The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

Mast cells play pivotal roles in innate host defenses against venom. Activated mast cells release large amounts of prostaglandin D 2 (PGD 2 ). However, the role of PGD 2 in such host defense remains unclear. We found that c-kit-dependent and c-kit-independent mast cell–specific hematopoietic prostaglandin D synthase ( H-pgds ) deficiency significantly exacerbated honey bee venom (BV)–induced hypothermia and increased mortality rates in mice. BV absorption via postcapillary venules in the skin was accelerated upon endothelial barrier disruption resulting in increased plasma venom concentrations. These results suggest that mast cell–derived PGD 2 may enhance host defense against BV and save lives by inhibiting BV absorption into circulation.

Delayed wound healing is a major problem in patients with diabetes, which significantly impairs their quality of life. Prostaglandin (PG) D2 is a major inflammatory lipid mediator synthesized by hematopoietic PGD2 synthase (HPGDS) from PGH2, a common precursor of all of PGs. We have previously shown that HPGDS produced PGD2 is involved in delayed wound healing in diabetic skin. In this study, we investigated the involvement of DP1 receptor in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice. C57BL/6 mice were injected intraperitoneal with 50 mg/kg of STZ daily for 5 days. Four weeks after the injection of STZ, a full thickness wound was created with an 8-mm diameter biopsy punch on the dorsal of mice. Wound healing was significantly decelerated in diabetic mice compared with non-diabetic mice. HPGDS mRNA was significantly increased in diabetic mouse skin compared to nondiabetic mouse skin. On the other hand, there was no significant change in the amount of DP1 receptor mRNA. Furthermore, immunohistochemically analysis revealed that HPGDS was expressed in epidermal Langerhans cells of diabetic mice, and the DP1 receptor was expressed in keratinocytes. These results suggest that in hyperglycemic skin, PGD2 produced by Langerhans cells acts on DP1 receptors on keratinocytes and may be involved in delayed wound healing.

Supplementary Table 1 from Hematopoietic Prostaglandin D Synthase Suppresses Intestinal Adenomas in <i>Apc</i>^Min/+ Mice

【Introduction】Exosomes are small (50-150 nm) membrane vesicles of endocytic origin, which are found in bodying fluids, and supporting their role in intercellular communication. Although recent studies have demonstrated that various biomarkers involved in the extent of pain from the serum exosomes, the effects of exosomes on pain have not been elucidated. We have previously demonstrated that increased expression of complement C5 on exosome bilayers from partial sciatic nerve ligation (PSNL) mouse sera enhances formalin-induced nociceptive behavior. In this study, to identify tissues producing complement C5 on exosome bilayers, we extracted 1) intracellular vesicles in tissues from PSNL and sham-operated groups, 2) organ-specific exosomes from serum-derived exosomes by immunoprecipitation, and compared the protein expression levels.

Delayed wound healing is a major problem in patients with diabetes melitus, which significantly impairs their quality of life. Prostaglandin (PG) D2 is a major inflammatory lipid mediator synthesized by hematopoietic PGD2 synthase (HPGDS) from PGH2, a common precursor of all of PGs. In the present study, we investigated the role of PGD2 in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice. C57BL/6 mice were injected with 50 mg/kg of STZ intraperitoneally daily for 5 days. Four weeks after the injection of STZ, a full thickness wound was created with an 8-mm diameter biopsy punch on the dorsal of mice showing the hyperglycemia (>300 mg/dL). Wound healing was significantly decelerated in diabetic mice compared with non-diabetic mice. The mRNAs of HPGDS, Cyclooxygenase (COX) 1, COX2, DP1 and DP2 receptors in mouse skin were measured by quantitative PCR. The skin of diabetic mice had significantly increased mRNAs of HPGDS and DP2 receptors as compared with the skin of non-diabetic mice. In addition, there was no significant change in the amount of DP1 receptors mRNA and COX1 mRNA, but the amount of COX2 mRNA tended to increase. In addition, immunohistochemical analysis revealed that HPGDS was upregulated in epidermal Langerhans cells of diabetic mice. These results suggest that in hyperglycemic skin, production of PGD2 is increased in Langerhans cell and may be involved in delayed inflammation via DP2 receptors.

