Potent and selective oxytocin receptor agonists without disulfide bridges.

Oxytocin (OT) is a neuropeptide involved in a wide variety of physiological actions, both peripherally and centrally. Many human studies have revealed the potential of OT to treat autism spectrum disorders and schizophrenia. OT interacts with the OT receptor (OTR) as well as vasopressin 1a and 1b receptors (V1aR, V1bR) as an agonist, and agonistic activity for V1aR and V1bR may have a negative impact on the therapeutic effects of OTR agonism in the CNS. An OTR-selective agonistic peptide, FE 202767, in which the structural differences from OT are a sulfide bond instead of a disulfide bond, and N-alkylglycine replacement for Pro at position 7, was reported. However, the effects of amino acid substitutions in OT have not been comprehensively investigated to compare OTR, V1aR, and V1bR activities. This led us to obtain a new OTR-selective analog by comprehensive amino acid substitutions of OT and replacement of the disulfide bond. A systematic amino acid scanning (Ala, Leu, Phe, Ser, Glu, or Arg) of desamino OT (dOT) at positions 2, 3, 4, 5, 7, and 8 revealed the tolerability for the substitution at positions 7 and 8. Further detailed study showed that trans-4-hydroxyproline (trans-Hyp) at position 7 and γ-methylleucine [Leu(Me)] at position 8 were markedly effective for improving receptor selectivity without decreasing the potency at the OTR. Subsequently, a combination of these amino acid substitutions with the replacement of the disulfide bond of dOT analogs with a sulfide bond (carba analog) or an amide bond (lactam analog) yielded several promising analogs, including carba-1-[trans-Hyp7,Leu(Me)8]dOT (14) with a higher potency (7.2pM) at OTR than that of OT and marked selectivity (>10,000-fold) over V1aR and V1bR. Hence, we investigated comprehensive modification of OT and obtained new OT analogs that exhibited high potency at OTR with marked selectivity. These OTR-selective agonists could be useful to investigate OTR-mediated effects on psychiatric disorders.

Design, synthesis, and biological activities of 1-aryl-1,4-diazepan-2-one derivatives as novel triple reuptake inhibitors.

A novel series of triple reuptake inhibitors were explored by ligand-based drug design. A cyclic structure was designed from cyclopropane derivative 5 using the core structure of reported monoamine reuptake inhibitors, leading to the formation of the 1-aryl-1,4-diazepan-2-one derivative 23j-S. Compound 23j-S was shown to act as a potent TRI with an excellent ADME-Tox profile. Oral administration of 23j-S significantly enhanced norepinephrine, dopamine, and serotonin levels in the mouse prefrontal cortex and showed significant antidepressant-like activity in tail suspension tests in mouse.

Novel triple reuptake inhibitors with low risk of CAD associated liabilities: design, synthesis and biological activities of 4-[(1S)-1-(3,4-dichlorophenyl)-2-methoxyethyl]piperidine and related compounds.

A novel triple reuptake inhibitor with low potential of liabilities associated with cationic amphiphilic drug (CAD) was identified following an analysis of existing drugs. Low molecular weight (MW < ca. 300), low aromatic ring count (number = 1) and reduced lipophilicity (ClogP < 3.5) were hypothesized to be key factors to avoid the CAD associated liabilities (CYP2D6 inhibition, hERG inhibition and phospholipidosis). Based on the hypothesis, a series of piperidine compounds was designed with consideration of the common characteristic features of CNS drugs. Optimization of the side chain by adjusting overall lipophilicity suggested that incorporation of a methoxymethyl group could provide compounds with a balance of both potent reuptake inhibition and low liability potential. Compound (S)-3a showed a potent antidepressant-like effect in the mice tail suspension test (MED = 10 mg/kg, p.o.), proportional monoamine transporter occupancies and enhancement of monoamine concentrations in mouse prefrontal cortex.

Molecular mechanisms underlying hypothermia-induced neuroprotection.

Stroke is a dynamic event in the brain involving heterogeneous cells. There is now compelling clinical evidence that prolonged, moderate cerebral hypothermia initiated within a few hours after severe ischemia can reduce subsequent neuronal death and improve behavioral recovery. The neuroprotective role of hypothermia is also well established in experimental animals. However, the mechanism of hypothermic neuroprotection remains unclear, although, presumably involves the ability of hypothermia to suppress a broad range of injurious factors. In this paper, we addressed this issue by utilizing comprehensive gene and protein expression analyses of ischemic rat brains. To predict precise target molecules, we took advantage of the therapeutic time window and duration of hypothermia necessary to exert neuroprotective effects. We proposed that hypothermia contributes to protect neuroinflammation, and identified candidate molecules such as MIP-3α and Hsp70 that warrant further investigation as targets for therapeutic drugs acting as "hypothermia-like neuroprotectants."

Macrophage inflammatory protein-3alpha plays a key role in the inflammatory cascade in rat focal cerebral ischemia.

