Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of Its Conjugated Peptide.

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling. This tunability will allow optimization towards the intended use of the peptides to be released. Furthermore, because glycosylation is a reliable method of greatly increasing the solubility of a peptide, the presented glycosylated linkers are expected to permit the preparation of antibodies in aqueous buffers even in the case of sparingly soluble antigen peptides.

Manganese ion concentration affects production of human core 3 O-glycan in Saccharomyces cerevisiae.

BACKGROUND: Production of various mucin-like glycoproteins could be useful for development of antibodies specific to disease-related glycoproteins as well as for the biosynthesis of clinically useful glycoproteins. A Saccharomyces cerevisiae strain capable of in vivo production of mucin-type core 1 structure (Galß1-3GalNAcα1-O-Ser/Thr) has been reported, but a strain producing core 3 structure (GlcNAcß1-3GalNAcα1-O-Ser/Thr) has not been constructed. METHODS: To generate core 3-producing strain, genes encoding uridine diphosphate (UDP)-Gal-4-epimerase, UDP-GalNAc transporter, UDP-GlcNAc transporter, and two glycosyltransferases were integrated into the genome. A Mucin-1-derived acceptor peptide (MUC1ap) was expressed as an acceptor. The amount of the resulting modified peptide was analyzed by HPLC. RESULTS: Introduction of a codon-optimized UDP-GlcNAc:ßGal ß-1,3-N-acetylglucosaminyltransferase 6 (ß3Gn-T6) gene yielded increases in ß3Gn-T6 activity but did not alter the level of core 3 production. The highest in vitro activity of ß3Gn-T6 was observed at Mn(2+) concentrations of 10mM and above. Supplementation of MnCl2 to the culture medium yielded increases of up to 25% in the accumulation of core 3 on the MUC1ap. The yeast invertase from the core 3-producing strain was less extensively N-glycosylated; however, it was partially restored by the addition of MnCl2 to the medium. CONCLUSIONS: Physiological Mn(2+) concentration in S. cerevisiae was insufficient to facilitate optimal synthesis of core 3. Mn(2+) supplementation led to up-regulation of reaction of glycosylation in the Golgi, resulting in increases of core 3 production. GENERAL SIGNIFICANCE: This study reveals that control of Mn(2+) concentration is important for production of specific mammalian-type glycans in S. cerevisiae.

Discovery of novel phenylpyridone derivatives as potent and selective MCH1R antagonists.

The design, synthesis and structure-activity relationships of a novel class of N-phenylpyridone MCH1R antagonists are described. The core part of the N-phenylpyridone structure was newly designed and the side chain moieties that were attached to the core part were extensively explored. As a result of optimization of the N-phenylpyridone leads, we successfully developed the orally available, and brain-penetrable MCH1R selective antagonist 7c, exhibiting excellent anti-obese effect in diet-induced obese (DIO) mice.

NPY Y2 receptor agonist PYY(3-36) inhibits diarrhea by reducing intestinal fluid secretion and slowing colonic transit in mice.