The objective of this review is to evaluate the anti-dementia activities of saffron and its combination with Kampo medicine. The Kampo formula Kamiuntanto composed of 13 crude drugs is well known for its anti-dementia activity. A significant increase in choline acetyltransferase activity and mRNA levels were observed. Polygala radix was identified as the most essential component drug in Kamiuntanto, probably due to the saponins, tenuifolin, and sinapinic acid. Ginseng was also identified as an essential Kamiuntanto component in terms of its synergistic functions with Polygala radix. Saffron, which was recommended in the Bencao Gangmu for memory and dementia, and is used as an anti-spasmodic, anti-catarrhal, and sedative herbal drug. Saffron and its major constituent, crocin were shown to enhance learning-memory, non-rapid eye movement (rem) sleep, and inhibit depression and neuronal cell death due to strong anti-oxidant and anti-inflammation activities. In addition based on the epidemiological studies such as the treatment of sleeping disorders and the clinical trials of saffron for Alzheimer patients, we demonstrated the indirect and direct anti-dementia activities of crocin and saffron.

Degradation of hematopoietic prostaglandin D-2 synthase (H-PGDS) by proteolysis-targeting chimeras (PROTACs) is expected to be important in the treatment of allergic diseases and Duchenne's muscular dystrophy. We recently reported that PROTAC(H-PGDS)-7 (PROTAC 1), which is composed of H-PGDS inhibitor (TFC-007) and cereblon (CRBN) E3 ligase ligand (pomalidomide), showed potent H-PGDS degradation activity. Here, we investigated the structure-activity relationships of PROTAC 1, focusing on the C4- or C5-conjugation of pomalidomide, in addition, the H-PGDS ligand exchanging from TFC-007 with the biaryl ether to TAS-205 with the pyrrole. Three new PROTACs were evaluated for H-PGDS affinity, H-PGDS degrading activity, and inhibition of prostaglandin D-2 production. All compounds showed high H-PGDS degrading activities, but PROTAC(H-PGDS)-4-TAS-205 (PROTAC 3) was slightly less active than the other compounds. Molecular dynamics simulations suggested that the decrease in activity of PROTAC 3 may be due to the lower stability of the CRBN-PROTAC-H-PGDS ternary complex.

Duchenne muscular dystrophy (DMD) is a severe muscle disease caused by mutations in the dystrophin gene. We have found that hematopoietic prostaglandin (PG) D synthase (HPGDS) was induced in grouped necrotic muscle fibers in DMD patients and also in model mice (mdx). DMD also affects cardiac muscle and cardiac failures are the most common causes of death in DMD patients. In this study, we developed a novel specific degrader for HPGDS protein composed of the HPGDS inhibitor and E3 ligase ligand and investigated beneficial role of HPGDS degradation to cardiac function and morphology in heart of mdx mice.

Tetranor-Prostaglandin D Metabolite (tetranor-PGDM) is a metabolite of PGD2. Urinary tetranor-PGDM level is increased in some diseases, including food allergy. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength and pH. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA.

Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = ∼0.6 μM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 μM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.

Hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target for the development of therapeutic agents for Duchenne muscular dystrophy (DMD) and other H-PGDS-related diseases. We have recently developed the H-PGDS degrader PROTAC(H-PGDS)-1 , which is a chimeric molecule in which TFC-007 (that binds to H-PGDS) and pomalidomide (that binds to cereblon [CRBN]) were conjugated to the PEG5 linker. Herein, using a docking simulation of the ternary complex of the H-PGDS degrader, H-PGDS, and CRBN, we have succeeded in developing PROTAC(H-PGDS)-7, a new H-PGDS degrader that does not contain a linker. PROTAC(H-PGDS)-7 showed potent and selective degradation activity (DC 50 = 17.3 pM), and potent suppression of prostaglandin D 2 (PGD 2 ) production in KU812 cells. Additionally, in a DMD model using mdx mice with cardiac hypertrophy, PROTAC(H-PGDS)-7 showed better inhibition of inflammatory cytokines than TFC-007. PROTAC(H-PGDS)-7 is expected to be a promising candidate for the treatment of DMD and other H-PGDS-related diseases.

