Sub-chronic and mild social defeat stress exposure to C57BL/6J mice increases visceral fat mass and causes accumulation of cholesterol and bile acids in the liver.

Major depressive disorder is accompanied by a high metabolic illness comorbidity and patients with atypical depression are a subgroup with particularly high risk of obesity, dyslipidemia, and metabolic syndrome; however, the underlying mechanisms have not been fully elucidated. In this study, we examined visceral fat deposition, lipid profiles in the liver, and gut microbiota in sub-chronic and mild social defeat stress (sCSDS)-exposed C57BL/6J mice, which exhibit atypical depression-like phenotypes, i.e., increased body weight and food and water intake. We found that visceral fat mass and levels of hepatic cholesterol and bile acids in sCSDS-exposed mice were significantly increased compared to those in controls. The expression of hepatic small heterodimer partner, a negative regulator of cholesterol metabolism, was significantly elevated in sCSDS-exposed mice. We also found that gut microbial diversity and composition including lower relative abundance of Bacteroides spp. and Bifidobacterium spp. in sCSDS-exposed mice were different from those in controls. In addition, relative abundance of Bacteroides spp. and Bifidobacterium spp. was significantly and negatively correlated with body weight, visceral fat mass, and hepatic cholesterol and bile acids levels. These results indicate that sCSDS-exposure induces dysbiosis, and thereby contributes to metabolic disorder development.

Altered Expression of Astrocytic ATP Channels and Ectonucleotidases in the Cerebral Cortex and Hippocampus of Chronic Social Defeat Stress-Susceptible BALB/c Mice.

The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5'nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.

Alteration of Sweet and Bitter Taste Sensitivity with Development of Glucose Intolerance in Non-insulin-Dependent Diabetes Mellitus Model OLETF Rats.

Patients with diabetes exhibit altered taste sensitivity, but its details have not been clarified yet. Here, we examined alteration of sweet taste sensitivity with development of glucose intolerance in Otsuka Long-Evans Tokushima Fatty (OLETF) rats as a model of non-insulin-dependent diabetes mellitus. Compared to the cases of Long Evans Tokushima Otsuka (LETO) rats as a control, glucose tolerance of OLETF rats decreased with aging, resulting in development of diabetes at 36-weeks-old. In brief-access tests with a mixture of sucrose and quinine hydrochloride, OLETF rats at 25 or more-weeks-old seemed to exhibit lower sweet taste sensitivity than age-matched LETO ones, but the lick ratios of LETO, but not OLETF, rats for the mixture and quinine hydrochloride solutions decreased and increased, respectively, aging-dependently. Expression of sweet taste receptors, T1R2 and T1R3, in circumvallate papillae (CP) was almost the same in LETO and OLETF rats at 10- and 40-weeks-old, while expression levels of a bitter taste receptor, T2R16, were greater in 40-weeks-old rats than in 10-weeks-old ones in both strains. There was no apparent morphological alteration in taste buds in CP between 10- and 40-weeks-old LETO and OLETF rats. Metagenomic analysis of gut microbiota revealed strain- and aging-dependent alteration of mucus layer-regulatory microbiota. Collectively, we concluded that the apparent higher sweet taste sensitivity in 25 or more-weeks-old OLETF rats than in age-matched LETO rats was due to the aging-dependent increase of bitter taste sensitivity in LETO rats with alteration of the gut microbiota.

Precision engineered peptide targeting leukocyte extracellular traps mitigate acute kidney injury in Crush syndrome.

