggesting that the bent form of 1 would be favorable for the agonist activity. The conformational analysis of 1,7-naphthalene derivatives indicated that the twisting of the amide unit out from the naphthalene plane is important for the enhancement of activity. The introduction of a methyl group on the 2-position of 1,7-naphthalene ring effectively increased the activity, which led to the discovery of the potent OX2R agonist 28c (EC50 = 9.21 nM for OX2R, 148 nM for OX1R). The structure-activity relationship results were well supported by a comparison of the docking simulation results of the most potent derivative 28c with an active state of agonist-bound OX2R cryo-EM SPA structure. These results suggested important information for understanding the active conformation and orientation of pharmacophores in the orexin receptor agonists, which is expected as a chemotherapeutic agent for the treatment of narcolepsy.

Structurally diverse small compounds are utilized to obtain hit compounds that have suitable pharmacophores in appropriate three-dimensional conformations for the target drug receptors. We have focused on the 1,3,5-trioxazatriquinane skeleton, which has a high degree of three-dimensional properties, leading to a novel small-scale focused library based on 1,3,5- trioxazatriquinane. In the library screening for the orexin receptor, some of the compounds showed orexin receptor antagonistic activity with a high hit rate of 7%. By optimizing the hit compounds, we discovered a potent dual orexin receptor antagonist, 38b, and a selective orexin 1 receptor antagonist, 41b carrying the same plane structure. Both compounds showed reasonable brain permeability and beneficial effects when administered intraperitoneally to wild-type mice. Docking simulations of their eutomers, (–)-38b and (+)-41b, with orexin receptors suggested that the interaction between the 1,3,5-trioxazatriquinane core structure and the hydrophobic subpocket in orexin receptors enables a U-shape structure, which causes tight van der Waals interactions with the receptors similar to SB-334867, a selective orexin 1 receptor antagonist. These results indicate that the 1,3,5-trioxazatriquinanes bearing multiple effective residues (TriMERs) could serve as a privileged structure for G-protein coupled receptors (GPCRs) and the TriMER library approach might be useful for the hit discovery process targeting other GPCRs not only opioid and orexin receptors.

Retinal stem cell differentiation is orchestrated by transcription cascades, and is prone to be influenced by stress factors. While threshold radiation dose for human mental retardation is reported, the effects on retinal transcription factors are unknown. To elucidate the effects of low-dose radiation on retinal differentiation, stem cells were irradiated. Genome-wide gene expression and histone modification were observed. After a month, the expression of PAX6, an essential transcription factor for retinal ganglion cell, was increased by 30 mGy but decreased by 180 mGy. POU5F1, POU2F1, SOX2, and PAX6 were predicted to be involved in regulation of PAX6. Further, upstream of POU5F1/POU2F1/ SOX2 was detected. Altered Rho family could be responsible for activity of SRF/STAT3/RELA, which were upstream of POU2F1/SOX2. These cascades could contribute to the regulation of PAX6 and unstable retinal development under stressful environment. Identification of the hierarchical regulations in the differentiation will help elucidate the retinal cell maintenance.

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX1R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX1R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX1R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.

The 14-dehydration- and 14-H derivatives of the orexin 1 receptor (OX1R) antagonist YNT-707 (2) were synthesized. The obtained derivatives showed higher affinities for OX1R than the corresponding 14-hydroxy derivatives. The conformational analysis suggested that the 17-sulfonamide groups in the derivatives without the 14-hydroxy group have a greater tendency to be oriented toward the upper side of the D-ring compared with the 14-hydroxy derivatives. Additionally, the 14-dehydration-derivative with 6α-amide side chain showed significantly higher affinity than the 14-hydroxy derivative, while the corresponding 14-H derivative showed only slightly higher affinity. Thus, the 14-hydroxy group strongly affects the affinity of the antagonist for the OX1R.