Peptide YY (PYY)(3-36), a neuropeptide Y (NPY) Y2 receptor agonist, is a powerful inhibitor of intestinal secretion. Based on this anti-secretory effect, NPY Y2 receptor agonists may be useful as novel anti-diarrheal agents, but anti-diarrheal efficacy has yet to be determined. We therefore examined the anti-diarrheal efficacy of PYY(3-36) and a selective Y2 receptor agonist, N-acetyl-[Leu28, Leu31]-NPY(24-36), in experimental mouse models of diarrhea. Intraperitoneal administration of PYY(3-36) (0.01-1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) significantly inhibited diarrhea (increase in wet fecal weight and diarrhea score) induced by dimethyl-prostaglandin E2, 5-hydroxytryptamine, and castor oil. Anti-diarrheal activities of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) were comparable to the effects of loperamide (1mg/kg), a widely used anti-diarrheal drug. To clarify the anti-diarrheal mechanisms of NPY Y2 receptor agonists, we investigated the effects of PYY(3-36) and N-acetyl-[Leu28, Leu31]-NPY(24-36) on intestinal fluid secretion and colonic transit. PYY(3-36) (1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) significantly reduced dimethyl-prostaglandin E2-induced intestinal fluid accumulation in conscious mice, suggesting that NPY Y2 receptor agonists inhibit diarrhea, at least in part, by reducing intestinal secretion. In addition, PYY(3-36) (0.01-1mg/kg) and N-acetyl-[Leu28, Leu31]-NPY(24-36) (10mg/kg) potently inhibited normal fecal output, suggesting that NPY Y2 receptor activation inhibits colonic motor function and NPY Y2 receptor agonists inhibit diarrhea partly by slowing colonic transit. These results indicate that NPY Y2 receptor agonists inhibit diarrhea in mice by not only reducing intestinal fluid secretion, but also slowing colonic transit, and illustrate the therapeutic potential of NPY Y2 receptor agonists as effective treatments for diarrhea.

Pancreatic polypeptide enhances colonic muscle contraction and fecal output through neuropeptide Y Y4 receptor in mice.

Pancreatic polypeptide is released mainly from the pancreas, and is thought to be one of the major endogenous agonists of the neuropeptide Y Y(4) receptor. Pancreatic polypeptide has been shown to stimulate colonic muscle contraction, but whether pancreatic polypeptide has in vivo functional activity with respect to colonic transit is unclear. The present report investigated the effects of pancreatic polypeptide on fecal output as an index of colonic transit as well as intestinal motor activity, using wild-type (WT) and neuropeptide Y Y(4) receptor-deficient (KO) mice. Peripheral administration of pancreatic polypeptide increased fecal weight and caused diarrhea in WT mice in a dose-dependent manner (0.01-3mg/kg s.c.). Pancreatic polypeptide-induced increases in fecal weight and diarrhea completely disappeared in KO mice, while basal fecal weights did not differ between WT and KO mice. In longitudinal and circular muscles of mouse isolated colon, pancreatic polypeptide (0.01-1 microM) increased basal tone and frequency of spontaneous contraction in WT mice, but not in KO mice. Atropine did not affect pancreatic polypeptide-induced fecal output or increase in colonic muscle tone, indicating that the actions of pancreatic polypeptide are not mediated through cholinergic mechanisms. The present findings demonstrate that pancreatic polypeptide enhances colonic contractile activity and fecal output through neuropeptide Y Y(4) receptor, and a neuropeptide Y Y(4) receptor agonist might offer a novel therapeutic approach to ameliorate constipation.

Melanin-concentrating hormone 1-receptor antagonist suppresses body weight gain correlated with high receptor occupancy levels in diet-induced obesity mice.

Melanin-concentrating hormone (MCH), which is a neuropeptide expressed in the hypothalamus of the brain, is involved in regulating feeding behavior and energy homeostasis via the MCH(1) receptor in rodents. It is widely considered that MCH(1) receptor antagonists are worthy of development for medical treatment of obesity. Here we report on the development of an ex vivo receptor occupancy assay using a new radiolabeled MCH(1) receptor antagonist, [(35)S]-compound D. An MCH(1) receptor antagonist inhibited the binding of [(35)S]-compound D to brain slices in a dose-dependent manner. The result showed a good correlation between the receptor occupancy levels and plasma or brain levels of the MCH(1) receptor antagonist, suggesting that the ex vivo receptor binding assay using this radioligand is practical. Quantitative analysis in diet-induced obese mice showed that the efficacy of body weight reduction correlated with the receptor occupancy levels at 24h. Furthermore, more than 90% occupancy levels of MCH(1) receptor antagonists during 24h post-dosing are required for potent efficacy on body weight reduction. The present occupancy assay could be a useful pharmacodynamic marker to quantitatively estimate anti-obese efficacy, and would accelerate the development of MCH(1) receptor antagonists for treatment of obesity.