Crush syndrome induced by skeletal muscle compression causes fatal rhabdomyolysis-induced acute kidney injury (RIAKI) that requires intensive care, including hemodialysis. However, access to crucial medical supplies is highly limited while treating earthquake victims trapped under fallen buildings, lowering their chances of survival. Developing a compact, portable, and simple treatment method for RIAKI remains an important challenge. Based on our previous finding that RIAKI depends on leukocyte extracellular traps (ETs), we aimed to develop a novel medium-molecular-weight peptide to provide clinical treatment of Crush syndrome. We conducted a structure-activity relationship study to develop a new therapeutic peptide. Using human peripheral polymorphonuclear neutrophils, we identified a 12-amino acid peptide sequence (FK-12) that strongly inhibited neutrophil extracellular trap (NET) release in vitro and further modified it by alanine scanning to construct multiple peptide analogs that were screened for their NET inhibition ability. The clinical applicability and renal-protective effects of these analogs were evaluated in vivo using the rhabdomyolysis-induced AKI mouse model. One candidate drug [M10Hse(Me)], wherein the sulfur of Met10 is substituted by oxygen, exhibited excellent renal-protective effects and completely inhibited fatality in the RIAKI mouse model. Furthermore, we observed that both therapeutic and prophylactic administration of M10Hse(Me) markedly protected the renal function during the acute and chronic phases of RIAKI. In conclusion, we developed a novel medium-molecular-weight peptide that could potentially treat patients with rhabdomyolysis and protect their renal function, thereby increasing the survival rate of victims affected by Crush syndrome.

Establishment of One-Pot Disulfide-Driven Cyclic Peptide Synthesis with a 3-Nitro-2-pyridinesulfenate.

We have developed a new one-pot disulfide-driven cyclic peptide synthesis. The entire process is carried out in the solid phase, thus eliminating complicated work up procedures to remove by-products and unreacted reagents and enabling production of high-purity cyclic disulfide peptides by simple cleavage of a peptidyl resin. The one-pot synthesis of oxytocin was accomplished in this way with an isolated yield of 28% over 13 steps. These include peptide chain elongation from an initial resin, sulfenylation of the protected side chain of a cysteine (Cys) residue, disulfide ligation between thiols in an additional peptide fragment and a 3-nitro-2-pyridinesulfenyl-protected cysteine (Cys(Npys))-containing peptide resin, subsequent intramolecular amide bond formation of the disulfide-connected fragments by an Ag+-promoted thioester method, followed by deprotection and HPLC purification.

Combination therapy with anamorelin and a myostatin inhibitor is advantageous for cancer cachexia in a mouse model.

Cancer cachexia is a multifactorial disease that causes continuous skeletal muscle wasting. Thereby, it seems to be a key determinant of cancer-related death. Although anamorelin, a ghrelin receptor agonist, has been approved in Japan for the treatment of cachexia, few medical treatments for cancer cachexia are currently available. Myostatin (MSTN)/growth differentiation factor 8, which belongs to the transforming growth factor-ß family, is a negative regulator of skeletal muscle mass, and inhibition of MSTN signaling is expected to be a therapeutic target for muscle-wasting diseases. Indeed, we have reported that peptide-2, an MSTN-inhibiting peptide from the MSTN prodomain, alleviates muscle wasting due to cancer cachexia. Herein, we evaluated the therapeutic benefit of myostatin inhibitory D-peptide-35 (MID-35), whose stability and activity were more improved than those of peptide-2 in cancer cachexia model mice. The biologic effects of MID-35 were better than those of peptide-2. Intramuscular administration of MID-35 effectively alleviated skeletal muscle atrophy in cachexia model mice, and the combination therapy of MID-35 with anamorelin increased food intake and maximized grip strength, resulting in longer survival. Our results suggest that this combination might be a novel therapeutic tool to suppress muscle wasting in cancer cachexia.

Short peptides derived from hGAPDH exhibit anti-cancer activity.

Peptides have become an attractive drug discovery modality alongside small molecule compounds and high molecular weight biomolecules because they bind strongly to their target molecules. Previously, we found that secreted extracellular human GAPDH exhibits inhibitory activity against cancer cell growth. We sought to identify the minimal peptide sequence required for GAPDH activity in an effort to develop a small GAPDH-derived peptide with anti-cancer activity. Moreover, derivatives of the identified peptide, in which some amino acid residues were substituted with unnatural amino acids, were found to show stronger anti-cancer activity than non-substituted peptides.

Prophylactic Administration of Magnesium Oxide Prevents Dextran Sulfate Sodium-Induced Colonic Injury in Mice.

