Individual resolvin E family members work distinctly and in a coordinated manner in the resolution of inflammation.

Three main E-type resolvins (RvEs): RvE1, RvE2, and RvE3, have roles in the resolution of inflammation as anti-inflammatory activities. To investigate the roles of each RvE in the resolution of inflammation, timing of interleukin (IL)- 10 release and IL-10 receptor expressions, and phagocytosis evoked by each RvE in differentiated human monocytes, macrophage-like U937 cells were examined. Here, we show that RvEs enhance the expression of IL-10, and IL-10 receptor-mediated signaling pathways and IL-10-mediated-signaling-independent resolution of inflammatory effects by activating the phagocytotic function. Thus, RvE2 mainly evoked an IL-10-mediated anti-inflammatory function, whereas RvE3 principally activated phagocytotic activity of macrophages, which may be involved in tissue repair. On the other hand, RvE1 showed both functions, although not prominent but rather acting as a relief mediator that takes over the RvE2 function and passes over to the RvE3 function. Therefore, each RvE may act as an important role/stage-specific mediator in a coordinated manner with other RvEs in the processes of the resolution of inflammation.

Intranasal Administration of Resolvin E1 Produces Antidepressant-Like Effects via BDNF/VEGF-mTORC1 Signaling in the Medial Prefrontal Cortex.

Intracerebroventricular infusion of resolvin E1 (RvE1), a bioactive metabolite derived from eicosapentaenoic acid, exerts antidepressant-like effects in a mouse model of lipopolysaccharide (LPS)-induced depression; these effects are blocked by systemic injection of rapamycin, a mechanistic target of rapamycin complex 1 (mTORC1) inhibitor. Additionally, local infusion of RvE1 into the medial prefrontal cortex (mPFC) or dorsal hippocampal dentate gyrus (DG) produces antidepressant-like effects. To evaluate the potential of RvE1 for clinical use, the present study examined whether treatment with RvE1 via intranasal (i.n.) route, a non-invasive route for effective drug delivery to the brain, produces antidepressant-like effects in LPS-challenged mice using tail suspension and forced swim tests. Intranasal administration of RvE1 significantly attenuated LPS-induced immobility, and these antidepressant-like effects were completely blocked by an AMPA receptor antagonist or L-type voltage-dependent Ca2+ channel blocker. The antidepressant-like effects of both i.n. and intra-mPFC administrations of RvE1 were blocked by intra-mPFC infusion of a neutralizing antibody (nAb) for brain-derived neurotrophic factor (BDNF) or vascular endothelial growth factor (VEGF). Intra-mPFC infusion of rapamycin completely blocked the antidepressant-like effects of both i.n. and intra-mPFC administrations of RvE1 as well as those of intra-mPFC infusion of BDNF and VEGF. Moreover, i.n. RvE1 produced antidepressant-like effects via mTORC1 activation in the mPFC of a mouse model of repeated prednisolone-induced depression. Intra-dorsal DG infusion of BDNF and VEGF nAbs, but not rapamycin, blocked the antidepressant-like effects of i.n. RvE1. These findings suggest that i.n. administration of RvE1 produces antidepressant-like effects through activity-dependent BDNF/VEGF release in the mPFC and dorsal DG, and mTORC1 activation in the mPFC, but not in the dorsal DG. Thus, RvE1 can be a promising candidate for a novel rapid-acting antidepressant.

Synthesis of γ-Aminobutyric Acid (GABA) Analogues Conformationally Restricted by Bicyclo[3.1.0]hexane/hexene or [4.1.0]Heptane/heptene Backbones as Potent Betaine/GABA Transporter Inhibitors.

Novel γ-aminobutyric acid (GABA) analogues 3-5, having a bicyclo[3.1.0]hexene, [4.1.0]heptane, or [4.1.0]heptene backbone, respectively, were designed from the bioactive form analysis of the previous inhibitor 2 with a bicyclo[3.1.0]hexane backbone. Compounds 3-5 and 2 were synthesized from a common 1,7-diene intermediate 6 using ring-closing metathesis (RCM) to construct the key bicyclo backbones. Compounds 3-5 strongly inhibit betaine/GABA transporter 1 (BGT1) uptake, but compound 4 stands out with its selective low micromolar potency.