The D-nor-nalfurafine derivatives, which were synthesized by contraction of the six-membered D-ring in nalfurafine (1), had no affinity for orexin 1 receptors (OX1Rs). The 17N-lone electron pair in 1 oriented toward the axial direction, while that of D-nor-derivatives was directed in the equatorial configuration. The axial lone electron pair can form a hydrogen bond with the 14-hydroxy group, which could push the 6-amide side chain toward the downward direction with respect to the C-ring. The resulting conformation would be an active conformation for binding with OX1R. The dual affinities of 1 for OX1R and κ opioid receptor (KOR) led us to elucidate the mechanism by which only 1 showed no aversion but U-50488H. Actually, 1 selectively induced severe aversion in OX1R knockout mice, but not in wild-type mice. These results well support that OX1R suppresses the aversion of 1. This is the elucidation of long period puzzle which 1 showed no aversion in KOR.

Cortical neurons fire intermittently and synchronously during non-rapid eye movement sleep (NREMS), in which active and silent periods are referred to as ON and OFF periods, respectively. Neuronal firing rates during ON periods (NREMS-ON-activity) are similar to those of wakefulness (W-activity), raising the possibility that NREMS-ON neuronal-activity is fragmented W-activity. To test this, we investigated the patterning and organization of cortical spike trains and of spike ensembles in neuronal networks using extracellular recordings in mice. Firing rates of neurons during NREMS-ON and W were similar, but showed enhanced bursting in NREMS with no apparent preference in occurrence, relative to the beginning or end of the on-state. Additionally, there was an overall increase in the randomness of occurrence of sequences comprised of multi-neuron ensembles in NREMS recorded from tetrodes. In association with increased burst firing, somatic calcium transients were increased in NREMS. The increased calcium transients associated with bursting during NREM may activate calcium-dependent, cell-signaling pathways for sleep related cellular processes.

The essential structure of the orexin 1 receptor (OX 1 R) antagonist YNT-707 ( 2 ) was clarified, particularly the roles to OX 1 R antagonist activities of the 3-OMe, the 4,5-epoxy ring, the 14-hydroxy group, and the orientation of the 6-amide side chain. The 3-OMe and 17-sulfonamide group were shown to be essential for the OX 1 R antagonistic activity. The 4,5-epoxy ring plays an important role for the active orientation of the 6-amide group. The 14-hydroxy group could lower the activity of the 6β-amide isomer by the interaction of the 14-hydroxy group with the 6-amide group, which could orient the 6-amide group toward the upper side of the C-ring. Finally, we proposed the difference in the active conformation between OX 1 R and κ opioid receptor (KOR), especially in the orientation of the 6-amide group which is expected to be a useful guide for medicinal chemists to design OX 1 R ligands.

β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an NADPH-dependent reductase that converts cortisone to cortisol in adipose tissue. We previously reported that GH and IGF-I decrease 11β-HSD1 activity and mRNA levels in adipocytes. Hexose-6-phosphate dehydrogenase (H6PDH) is involved in the production of NADPH, which is a coenzyme for 11β-HSD1. The aim of the present study was to clarify further the mechanism of repression of 11β-HSD1 activity by GH using linsitinib, an IGF-I receptor inhibitor. The suppression of 11β-HSD1 mRNA by IGF-I was attenuated in the presence of 1 μM linsitinib (17.2% vs. 53.3% of basal level, P

Nalfurafine, a κ-selective opioid receptor agonist, unexpectedly showed a selective antagonist activity toward the orexin 1 receptor (OX1R) (Ki = 250 nM). Modification of the 17-amino side chain of the opioid ligand to an arylsulfonyl group and the 6-furan acrylamide chain to 2-pyridyl acrylamide led to compound 71 with improvement of the antagonist activity (OX1R, Ki = 1.36 nM; OX2R, not active) without any detectable affinity for the opioid receptor. The dihydrosulfate salt of 71, freely soluble in water, attenuated the physical dependence of morphine. Furthermore, all of the active nalfurafine derivatives in this study had almost no activity for OX2R, which led to high OX1R selectivity. These results suggest that nalfurafine derivatives could be a useful series of lead compounds to develop highly selective OX1R antagonists.

Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of therapeutic options for postprandial hyperglycemia. Previously, we reported potent and selective SGLT1 inhibitors 1 and 2 showing efficacy in oral carbohydrate tolerance tests in diabetic rat models. In a pharmacokinetic (PK) study of 2, excessive systemic exposure to metabolites of 2 was observed, presumably due to the high permeability of its aglycone (2a). To further improve SGLT1 inhibitory activity and reduce aglycone permeability, a series of 4-benzyl-5-isopropyl-1H-pyrazol-3-yl β-D-glycopyranoside derivatives bearing novel hydrophilic substitution groups on the phenyl ring were synthesized and their inhibitory activity toward SGLTs was evaluated. Optimized compound 14c showed an improved profile satisfying both higher activity and lower permeability of its aglycone (22f) compared with initial leads 1 and 2. Moreover, the superior efficacy of 14c in various carbohydrate tolerance tests in diabetic rat models was confirmed compared with acarbose, an α-glucosidase inhibitor (α-GI) widely used in the clinic.

The high-affinity sodium glucose cotransporter (SGLT1) plays a critical role in glucose absorption from the gastrointestinal tract. We have developed 3-(3-{4-[3-(β-D-glucopyranosyloxy)-5-isopropyl-1H-pyrazol-4-ylmethyl]-3-methylphenoxy}propylamino)propionamide (KGA-2727), which has a pyrazole-O-glucoside structure, as the first selective SGLT1 inhibitor. KGA-2727 inhibited SGLT1 potently and highly selectively in an in vitro assay using cells transiently expressing recombinant SGLTs. In a small intestine closed loop absorption test with normal rats, KGA-2727 inhibited the absorption of glucose but not that of fructose. After oral intake of starch along with KGA-2727 in normal rats, the residual content of glucose in the gastrointestinal tract increased. In the oral glucose tolerance test with streptozotocin-induced diabetic rats, KGA-2727 attenuated the elevation of plasma glucose after glucose loading, indicating that KGA-2727 improved postprandial hyperglycemia. In Zucker diabetic fatty (ZDF) rats, chronic treatments with KGA-2727 reduced the levels of plasma glucose and glycated hemoglobin. Furthermore, KGA-2727 preserved glucose-stimulated insulin secretion and reduced urinary glucose excretion with improved morphological changes of pancreatic islets and renal distal tubules in ZDF rats. In addition, the chronic treatment with KGA-2727 increased the level of glucagon-like peptide-1 in the portal vein. Taken together, our data indicate that the selective SGLT1 inhibitor KGA-2727 had antidiabetic efficacy and allow us to propose KGA-2727 as a candidate for a novel and useful antidiabetic agent.

Ta/Si (100) and Cu/Ta/Si (100) film structures were fabricated by using ion beam deposition with a modified RF sputter-type ion source, in which a strong RF discharge was introduced in order to enhance the plasma density. For Ta/Si structures, Ta films were deposited at various bias voltages. When the substrate bias voltage was not applied, the Ta film showed a columnar structure and had a high resistivity of 2600 nΩm. On the other hand, when the substrate bias voltage of -50- - 200 V was applied, the cross-sectional observation did not show columnar structure at all. In this case, film deposition was considered to be sufficient migration energy by the accelerated Ta + ions. In particular, Ta films deposited at a bias voltage of -125 V had a very small resistivity of 360 nΩm. Thermal stability of Cu(100 nm)/Ta(50 nm)/Si films, where Ta plays a role of diffusion barrier, was evaluated after annealing in H 2 atmosphere for 60 min at various temperatures. Non-columnar structure Ta films deposited at substrate bias voltages of -50 V and -125 V were found to be stable up to 600°C, while columnar structure Ta films deposited at zero bias voltage degraded at 300°C. This result indicates that the thermal stability of the Ta films is mainly governed by the film microstructure of the deposited layer.

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.

