Takahiko Yano

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1–3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

Localization and neuronal response of RFamide related peptides in the rat central nervous system

RFamide related peptides (RFRP)-1 and RFRP-3 are neuropeptides derived from the same preproprotein. We have examined the distribution of RFRP-1 and RFRP-3 immunoreactivities (irs) in the rat central nervous system using specific antibodies. Neuronal cell bodies containing both RFRP-1 and RFRP-3 were detected within the caudal portion of the hypothalamus, the periventricular nucleus (PerVN), and the portion around or above the ventromedial nucleus of the hypothalamus. Both immunohistochemical and in situ hybridization analyses showed that neurons containing RFRP immunoreactivity and mRNA were distinct from those of neuropeptide FF, which contains the same structure at the C-terminus, Pro-Glu-Arg-Phe-NH2, as RFRP-3. Fibers containing both RFRP-1 and RFRP-3 were widely distributed in the brain: the lateral septal nucleus in the telencephalon, the paraventricular thalamic nucleus, various hypothalamic nuclei, the periaqueductal gray in the midbrain, the parabrachial nucleus in the pons, and the nucleus tractus solitarius (NTS) in the medulla oblongata. Only RFRP-1-ir was detected within the posterior gray horn in the spinal cord. Only RFRP-3-ir was detected in several thalamic nuclei and the spinal cord, especially at the posterior intermediate sulcus and within the anterior gray horn. Intracerebroventricular administration of RFRPs induced c-Fos expression in the anterior portion of the NTS, locus coeruleus, the nucleus of incertus, supraoptic nucleus, PerVN and the arcuate nucleus of the hypothalamus. These results show that RFRP-1 and RFRP-3 are widely distributed in the rat central nervous system and might be involved in various functions such as the neuroendocrine system or pain modulation.

Intracerebroventricular administration of urotensin II promotes anxiogenic-like behaviors in rodents

We identified urotensin II (U-II) as the endogenous ligand for the orphan G-protein-coupled receptor GPR14 or SENR. Both U-II and GPR14 are expressed not only in peripheral tissues but also in the brain of rodents, suggesting that U-II plays a physiological role in the central nervous system. In the present study, we investigated the central effects of U-II in rodents. Intracerebroventricular administration of U-II induced anxiogenic-like behaviors in the elevated plus maze test and the hole-board test in mice in a dose-dependent manner, as did corticotropin releasing factor (CRF). The effective doses of U-II were 10-100-fold higher than these of CRF in these tests. Our results suggest that U-II is a candidate for the mediator of some aspect of stress or anxiety in the central nervous system.

Gonadal regulation of PrRP mRNA expression in the nucleus tractus solitarius and ventral and lateral reticular nuclei of the rat

We investigated the prolactin-releasing peptide (PrRP) gene expression quantitatively in the rat brain and the involvement of estrogen and progesterone using in situ hybridization. The strongest signals were observed in the nucleus tractus solitarius (NTS), which showed approximately 70% of total PrRP mRNA in the brain. Moderate expression was observed in the ventral and lateral reticular nuclei (VLRN) of the medulla oblongata. PrRP mRNA signals in the hypothalamic ventromedial- and dorsomedial nuclei showed only 5% of total signals. The PrRP mRNA expression among female rats showing normal gonadal cycle and male rats showed that the highest levels were in female rats in proestrus. Administration of estrogen or progesterone after ovariectomy induced an increase in PrRP mRNA expression in the NTS. PrRP mRNA content in the NTS increased with the progress of the pregnancy and reached a peak on the 14th day, the mid-period of pregnancy, when plasma progesterone increases. We also observed the colocalization of PrRP and estrogen receptor alpha in the neurons distributed in the NTS by double labeling immunocytochemistry. These findings indicate that PrRP gene expression is regulated by gonadal steroid hormones in the medulla oblongata, and parts of PrRP synthesizing neurons are considered to be directly influenced by estrogen in the NTS.