Discovery of trans-N-[1-(2-fluorophenyl)-3-pyrazolyl]-3-oxospiro[6-azaisobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide, a potent and orally active neuropeptide Y Y5 receptor antagonist.

A series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide derivatives were synthesized to identify potent NPY Y5 receptor antagonists. Of the compounds, 21j showed high Y5 binding affinity, metabolic stability and brain and cerebrospinal fluid (CSF) penetration, and low susceptibility to P-glycoprotein transporters. Oral administration of 21j significantly inhibited the Y5 agonist-induced food intake in rats with a minimum effective dose of 1mg/kg. This compound was selected for proof-of-concept studies in human clinical trials.

Identification of positron emission tomography ligands for NPY Y5 receptors in the brain.

A series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide derivatives were synthesized and profiled for NPY Y5 binding affinity, brain and CSF penetrability in rats, and susceptibility to human and mouse P-glycoprotein transporters in order to develop a PET ligand. Compound 12b exhibited an acceptable profile for a PET ligand, and [(11)C]12b was successfully utilized in clinical settings as a Y5 PET ligand.

Discovery of novel diarylketoxime derivatives as selective and orally active melanin-concentrating hormone 1 receptor antagonists.

Optimization of the lead 2a led to the identification of a novel diarylketoxime class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Our focus was directed toward improvement of hERG activity and metabolic stability. The representative derivative 4b showed potent and dose-dependent body weight reduction in diet-induced obese (DIO) C57BL/6J mice after oral administration. The synthesis and structure-activity relationships of the novel diarylketoxime MCH-1R antagonists are described.

Discovery of novel phenethylpyridone derivatives as potent melanin-concentrating hormone 1 receptor antagonists.

Novel phenethylpyridone derivatives were identified as potent human melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. A search for surrogates for the 4-(2-aminoethoxy)phenyl moiety of 1 resulted in discovery of 2-[4-(aminomethyl)phenyl]ethyl substructure as in 6a. Successive optimization of the right-hand moiety led to the identification of a number of potent derivatives.

Discovery of pyridone-containing imidazolines as potent and selective inhibitors of neuropeptide Y Y5 receptor.

A series of 2-pyridone-containing imidazoline derivatives was synthesized and evaluated as neuropeptide Y Y5 receptor antagonists. Optimization of the 2-pyridone structure on the 2-position of the imidazoline ring led to identification of 1-(difluoromethyl)-5-[(4S,5S)-4-(4-fluorophenyl)-4-(6-fluoropyridin-3-yl)-5-methyl-4,5-dihydro-1H-imidazol-2-yl]pyridin-2(1H)-one (7m). Compound 7m displayed statistically significant inhibition of food intake in an agonist-induced food intake model in SD rats and no adverse cardiovascular effects in anesthetized dogs. In addition, markedly higher brain penetrability and a lower plasma Occ90 value were observed in P-gp-deficient mdr1a (-/-) mice compared to mdr1a (+/+) mice after oral administration of 7m.

Discovery of imidazo[1,2-a]pyridines as potent MCH1R antagonists.

A series of imidazo[1,2-a]pyridine derivatives was identified and evaluated for MCH1R binding and antagonistic activity. Introduction of a methyl substituent at the 3-position of imidazo[1,2-a]pyridine provided compounds with a significant improvement in MCH1R affinity. Representative compounds in this series exhibited good potency and brain exposure in rats.

Synergistic interaction between neuropeptide Y1 and Y5 receptor pathways in regulation of energy homeostasis.