The sake yeast Saccharomyces cerevisiae Kyokai no. 7 (K-7) is capable of producing more than 20% (v/v) ethanol in culture. However, the activity of its alcohol dehydrogenase (ADH), which is a key enzyme in alcohol production, is less than that of the laboratory yeast S. cerevisiae X2180-1A. The ADH activities of brewery yeasts which were isolated and bred for the purpose of ethanol production were compared with those of laboratory yeasts. The ADH activities of a number of the brewery yeasts were found to be less than those determined for the laboratory yeasts. The level of ADH activity of each clone derived from a brewery yeast was also lower than that of each clone derived from laboratory yeasts when expressed in Escherichia coli cells. In the ADH1 gene of K-7, the region between the TATA ☐ and ATG start codon and the region of polyadenylation in this nucleotide sequence were consistent with previous data. The ratio of intracellular ADH activity of K-7 to that of X2180-1A was about 1 : 2. On the other hand, the ratio of ADH activity of the clones derived from K-7 and X2180-1A, expressed under the control of the same promoter and in the same host, was about 1 : 1.3. The amino acid sequence of K-7 ADH1 was compared with previous data. The Glu 128 of K-7 ADH1 was unique among yeast ADH1s. Site-directed mutagenesis was carried out using ADH1 DNAs isolated from the sake yeast K-7 and the laboratory yeast X2180-1A, in order to determine amino acid alterations which affect activity. No single amino acid substitutions resulted in a change in the level of ADH activity. However, two double substitutions E128Q and E148Q or E128Q and V152I increased ADH activity to levels similar to that of X2180-1A Adh1. The substitutions of X2180-1A ADH1 to Q128E and Q148E or Q128E and I152V reduced ADH1 activity to the levels observed for K-7 Adh1.

Sodium glucose co-transporter 1 (SGLT1) plays a dominant role in the absorption of glucose in the gut and is considered a promising target in the development of treatments for postprandial hyperglycemia. A series of 4-benzyl-1H-pyrazol-3-yl β- d -glucopyranoside derivatives have been synthesized, and its inhibitory activity toward SGLTs has been evaluated. By altering the substitution groups at the 5-position of the pyrazole ring, and every position of the phenyl ring, we studied the structure–activity relationship (SAR) profiles and identified a series of potent and selective SGLT1 inhibitors. Representative derivatives showed a dose-dependent suppressing effect on the escalation of blood glucose levels in oral mixed carbohydrate tolerance tests (OCTT) in streptozotocin–nicotinamide-induced diabetic rats (NA-STZ rats).

Highly specific dockerin–cohesin interaction intrinsically involved in the cellulosome formation in Clostridium josui was applied for the construction of an affinity tag purification system. Amino acid substitutions were introduced into the dockerin domain of C. josui Cel8A at positions 11, 12, 44, and 45 and mutant dockerin domains were examined for their ability as an affinity tag: mutant dockerin-tagged proteins were adsorbed onto a cohesin (Coh2)-coupled Sepharose in the presence of Ca 2+ and desorbed from the protein and Coh2-Sepharose complex by the addition of a chelating agent, EGTA. Single-step purification tests showed that substitution of glycine or serine for isoleucine at position 45 markedly improved the recovery of the recombinant proteins from the proteins and Coh2-Sepharose complex. Surface plasmon resonance analysis of the interaction between the I45G mutant and Coh2 indicated that the mutation decreased binding rate and increased dissociation rate, resulting in decrease in dissociation constant. When model proteins such as JNK3, MAP2K3, IL-8, and pro-IL-18 were expressed as I45G dockerin-tagged proteins in the baculovirus expression system and purified by the single-step purification, purity of all the I45G dockerin-tagged proteins tested was higher than 90%. Furthermore, insertion of a thrombin cleavage site between the dockerin tag and target proteins enabled rapid removal of the tag from the target proteins by thrombin protease. This system, named the Dock tag purification system, can be widely utilized and contributes to various fields in academic and application researches.