Developmental expression of prolactin releasing peptide in the rat brain: localization of messenger ribonucleic acid and immunoreactive neurons

Prolactin releasing peptide (PrRP) was recently identified as the stimulator of prolactin release from the anterior pituitary. PrRP mRNA is expressed in the medulla oblongata and the hypothalamus in the rat brain. The fibers containing PrRP are widely distributed in the brain, therefore, it was postulated that PrRP may act as a neurotransmitter or neuromodulator as well as an endocrine substance. To clarify the developmental changes in the expression of PrRP during brain development, we examined PrRP in rat fetuses and neonates using in situ hybridization and immunohistochemistry. The PrRP mRNA was expressed in the nucleus of the solitary tract (NTS) at embryonic day 18 (E18) and in the ventral and lateral reticular nucleus (VLRN) of the caudal medulla oblongata at E20. The PrRP mRNA in the hypothalamus was first expressed at postnatal day 13 (P13). Reverse transcription-polymerase chain reaction analysis (RT-PCR) for PrRP revealed that PCR product, a 268 bp band, was detected from either E18 in the medulla or P13 in the hypothalamus. Immunodetection with monoclonal antibody against prepro-PrRP revealed intensive staining of cells in the NTS at E18, in the VLRN at E20 or in the dorsomedial hypothalamus at P13. Immunohistochemistry using monoclonal antibody against mature PrRP at P6 showed PrRP fibers to be distributed in the paraventricular hypothalamic nucleus, periventricular hypothalamic nucleus, medial preoptic area, basolateral amygdaloid nucleus, dorsomedial hypothalamus, ventromedial hypothalamus, periventricular nucleus of the thalamus and bed nucleus of the stria terminalis as previously shown in the adult rat. PrRP fibers were also found in the optic chiasm, dorsal endopiriform nucleus, cingulum, intermediate reticular nucleus, and caudal ventrolateral reticular nucleus at P6 and P9. However, PrRP fibers were never found in the above regions in the adult animal. These findings suggest that PrRP fibers originating in the medulla oblongata have been widely distributed in the rat brain during the early postnatal day and PrRP may play various roles in the brain development.

Discovery of pyrrolo[2,3-d]pyrimidin-4-ones as corticotropin-releasing factor 1 receptor antagonists with a carbonyl-based hydrogen bonding acceptor

A new class of pyrrolo[2,3-d]pyrimidin-4-one corticotropin-releasing factor 1 (CRF(1)) receptor antagonists has been designed and synthesized. In general, reported CRF(1) receptor antagonists possess a sp(2)-nitrogen atom as hydrogen bonding acceptor (HBA) on their core scaffolds. We proposed to use a carbonyl group of pyrrolo[2,3-d]pyrimidin-4-one derivatives as a replacement for the sp(2)-nitrogen atom as HBA in classical CRF(1) receptor antagonists. As a result, several pyrrolo[2,3-d]pyrimidin-4-one derivatives showed CRF(1) receptor binding affinity with IC(50) values in the submicromolar range. Ex vivo (125)I-sauvagine binding studies showed that 2-(dipropylamino)-3,7-dimethyl-5-(2,4,6-trimethylphenyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (16b) (30 mg/kg, p.o.) was able to penetrate into the brain and inhibit radioligand binding to CRF(1) receptors (frontal cortex, olfactory bulb, and pituitary) in mice. We identified pyrrolo[2,3-d]pyrimidin-4-one derivatives as the first CRF(1) antagonists with a carbonyl-based HBA.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 μM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 μmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

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.

Discovery of 1,2,3,4-tetrahydropyrimido[1,2-a]benzimidazoles as novel class of corticotropin releasing factor 1 receptor antagonists

A new class of corticotropin releasing factor 1 (CRF1) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a-e were designed as CRF1 receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF1 receptor antagonist. The synthesized tricyclic derivatives 2a-e showed CRF1 receptor binding activity with IC50 values of less than 400 nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF1 receptor binding activity (IC50 = 7.1 nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF1 receptor binding activity (IC50 = 58 nM) with good oral bioavailability (F = 68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [125I]-CRF binding in the frontal cortex (5 and 10 mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10 mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF1 receptors in the brain and exhibits the potential to be further examined for clinical studies.

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats

&NA; The aim of this study was to investigate peripheral and central roles of corticotropin‐releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic‐pituitary‐adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF1 receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF‐induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF1 receptor binding affinities (15 and 10 nM) and functional activities in reporter gene assay (15 and 9.5 nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10 mg/kg inhibited [125I]‐CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [125I]‐CRF binding, indicating that only Compound A inhibited central [125I]‐CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge‐induced anxiety behavior and c‐fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF1 receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders. HighlightsEndocrinological and behavioral regulations by peripheral and central CRF challenges.Effects of two original CRF1 receptor antagonists with distinct [125I]‐CRF binding inhibition in the cortex on CRF‐induced endocrinological and behavioral responses.The central actions of CRF1 receptor antagonists are indicated to be critical for regulating CRF‐induced behavioral changes.