Resolvin E3 ameliorates high-fat diet-induced insulin resistance via the phosphatidylinositol-3-kinase/Akt signaling pathway in adipocytes.

Obesity-associated type 2 diabetes mellitus is associated with the development of insulin resistance. Among several metabolites, resolvins that are metabolites of eicosapentaenoic acid have been shown to exert insulin-sensitizing effects; however, the role of resolvin E3 (RvE3) in glucose metabolism has not been studied. In this study, the effect of RvE3 on glucose metabolism in mice with high-fat diet-induced obesity and 3T3L1 adipocytes was studied. C57BL/6 mice fed a high-fat diet were administered RvE3, for which insulin tolerance, oral glucose tolerance tests, and the homeostasis model assessment of insulin resistance, were performed. RvE3 treatment significantly improved insulin sensitivity and glucose tolerance and regulated protein kinase B (Akt) phosphorylation in the adipose tissue. Moreover, RvE3 treatment enhanced the insulin-stimulated glucose transporter 4 (Glut4) translocation, glucose uptake, phosphatidylinositol-3-kinase (PI3K) activity, and Akt phosphorylation in 3T3L1 adipocytes, whereas a PI3K inhibitor inhibited the enhanced insulin-stimulated glucose uptake induced by RvE3. These findings indicate that RvE3 likely improves insulin sensitivity, resulting in the upregulation of glucose uptake in adipocytes by activating the PI3K/Akt signaling pathways. Collectively, the findings of this study show that RvE3 may play a role in glucose homeostasis and could be used as a potential therapeutic target for developing treatments for obesity-associated diabetes.

Resolvin E1 Attenuates Chronic Pain-Induced Depression-Like Behavior in Mice: Possible Involvement of Chemerin Receptor ChemR23.

The antidepressant effect of eicosapentaenoic acid-derived bioactive lipid, resolvin E1 (RvE1), was examined in a murine model of chronic pain-induced depression using a tail suspension test. Because RvE1 reportedly possesses agonistic activity on a chemerin receptor ChemR23, we also examined the antidepressant effect of chemerin. Two weeks after surgery for unilateral spared nerve injury to prepare neuropathic pain model mice, immobility time was measured in a tail suspension test. Chronic pain significantly increased immobility time, and this depression-like behavior was attenuated by intracerebroventricular injection of RvE1 (1 ng) or chemerin (500 ng). These results demonstrate that RvE1 exerts an antidepressant effect in a murine model of chronic pain-induced depression, which is likely to be via ChemR23. RvE1 and its receptor may be promising targets to develop novel antidepressants.

Structure-activity Relationship of Indomethacin Derivatives as IDO1 Inhibitors.

BACKGROUND/AIM: Indoleamine 2,3-dioxygenase (IDO) is regarded as an important molecular target for cancer immune therapy. This study aimed to examine the IDO1 inhibitory activity of newly synthesized indomethacin derivatives to develop an IDO1 inhibitor. MATERIALS AND METHODS: The inhibitory effects of indole-containing compounds against recombinant human IDO1 (rhIDO1) were evaluated. RESULTS: While some drugs including those with an indole scaffold could inhibit rhIDO1, simple indole compounds were inactive. A total of 27 indomethacin derivatives, including 18 newly synthesized derivatives, were evaluated. Numerous derivatives showed enhanced IDO1 inhibitory activity. The functional group at the 3-position had a strong effect on IDO1 inhibitory activity. The IDO1 inhibitory activity was not directly correlated with tumor cell cytotoxicity. CONCLUSION: We report the finding of novel IDO1 inhibitors and the structure-activity relationship based on indomethacin derivatives. Our findings will be beneficial for the development of IDO1 inhibitors for cancer immune therapy.

A Macrocyclic Peptide Library with a Structurally Constrained Cyclopropane-containing Building Block Leads to Thiol-independent Inhibitors of Phosphoglycerate Mutase.

Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator whose structure is more constrained than the ordinary N-chloroacetyl-α-amino acid initiators. The use of such an initiator has led to a macrocycle library with significantly suppressed population of lariat-shaped species compared with the conventional libraries. We previously used a conventional library and identified a small lariat thioether-macrocycle with a tail peptide with a C-terminal free Cys whose sidechain plays an essential role in potent inhibitory activity against a parasitic model enzyme, phosphoglycerate mutase. On the other hand, the cyclopropane-containing macrocycle library has yielded a larger thioether-macrocycle lacking a free Cys residue, which exhibits potent inhibitory activity to the same enzyme with a different mode of action. This result indicates that such a cyclopropane-containing macrocycle library would allow us to access mechanistically distinct macrocycles.

Design and Synthesis of Benzene Congeners of Resolvin E2, a Proresolving Lipid Mediator, as Its Stable Equivalents.

Resolvins (Rvs) are highly potent anti-inflammatory lipid mediators that are chemically and biologically unstable because of their polyunsaturated structures. To address this issue, we designed benzene congeners of RvE2, i.e., o-, m-, and p-BZ-RvE2s, as stable equivalents of RvE2 by replacing the unstable skipped diene moiety with a benzene ring on the basis of computational conformation studies and synthesized these congeners via a short common route through two Stille couplings. o-BZ-RvE2 exhibited more potent anti-inflammatory activity and much higher metabolic stability than RvE2. Thus, o-BZ-RvE2 was identified as a stable equivalent of RvE2, which is useful as a lead for anti-inflammatory drugs with a new mechanism of action as well as a biotool for investigating RvE2-mediated inflammation resolving pathways.

[Medicinal Chemical Studies Based on the Theoretical Design of Bioactive Compounds].

I have engaged in medicinal chemical studies based on the theoretical design of bioactive compounds. First, I present a three-dimensional structural diversity-oriented conformational restriction strategy for developing bioactive compounds based on the characteristic steric and stereoelectronic features of cyclopropane. Using this strategy, various biologically active small molecule compounds, such as receptor agonists/antagonists and enzyme inhibitors, were effectively developed. The strategy was also applied to develop versatile peptidomimetics and membrane-permeable cyclic peptides. Next, studies on Ca2+-mobilizing second messengers, cyclic ADP-ribose (cADPR) and myo-inositol trisphosphates (IP3), are described. In these studies, stable equivalents of cADPR were developed, since biological studies of cADPR have been limited due to its instability. Various potent IP3 receptor ligands, which were designed using the d-glucose structure as a bioisostere of the myo-inositol moiety of IP3, have been identified. Organic chemistry studies have also been extensively performed, because excellent organic chemistry is essential for promoting high-level medicinal chemical studies. For examples, new methods for the synthesis of chiral cyclopropanes, new radical reactions with silicon tethers, and kinetic anomeric effect-dependent stereoselective glycosidations have been developed.

Development of a novel class of peroxisome proliferator-activated receptor (PPAR) gamma ligands as an anticancer agent with a unique binding mode based on a non-thiazolidinedione scaffold.

We previously identified dibenzooxepine derivative 1 as a potent PPARγ ligand with a unique binding mode owing to its non-thiazolidinedione scaffold. However, while 1 showed remarkably potent MKN-45 gastric cancer cell aggregation activity, an indicator of cancer differentiation-inducing activity induced by PPARγ activation, we recognized that 1 was metabolically unstable. In the present study, we identified a metabolically soft spot, and successfully discovered 3-fluoro dibenzooxepine derivative 9 with better metabolic stability. Further optimization provided imidazo[1,2-a]pyridine derivative 17, which showed potent MKN-45 gastric cancer cell aggregation activity and excellent PK profiles compared with 9. Compound 17 exerted a growth inhibitory effect on AsPC-1/AG1 pancreatic tumor in mice. Furthermore, the decrease in the hematocrit (an indicator of localized edema, a serious adverse effect of PPARγ ligands) was tolerable even with oral administration at 200 mg/kg in healthy mice.

Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

We investigated the effects of resolvin E (RvE) 1, RvE2, and RvE3 on IL-4- and IL-33-stimulated bone marrow-derived dendritic cells (BMDCs) from house dust mite (HDM)-sensitized mice. We also investigated the role of RvE3 in a murine model of HDM-induced airway inflammation. In vitro, BMDCs from HDM-sensitized mice were stimulated with IL-4 and IL-33 and then treated with RvE1, RvE2, RvE3, or vehicle. RvE1, RvE2, and RvE3 suppressed IL-23 release from BMDCs. In vivo, RvE3 administrated to HDM-sensitized and challenged mice in the resolution phase promoted a decline in total numbers of inflammatory cells and eosinophils, reduced levels of IL-23 and IL-17 in lavage fluid, and suppressed IL-23 and IL-17A mRNA expression in lung and peribronchial lymph nodes. RvE3 also reduced resistance in the lungs of HDM-sensitized mice. A NanoBiT ß-arrestin recruitment assay using human embryonic kidney 293 cells revealed that pretreatment with RvE3 suppressed the leukotriene B4 (LTB4)-induced ß-arrestin 2 binding to LTB4 receptor 1 (BLT1R), indicating that RvE3 antagonistically interacts with BLT1R. Collectively, these findings indicate that RvE3 facilitates the resolution of allergic airway inflammation, partly by regulating BLT1R activity and selective cytokine release by dendritic cells. Our results accordingly identify RvE3 as a potential therapeutic target for the management of asthma.-Sato, M., Aoki-Saito, H., Fukuda, H., Ikeda, H., Koga, Y., Yatomi, M., Tsurumaki, H., Maeno, T., Saito, T., Nakakura, T., Mori, T., Yanagawa, M., Abe, M., Sako, Y., Dobashi, K., Ishizuka, T., Yamada, M., Shuto, S., Hisada, T. Resolvin E3 attenuates allergic airway inflammation via the interleukin-23-interleukin-17A pathway.

Development of a Highly Potent Analogue and a Long-Acting Analogue of Oxytocin for the Treatment of Social Impairment-Like Behaviors.

The nonapeptide hormone oxytocin (OT) has pivotal brain roles in social recognition and interaction and is thus a promising therapeutic drug for social deficits. Because of its peptide structure, however, OT is rapidly eliminated from the bloodstream, which decreases its potential therapeutic effects in the brain. We found that newly synthesized OT analogues in which the Pro7 of OT was replaced with N-( p-fluorobenzyl)glycine (2) or N-(3-hydroxypropyl)glycine (5) exhibited highly potent binding affinities for OT receptors and Ca2+ mobilization effects by selectively activating OT receptors over vasopressin receptors in HEK cells, where 2 was identified as a superagonist ( EMax = 131%) for OT receptors. Furthermore, the two OT analogues had a remarkably long-acting effect, up to 16-24 h, on recovery from impaired social behaviors in two strains of CD38 knockout mice that exhibit autism spectrum disorder-like social behavioral deficits, whereas the effect of OT itself rapidly diminished.

A Covalent Inhibitor for Glutathione S-Transferase Pi (GSTP1-1 ) in Human Cells.

Glutathione S-transferase π (GSTP1-1 ) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP1-1 both in vitro and in cellulo with a long duration of action.

Development of Dihydrodibenzooxepine Peroxisome Proliferator-Activated Receptor (PPAR) Gamma Ligands of a Novel Binding Mode as Anticancer Agents: Effective Mimicry of Chiral Structures by Olefinic E/ Z-Isomers.

A novel class of PPARγ ligand 1 (EC50 = 197 nM) with a dibenzoazepin scaffold was identified through high-throughput screening campaign. To avoid the synthetically troublesome chiral center of 1, its conformational analysis using the MacroModel was conducted, focusing on conformational flip of the tricyclic ring and the conformational restriction by the methyl group at the chiral center. On the basis of this analysis, scaffold hopping of dibenzoazepine into dibenzo[ b, e]oxepine by replacing the chiral structures with the corresponding olefinic E/ Z isomers was performed. Consequently, dibenzo[ b, e]oxepine scaffold 9 was developed showing extremely potent PPARγ reporter activity (EC50 = 2.4 nM, efficacy = 9.5%) as well as differentiation-inducing activity against a gastric cancer cell line MKN-45 that was more potent than any other well-known PPARγ agonists in vitro (94% at 30 nM). The X-ray crystal structure analysis of 9 complexed with PPARγ showed that it had a unique binding mode to PPARγ ligand-binding domain that differed from that of any other PPARγ agonists identified thus far.