The low-affinity sodium glucose cotransporter (SGLT2) is responsible for most of the glucose reabsorption in the kidney and has been highlighted as a novel therapeutic target for the treatment of diabetes. We discovered sergliflozin etabonate, a novel selective SGLT2 inhibitor, and found that selective inhibition of SGLT2 increased urinary glucose excretion and consequently decreased plasma glucose levels. In this report, we examined the antihyperglycemic effects of sergliflozin etabonate in normal and diabetic rats in comparison with those of a sulfonylurea (gliclazide) and an alpha-glucosidase inhibitor (voglibose). Sergliflozin etabonate increased urinary glucose excretion in a dose-dependent manner, and inhibited the increase in plasma glucose after sucrose loading independently of insulin secretion in normal rats. Sergliflozin etabonate also improved postprandial hyperglycemia in neonatal streptozotocin-induced diabetic rats; whereas gliclazide did not improve it. In rats with mild or moderate streptozotocin-induced diabetes, the degree of the antihyperglycemic effects of sergliflozin etabonate correlated with the severity of the diabetic condition. Sergliflozin etabonate did not affect the plasma glucose level of normal rats as seen with gliclazide. Chronic treatment with sergliflozin etabonate reduced the levels of glycated hemoglobin and fasting plasma glucose, and improved the glycemic response after glucose loading in Zucker fatty rats. In addition, sergliflozin etabonate did not affect the body weight or food intake. These data indicate that sergliflozin etabonate could improve glycemic control without its use resulting in insulin secretion, hypoglycemia, and body weight gain, and may provide a unique approach to the treatment of diabetes.

The low-affinity sodium glucose cotransporter (SGLT2) plays an important role in renal glucose reabsorption and is a remarkable transporter as a molecular target for the treatment of diabetes. We have discovered remogliflozin etabonate, which is a novel category of selective SGLT2 inhibitors. Remogliflozin etabonate is a prodrug based on benzylpyrazole glucoside and is metabolized to its active form, remogliflozin, in the body. We identified remogliflozin to be a potent and highly selective SGLT2 inhibitor by examining COS-7 cells transiently expressing either high-affinity sodium glucose cotransporter (SGLT1) or SGLT2. Orally administered remogliflozin etabonate increased urinary glucose excretion in a dose-dependent manner in both mice and rats. By increasing urinary glucose excretion, remogliflozin etabonate inhibited the increase in plasma glucose after glucose loading without stimulating insulin secretion in normal rats. Remogliflozin etabonate also showed antihyperglycemic effects in both streptozotocin-induced diabetic rats in oral glucose tolerance and in db/db mice in the fed condition. Chronic treatment with remogliflozin etabonate reduced the levels of fasting plasma glucose and glycated hemoglobin, and it ameliorated glucosuria in db/db mice. In high-fat diet-fed Goto-Kakizaki rats, remogliflozin etabonate improved hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and insulin resistance. This study demonstrates that treatment with remogliflozin etabonate exhibits antidiabetic efficacy in several rodent models and suggests that remogliflozin etabonate may be a new and useful drug for the treatment of diabetes.

We established a rapid bioassay method for transmissibility of human prions to knock-in mouse expressing human/mouse chimeric prion protein (Ki-ChM mouse). In Ki-ChM mice, we detected accumulation of abnormal isoform (PrPSc) of prion protein in follicular dendritic cells (FDCs) in lymphoid organ within 14 days following intraperitoneal administration of human prions. Intraperitoneal administration of the inoculum provides an advantage over intracerebral injection, considering only a limited volume of 20µl can be inoculated with the intracerebral route. To overcome this limitation and to confirm dose dependent effect on PrPSc accumulation with intraperitoneal injection, we made an attempt to inoculate brain homogenate of human sporadic CJD (sCJD) with large dose and repetitive injection of a volume up to ∼5000µl. Fifty micro liter of 10−2, 10−3, and 10−4 diluted sCJD were inoculated respective groups intraperitoneally. Five hundred µl of 10−3 and 10−4 dilution of sCJD inoculated with one shot were shown as equivalent infectivity to 50µl of 10−2 and 10−3 dilution of them respectively. Five hundred micro liter of 10−4 dilution of sCJD inoculated with once a day during 10 days were shown as an equivalent infectivity to 50µl of 10−2 dilution of it. These results indicate that the detection of PrPSc in FDCs has increased in proportion to the amount of prion inoculated. It is conceivable that the prion of a low density piles up the intake frequency and the pathogenicity rises as well as a high density prion.