We previously demonstrated that per os administration and ad libitum ingestion of a magnesium chloride (MgCl2) solution had a prophylactic effect on dextran sulfate sodium (DSS)-induced colitis in mice, magnesium being considered to play a role in this preferable action. Magnesium oxide (MgO) is a commercially available magnesium formulation, but whether or not it prevents development of colitis is unknown. In this study, we investigated the effect of MgO administration on development of colitis in DSS-treated male C57BL/6J mice. Experimental colitis was induced by ad libitum ingestion of 1% (w/v) DSS, and the colitis severity was evaluated by disease activity index (DAI) scores, histological assessment and colonic expression of inflammatory cytokines. A 1 mg/mL MgO solution was administered to mice through ad libitum ingestion from a day before DSS treatment to the end of the experimental period of 12 d. In addition, the effects of DSS, MgO and their combination on the gut microbiota were investigated by 16S ribosomal RNA metagenome analysis. DSS-induced elevation of DAI scores was partially but significantly decreased by MgO administration, while MgO administration had no apparent effect on the shortened colonic length, elevated mRNA expression of colonic interleukin-1ß and tumor necrosis factor-α, increased accumulation of colonic mast cells, or altered features of the gut microbiota in DSS-treated mice. Overall, we demonstrated that MgO had a prophylactic effect on the development of colitis in DSS-treated mice by preventing histological colonic damage, but not colonic inflammation or alteration of the gut microbiota.

Peptide Tool-Driven Functional Elucidation of Biomolecules Related to Endocrine System and Metabolism.

The enhancement of basic research based on biomolecule-derived peptides has the potential to elucidate their biological function and lead to the development of new drugs. In this review, two biomolecules, namely "neuromedin U (NMU)" and "myostatin," are discussed. NMU, a neuropeptide first isolated from the porcine spinal cord, non-selectively activates two types of receptors (NMUR1 and NMUR2) and displays a variety of physiological actions, including appetite suppression. The development of receptor-selective regulators helps elucidate each receptor's detailed biological roles. A structure-activity relationship (SAR) study was conducted to achieve this purpose using the amidated C-terminal core structure of NMU for receptor activation. Through obtaining receptor-selective hexapeptide agonists, molecular functions of the core structure were clarified. Myostatin is a negative regulator of skeletal muscle growth and has attracted attention as a target for treating atrophic muscle disorders. Although the protein inhibitors, such as antibodies and receptor-decoys have been developed, the inhibition by smaller molecules, including peptides, is less advanced. Focusing on the inactivation mechanism by prodomain proteins derived from myostatin-precursor, a first mid-sized α-helical myostatin-inhibitory peptide (23-mer) was identified from the mouse sequence. The detailed SAR study based on this peptide afforded the structural requirements for effective inhibition. The subsequent computer simulation proposed the docking mode at the activin type I receptor binding site of myostatin. The resulting development of potent inhibitors suggested the existence of a more appropriate binding mode linked to their ß-sheet forming properties, suggesting that further investigations might be needed.

Strategic structure-activity relationship study on a follistatin-derived myostatin inhibitory peptide.

Myostatin, a negative regulator of muscle mass is a promising target for the treatment of muscle atrophic diseases. The novel myostatin inhibitory peptide, DF-3 is derived from the N-terminal α-helical domain of follistatin, which is an endogenous inhibitor of myostatin and other TGF-ß family members. It has been suggested that the optimization of hydrophobic residues is important to enhance the myostatin inhibition. This study describes a structure-activity relationship study focused on hydrophobic residues of DF-3 and designed to obtain a more potent peptide. A methionine residue in DF-3, which is susceptible to oxidation, was successfully converted to homophenylalanine in DF-100, and a new derivative DF-100, with four amino acid substitutions in DF-3 shows twice the potent inhibitory ability as DF-3. This report provides a new platform of a 14-mer peptide muscle enhancer.

Development of functionalized peptides for efficient inhibition of myostatin by selective photooxygenation.

For the inhibition of myostatin, which is an attractive strategy for the treatment of muscle atrophic disorders including muscular dystrophy, myostatin-binding peptides were synthesized with an on/off-switchable photooxygenation catalyst at different positions on the peptide chain. These functionalized peptides oxygenated and inactivated myostatin upon irradiation with near-infrared light. Among the peptides tested, a peptide (5) with the catalyst moiety at the 16 position induced myostatin-selective photooxygenation, and efficiently inhibited myostatin. These peptides exhibited low phototoxicity. Such functionalized peptides would provide a precedented strategy for myostatin-targeting therapy, in which myostatin is irreversibly and catalytically inactivated by photooxygenation.