Although hypothermia is one of the most robust neuroprotectants clinically available, its underlying mechanisms remain unclear. Through microarray gene expression analysis, we previously identified several key molecules potentially involved in the efficacy of hypothermia in a 2h middle cerebral artery occlusion (MCAO) rat model, including cytokine and chemokine genes. The present study demonstrated that the expressions of 2 genes, macrophage inflammatory protein-3alpha (MIP-3alpha) and its receptor, CC-chemokine receptor 6 (CCR6), were upregulated in the model and were suppressed by hypothermia. To investigate the role of cerebral MIP-3alpha, it was administered into the rat striatum; dose- and time-dependent induction of CCR6 gene expression was observed. Interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha injection also induced sequential expressions of MIP-3alpha and CCR6. MIP-3alpha was found to be produced by proinflammatory cytokines in rat astrocytes, while it was suppressed by hypothermia. In turn, MIP-3alpha stimulated IL-1beta and inducible nitric oxide synthase expressions in rat microglia and rat brains. Furthermore, intracerebroventricular administration of an anti-rat MIP-3alpha-neutralizing antibody significantly reduced the infarct in MCAO rat brains. These findings suggest that MIP-3alpha plays a pivotal role in inflammatory cascades in ischemic brains, and may be a novel therapeutic target for cerebral ischemia.

Hypothermia enhances heat-shock protein 70 production in ischemic brains.

Although moderate hypothermia is one of the most robust and effective techniques available for reducing ischemic injury, its key mechanism still remains unclear. Our proteomic analysis of the brains of rats treated with a 2-h middle cerebral artery occlusion showed that postischemic hypothermia markedly potentiated a sustained increase in heat-shock protein 70 (Hsp70). The elevated Hsp70 level was confirmed by enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemical staining. Expression of other Hsp proteins was unaffected by hypothermia. Interestingly, hypothermia did not increased, even decreased, the upregulation of hsp70 mRNA expression by ischemia, suggesting that Hsp70 abundance is controlled by an unknown posttranscriptional regulation. As Hsp70 exerts a protective role against ischemic damage, the specific increase in Hsp70 production may contribute to the neuroprotective effect of hypothermia.

Therapeutic time window of post-ischemic mild hypothermia and the gene expression associated with the neuroprotection in rat focal cerebral ischemia.

Hypothermia is the only neuroprotective therapy proven to be clinically effective. Identifying the molecules that play important roles in the efficacy of hypothermia, we developed a multi-channel computer-controlled system, in which the brain temperatures of freely moving rats were telemetrically monitored and maintained below 35 degrees C, and examined the time window necessary to exert its significant neuroprotective effects. Eight-week-old SD rats were subjected to a 2h middle cerebral artery occlusion (MCAO) with an intraluminal filament, and post-ischemic hypothermia was introduced at 0, 2, 4, or 6h after reperfusion until the rats were killed 2 days after MCAO. Since a significant protection was observed when hypothermia was started within 4h after reperfusion, it was concluded that the therapeutic time window of mild hypothermia lasts for 4h after reperfusion in our model. On the basis of the window, comprehensive gene expression analyses using oligonucleotide microarrays were conducted and identified potential genes related to the efficacy of hypothermia, which included inflammatory genes like osteopontin, early growth response-1, or macrophage inflammatory protein-3alpha. Therefore, the neuroprotective effects of post-ischemic mild hypothermia were strongly suggested to be mainly associated with the reduction of neuronal inflammation.

Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype.

The human MrgX3 gene, belonging to the mrgs/SNSRs (mas related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.

Expression of KiSS-1, a metastasis suppressor gene, in trophoblast giant cells of the rat placenta.

Metastin is encoded by a putative human metastasis suppressor gene KiSS-1, and is the cognate ligand of a G-protein-coupled receptor designated OT7T175. To study the physiological function(s) of metastin, we cloned rat and mouse KiSS-1 cDNAs both encoding 130-amino acid KiSS-1 proteins. Sequence analysis suggested that processing of the rat and mouse KiSS-1 proteins produces 52-amino-acid peptides, each with an amidated carboxyl terminal and with a single possible disulfide bond, corresponding to rat and mouse metastins. The carboxyl-terminal sequence of metastin, known to be essential for functional receptor interaction, was found to be highly conserved among humans and rodents. Real-time PCR analysis indicated that rat KiSS-1 mRNA showed the highest expression level in the cecum and colon. Since KiSS-1 mRNA and metastin are known to be abundant in human placenta, we further studied the localization of KiSS-1 and OT7T175 mRNAs in rat placenta by in situ hybridization. KiSS-1 and OT7T175 mRNAs were specifically detected in trophoblast giant cells at embryonic day 12.5, and the transcripts in the cells gradually decreased during placental maturation. These results suggest that metastin/OT7T175 signaling may participate in implantation of the mammalian embryo, placenta formation, and maintenance of pregnancy.

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.