Uptake and metabolism of mizoribine, an immunosuppressant, in L5178Y-R mouse lymphoma cells in vitro and peripheral blood mononuclear cells of rats and kidney transplant recipients in vivo.

The cellular uptake of mizoribine (MZR), an immunosuppressant, and metabolism of MZR to MZR-5'- monophosphate (MZRP), an active metabolite, were evaluated in L5178Y-R mouse lymphoma cells and peripheral blood mononuclear cells (PBMCs) of rats and kidney transplant recipients (KTRs, n = 22). Real-time PCR analysis revealed the expression of ENT1 and ENT2 mRNAs, but not of CNTs, in L5178Y-R cells and rat's PBMCs. In L5178Y-R cells, the uptake of MZR was suppressed by adenosine, a substrate for ENT1 and ENT2, but not by 5-(4-nitrobenzyl)-6-thioinosine (0.1 µM), an ENT1 inhibitor. Saturable metabolism of MZR to MZRP was observed. In rats, peak plasma concentrations of MZR and peak concentrations of MZR and MZRP in PBMCs were observed 3 h after oral administration. MZR disappeared from PBMCs in parallel with plasma MZR, but the disappearance of MZRP from PBMCs appeared to be slow. In KTRs, the mean plasma concentration of MZR 3-4 h after ingestion was 3.14 µg/ml and the mean MZRP concentration in PBMCs was 16.8% of MZR, reflecting the involvement of ENT in the uptake of MZR. A linear relationship was observed between plasma MZR concentrations ranging from 1 to 6 µg/ml and PBMC's MZRP concentrations ranging from 90 to 200 ng/ml.

Synthesis of Chiral cis-Cyclopropane Bearing Indole and Chromone as Potential TNFα Inhibitors.

Conformationally restricted analogues of SPD-304, the first small-molecule TNFα inhibitor, in which two heteroaryl groups, indole and chromone, are connected by chiral methyl- or ethyl- cis-cyclopropane, were designed. Synthesis of these molecules was achieved via Suzuki-Miyaura or Stille coupling reactions with chiral bromomethylenecyclopropane or iodovinyl- cis-cyclopropane as the substrate, both of which were prepared from chiral methylenecyclopropane as a common intermediate, constructing the heteroaryl-methyl or -ethyl- cis-cyclopropane structures as key steps. This study presents an efficient synthesis of a series of chiral cis-cyclopropane conjugates with two heteroaryl groups.

Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus.

Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.

Cyclic ADP-Carbocyclic-Ribose and -4-Thioribose, as Stable Mimics of Cyclic ADP-Ribose, a Ca2+-Mobilizing Second Messenger.

Cyclic ADP-ribose (cADPR), a general mediator involved in Ca2+ signaling, has the characteristic 18-membered ring consisting of an adenine, two riboses and a pyrophosphate, in which the two primary hydroxy groups of the riboses are linked by a pyrophosphate unit. This review focuses on chemical synthetic studies of cADPR analogues of biological importance. Although cADPR analogues can be synthesized by enzymatic and chemo-enzymatic methods using ADP-ribosyl cyclase, the analogues obtained by these methods are limited due to the substrate-specificity of the enzymes. Consequently, chemical synthetic methods providing a greater variety of cADPR analogues are required. Although early chemical synthetic studies demonstrated that construction of the large 18-membered ring structure is difficult, the construction was achieved using the phenylthiophosphate-type substrates by treating with AgNO3 or I2. This is now a general method for synthesizing these types of biologically important cyclic nucleotides. Using this method as the key step, the chemically and biologically stable cADPR mimic, cADP-carbocyclic-ribose (cADPcR) and -4-thioribose (cADPtR), were synthesized.