Targeted protein degradation by proteolysis-targeting chimera (PROTAC) is one of the exciting modalities for drug discovery and biological discovery. It is important to select an appropriate linker, an E3 ligase ligand, and a target protein ligand in the development; however, it is necessary to synthesize a large number of PROTACs through trial and error. Herein, using a docking simulation of the ternary complex of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, H-PGDS, and cereblon, we have succeeded in developing PROTAC(H-PGDS)-7 (6), which showed potent and selective degradation activity (DC50 = 17.3 pM) and potent suppression of prostaglandin D-2 production in KU812 cells. Additionally, in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy, compound 6 showed better inhibition of inflammatory cytokines than a potent H-PGDS inhibitor TFC-007. Thus, our results demonstrated that in silico simulation would be useful for the rational development of PROTACs.

Orexins/hypocretins are hypothalamic neuropeptides that promote and stabilize wakefulness by binding to the orexin receptor type-1 (OX1R) and type-2 (OX2R). Disruption of orexinergic signaling results in the sleep disorder narcolepsy in mice, rats, dogs, and humans. The orexin receptor antagonist suvorexant promotes sleep by blocking both OX1R and OX2R. Whereas suvorexant has been clinically approved for the treatment of insomnia because it is well tolerated in experimental animals as well as in human patients, a logical question remains as to why orexin receptor antagonists do not induce overt narcolepsy-like symptoms. Here we show that acute and chronic suvorexant promotes both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep without inducing cataplexy in mice. Interestingly, chronic suvorexant increases OX2R mRNA and decreases orexin mRNA and peptide levels, which remain low long after termination of suvorexant administration. When mice are chronically treated with suvorexant and then re-challenged with the antagonist after a 1-week washout, however, cataplexy and sleep-onset REM (SOREM) are observed, which are exacerbated by chocolate administration. Heterozygous orexin knockout mice, with lower brain orexin levels, show cataplexy and SOREM after acute suvorexant administration. Furthermore, we find that acute suvorexant can induce cataplexy and SOREM in wild-type mice when co-administered with chocolate under stress-free (temporally anesthetized) conditions. Taken together, these results suggest that suvorexant can inhibit orexin synthesis resulting in susceptibility to narcolepsy-like symptoms in mice under certain conditions.

Although hematopoietic prostaglandin D synthase (H-PGDS) is an attractive target fortreatment of a variety of diseases, including allergic diseases and Duchenne musculardystrophy, no H-PGDS inhibitors have yet been approved for treatment of thesediseases. Therefore, the development of novel agents having other mode of actions tomodulate the activity of H-PGDS is required. In this study, a chimeric small moleculethat degrades H-PGDS via the ubiquitin-proteasome system, PROTAC(H-PGDS)-1,was developed. PROTAC(H-PGDS)-1 is composed of two ligands, TFC-007 (thatbinds to H-PGDS) and pomalidomide (that binds to cereblon). PROTAC(H-PGDS)-1showed potent activity in the degradation of H-PGDS protein via theubiquitin-proteasome system and in the suppression of prostaglandin D2 (PGD2)production. Notably, PROTAC(H-PGDS)-1 was slightly more effective in thesuppression of PGD2 production than the known inhibitor, TFC-007. Thus, the H-PGDSdegrader—PROTAC(H-PGDS)-1—is expected to be useful in biological research andclinical therapies.

Tetranor-PGDM is a metabolite of PGD 2 . Urinary tetranor-PGDM levels were reported to be increased in some diseases, including food allergy, Duchenne muscular dystrophy, and aspirin-intolerant asthma. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength, pH, precision, and reliability. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited a half-maximal inhibition concentration (IC 50 ) of 1.79 ng/mL, limit of detection (LOD) of 0.0498 ng/mL, and range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The intra- and inter-assay variation for tetranor-PGDM was 3.9–6.0% and 5.7–10.4%, respectively. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA. The established EIA would be useful for routine detection and monitoring of tetranor-PGDM in research or diagnostic body fluids.