Proposal for the binding mode of the 23-mer inhibitory peptide to myostatin.

Inhibition of myostatin is a promising strategy for the treatment of amyotrophic disorders. Previously, we identified a minimum 23-mer peptide spanning positions 21-43 of a mouse myostatin precursor-derived prodomain and identified the nine key residues for effective myostatin inhibition through Ala scanning. We also reported the 23-mer peptides that show the propensity to form an α-helical structure around positions 32-36. Here, based on these findings, we conducted a docking simulation of a peptide-myostatin interaction. The results showed that by α-helix restraint docking of the 30-41 main chain, we obtained a proposed binding mode in which all nine of the key residues interact with myostatin. By analyzing the binding mode of four proposed docking models, we identified six of the myostatin residues that play an important role in the interaction with the peptide. This result provides a valuable insight into the relationship between myostatin and peptide interaction sites and may help in the design of future inhibitors.

Structural Revision of Natural Cyclic Depsipeptide MA026 Established by Total Synthesis and Biosynthetic Gene Cluster Analysis.

A revised structure of natural 14-mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid-phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell-based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin-1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

Peptide-2 from mouse myostatin precursor protein alleviates muscle wasting in cancer-associated cachexia.

Cancer cachexia, characterized by continuous muscle wasting, is a key determinant of cancer-related death; however, there are few medical treatments to combat it. Myostatin (MSTN)/growth differentiation factor 8 (GDF-8), which is a member of the transforming growth factor-ß family, is secreted in an inactivated form noncovalently bound to the prodomain, negatively regulating the skeletal muscle mass. Therefore, inhibition of MSTN signaling is expected to serve as a therapeutic target for intractable muscle wasting diseases. Here, we evaluated the inhibitory effect of peptide-2, an inhibitory core of mouse MSTN prodomain, on MSTN signaling. Peptide-2 selectively suppressed the MSTN signal, although it had no effect on the activin signal. In contrast, peptide-2 slightly inhibited the GDF-11 signaling pathway, which is strongly related to the MSTN signaling pathway. Furthermore, we found that the i.m. injection of peptide-2 to tumor-implanted C57BL/6 mice alleviated muscle wasting in cancer cachexia. Although peptide-2 was unable to improve the loss of heart weight and fat mass when cancer cachexia model mice were injected with it, peptide-2 increased the gastrocnemius muscle weight and muscle cross-sectional area resulted in the enhanced grip strength in cancer cachexia mice. Consequently, the model mice treated with peptide-2 could survive longer than those that did not undergo this treatment. Our results suggest that peptide-2 might be a novel therapeutic candidate to suppress muscle wasting in cancer cachexia.

Transnasal Delivery of the Peptide Agonist Specific to Neuromedin-U Receptor 2 to the Brain for the Treatment of Obesity.

Obesity and metabolic syndrome are threats to the health of large population worldwide as they are associated with high mortality, mainly linked to cardiovascular diseases. Recently, CPN-116 (CPN), which is an agonist peptide specific to neuromedin-U receptor 2 (NMUR2) that is expressed predominantly in the brain, has been developed as a new therapeutic candidate for the treatment of obesity and metabolic syndrome. However, treatment with CPN poses a challenge due to the limited delivery of CPN to the brain. Recent studies have clarified that the direct anatomical connection of the nasal cavity with brain allows delivery of several drugs to the brain. In this study, we confirm the nasal cavity as a promising CPN delivery route to the brain for the treatment of obesity and metabolic syndrome. According to the pharmacokinetic study, the clearance of CPN from the blood was very rapid with a half-life of 3 min. In vitro study on its stability in the serum and cerebrospinal fluid (CSF) indicates that CPN was more stable in the CSF than in the blood. The concentration of CPN in the brain was higher after nasal administration, despite its lower concentrations in the plasma than that after intravenous administration. The study on its pharmacological potency suggests the effective suppression of increased body weight in mice in a dose-dependent manner due to the direct activation of NMUR2 by CPN. This results from the higher concentration of corticosterone in blood after nasal administration of CPN as compared to nasal application of saline. In conclusion, the above findings indicate that the nasal cavity is a promising CPN delivery route to the brain to treat obesity and metabolic syndrome.