Lipocalin-type PG D synthase (L-PGDS) has two roles: it can be a PGD synthase, or it can be a carrier protein of hydrophobic small molecules. In this study, we investigated the dual roles of L-PGDS in acute lung injury by using L-PGDS-deficient and point-mutated mice, which lack PGD(2) producibility but maintain lipocalin ability. Hydrochloride (HCl) administration (0.1 M intratracheally for 6 h) caused hemorrhage and dysfunction in the wild-type (WT) mouse lung. These symptoms were accompanied by an increase in PGD(2) production. Both deficiency and point mutation of L-PGDS aggravated the HCl-induced hemorrhage and dysfunction. Although both the gene modifications decreased PGD(2) production, only L-PGDS-deficient mice, but not point mutation mice, lacked protein expressions of L-PGDS in the lungs. In the WT mice, HCl administration caused pulmonary edema, indexed as an increase in lung water content and protein leakage in bronchoalveolar lavage fluid. L-PGDS deficiency and point mutation similarly aggravated edema formation. HCl administration also stimulated mucin production and bronchoalveolar lavage fluid leukocyte infiltration in the WT mouse lungs. Of interest, L-PGDS deficiency, but not point mutation, exacerbated these manifestations. Consistently, only L-PGDS deficiency increased the mRNA expression of IL-33, which stimulates mucin production in the inflamed lung. These results show that L-PGDS attenuated HCl-induced acute lung injury progresses in two different ways: L-PGDS produced PGD(2), which inhibited pulmonary edema formation, whereas its lipocalin ability decreased mucin formation and inflammatory cell infiltration in the inflamed lung.

Delayed wound healing is a major problem in patients with diabetes, which significantly impairs their quality of life.Prostaglandin (PG) D 2 is a major inflammatory lipid mediator synthesized by hematopoietic PGD 2 synthase (H-PGDS) from PGH 2 , a common precursor of all of PGs.In the present study, we investigated the role of PGD 2 in cutaneous wound healing in streptozotocin (STZ)-induced diabetic mice.C57BL/6 mice were injected with 50 mg/kg of STZ intraperitoneally daily for 5 days.Four weeks after the injection of STZ, a full thickness wound was created with an 8-mm diameter biopsy punch on the dorsal of mice.Wound healing was significantly decelerated and cutaneous H-PGDS mRNA was significantly increased in diabetic mice compared with non-diabetic mice.Daily administration of H-PGDS inhibitor for 14 days was significantly promoted wound healing in diabetic mice.These results suggest that PGD 2 involved in delayed wound healing in STZ-induced diabetic mice.

STUDY OBJECTIVES:Excessive daytime sleepiness (EDS) is a frequent cause for consultation and a defining symptom of narcolepsy and idiopathic hypersomnia (IH). The associated mechanisms remain unclear. Lipocalin-type prostaglandin D synthase (LPGDS) is a plausible sleep-inducing candidate. This study is to compare cerebral spinal fluid (CSF) and serum LPGDS levels in patients group with hypersomnia of central origin, including those with narcolepsy type 1 (NT1) and type 2 (NT2) and IH, to those in healthy controls (Con). METHODS:Serum LPGDS, CSF LPGDS, and CSF hypocretin-1(Hcrt-1) levels were measured by ELISA in 122 narcolepsy patients (106 NT1 and 16 NT2), 27 IH, and 51Con. RESULTS:LPGDS levels in CSF (p = 0.02) and serum (p < 0.001) were 22%-25% lower in control subjects than in patients with EDS complaints, including NT1, NT2, and IH. In contrast to significant differences in CSF Hcrt-1 levels, CSF L-PGDS levels and serum L-PGDS were comparable among NT1, NT2, and IH (p > 0.05), except for slightly lower serum LPGDS in IH than in NT1 (p = 0.01). Serum L-PGDS correlated modestly and negatively to sleep latency on MSLT (r = -0.227, p = 0.007) in hypersomnia subjects. CONCLUSIONS:As a somnogen-producing enzyme, CSF/serum LPGDS may serve as a new biomarker for EDS of central origin and imply a common pathogenetic association, but would complement rather than replaces orexin markers.