Disulfide-Driven Cyclic Peptide Synthesis of Human Endothelin-2 with a Solid-Supported Npys-Cl.

We report here the synthesis of human endothelin-2, a peptide of 21 amino acid residues with two disulfide bonds, based on the novel idea of a disulfide-driven cyclic peptide synthesis (DdCPS). This synthesis has two steps: (1) a one-pot solid-phase disulfide ligation of two different sulfur-containing peptide fragments using an Npys-Cl resin and (2) intramolecular cyclization of the disulfide peptide via amide bond formation using a thioester ligation. Human endothelin-2 was obtained in a total yield of 2.2% with two such DdCPS procedures and subsequent deprotection and HPLC purification. This strategy is the basis of a new solid-phase assisted practical synthesis of cyclic disulfide peptides.

Discovery of a follistatin-derived myostatin inhibitory peptide.

Follistatin is well known as an inhibitor of transforming growth factor (TGF)-ß superfamily ligands including myostatin and activin A. Myostatin, a negative regulator of muscle growth, is a promising target with which to treat muscle atrophic diseases. Here, we focused on the N-terminal domain (ND) of follistatin (Fst) that interacts with the type I receptor binding site of myostatin. Through bioassay of synthetic ND-derived fragment peptides, we identified DF-3, a new myostatin inhibitory 14-mer peptide which effectively inhibits myostatin, but fails to inhibit activin A or TGF-ß1, in an in vitro luciferase reporter assay. Injected intramuscularly, DF-3 significantly increases skeletal muscle mass in mice and consequently, it can serve as a platform for development of muscle enhancement based on myostatin inhibition.

Design and synthesis of peptidic partial agonists of human neuromedin U receptor 1 with enhanced serum stability.

Neuromedin U (NMU) activates two receptors (NMUR1 and NMUR2) and is a promising candidate for development of drugs to combat obesity. Previously, we obtained hexapeptides as selective full NMUR agonists. Development of a partial agonist which mildly activates receptors is an effective strategy which lead to an understanding of the functions of NMU receptors. In 2014, we reported hexapeptide 3 (CPN-124) as an NMUR1-selective partial agonist but its selectivity and serum stability were unsatisfactory. Herein, we report the development of a hexapeptide-type partial agonist (8, CPN-223) based on a peptide (3) but with higher NMUR1-selectivity and enhanced serum stability. A structure-activity relationship study of synthetic pentapeptide derivatives suggested that a hexapeptide is a minimum structure consistent with both good NMUR1-selective agonistic activity and serum stability.

Use of solid-supported 4-fluorophenyl 3-nitro-2-pyridinesulfenate in the construction of disulfide-linked hybrid molecules.

To construct disulfide-linked hybrid molecules systematically and efficiently, we established a more practical solid-phase disulfide ligation (SPDSL) system with enhanced utility. The group Npys-OPh(pF) shows reactivity similar to that of Npys-Cl, but it is more stable. An efficient synthesis of the cyclic peptide oxytocin and a peptide-sugar conjugate was accomplished as models. These results indicate that the Npys-OPh(pF) resin functions as a common synthetic platform in SPDSL.

A chemically stable peptide agonist to neuromedin U receptor type 2.

Neuromedin U (NMU) is a peptide with appetite suppressive activity and other physiological activities via activation of the NMU receptors NMUR1 and NMUR2. In 2014, we reported the first NMUR2 selective agonist, 3-cyclohexylpropionyl-Leu-Leu-Dap-Pro-Arg-Asn-NH2 (CPN-116). However, we found that CPN-116 in phosphate buffer is unstable because of Nα-to-Nß acyl migration at the Dap residue. In this study, the chemical stability of CPN-116 was evaluated under various conditions, and it was found to be relatively stable in buffers such as HEPES and MES. We also performed a structure-activity relationship study to obtain an NMUR2-selective agonist with improved chemical stability. Consequently, CPN-219 bearing a Dab residue in place of Dap emerged as a next-generation hexapeptidic NMUR2 agonist.