Neuropeptide Y plays a key role in the physiological control of energy homeostasis. Five neuropeptide Y receptor subtypes have been cloned, and multiple neuropeptide Y receptor subtypes are thought to mediate neuropeptide Y activity. However, interactions among neuropeptide Y receptor subtypes have not been elucidated to date. Herein, we examined the interaction between neuropeptide Y(1) and Y(5) receptors in feeding regulation by employing selective neuropeptide Y(1) and Y(5) receptor antagonists in C57BL/6 and neuropeptide Y(1) receptor knockout mice fed a high-fat diet. A single-dose of a neuropeptide Y(1) receptor antagonist (10-30 mg/kg) suppressed spontaneous food intake and reduced body weight in high-fat diet-fed C57BL/6 mice, while treatment with a neuropeptide Y(5) receptor antagonist did not significantly reduce food intake or body weight. Coadministration of a neuropeptide Y(1) receptor antagonist with a neuropeptide Y(5) receptor antagonist further suppressed food intake and reduced body weight. Next, we evaluated the chronic efficacy of a neuropeptide Y(5) receptor antagonist in high-fat diet-fed neuropeptide Y(1) receptor knockout mice in order to mimic chronic combination treatment with neuropeptide Y(1) and Y(5) receptor antagonists. The neuropeptide Y(5) receptor antagonist produced greater body weight reductions in high-fat diet-fed neuropeptide Y(1) receptor knockout mice than in wild-type C57BL/6 mice. These findings confirm an interaction between neuropeptide Y(1) and Y(5) receptors in the regulation of energy homeostasis, as blockade of both the neuropeptide Y(1) and Y(5) receptors produced a greater anti-obesity effect than blocking either receptor alone.

Cloning and characterization of rabbit neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are structurally related peptides that have numerous functions in both neural and endocrine signaling. These effects are mediated by the NPY receptor family and five members of this family have been cloned in mammals. To better characterize these receptor subtypes, we cloned and expressed the Y1, Y2, Y4 and Y5 receptor subtypes from the rabbit. Comparison of these sequences with human orthologs revealed that the Y1, Y2 and Y5 receptors have generally strong amino-acid sequence conservation, with 91-96% identity, while Y4 receptor showed relatively weak similarity with 82% identity, as with other species. Particularly in the transmembrane regions, Y1, Y2, and Y5 receptor subtypes showed remarkable conservation, with 98-99% amino acid identity. Competitive binding studies by NPY-family peptides and analogs showed that Y1, Y2 and Y5 receptors had similar pharmacological profiles between the respective rabbit and human receptor subtypes. Interestingly, all the tested peptides had a greater affinity for rabbit Y4 receptor than human Y4 receptor. These results suggest that rabbit and human Y1, Y2 and Y5 receptor subtypes are well conserved, whereas Y4 receptors are less well conserved.

Novel orally active NPY Y5 receptor antagonists: Synthesis and structure-activity relationship of spiroindoline class compounds.

Spiroindoline urea derivatives, designed to act as NPY Y5 receptor antagonists, were synthesized and their structure-activity relationships were investigated. Of these derivatives, compound 3a showed good Y5 binding affinity with favorable pharmacokinetic properties. Compound 3a significantly inhibited bPP Y5 agonist-induced food intake in rats, and suppressed body weight gain in DIO mice.

Discovery of novel spiro-piperidine derivatives as highly potent and selective melanin-concentrating hormone 1 receptor antagonists.

Optimization of high-throughput screening hit 1a led to the identification of a novel spiro-piperidine class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Compound 3c was identified as a highly potent and selective MCH-1R antagonist, which has an IC(50) value of 0.09 nM at hMCH-1R. The synthesis and structure-activity relationships of the novel spiro-piperidine MCH-1R antagonists are described.

Identification of 2-aminobenzimidazoles as potent melanin-concentrating hormone 1-receptor (MCH1R) antagonists.

A series of 2-aminobenzimidazole-based MCH1R antagonists was identified by core replacement of the aminoquinoline lead 1. Subsequent modification of the 2- and 5-positions led to improvement in potency and intrinsic clearance. Compound 25 exhibited good plasma and brain exposure, and attenuated MCH induced food intake at 30mg/kg PO in rats.

Aryl urea derivatives of spiropiperidines as NPY Y5 receptor antagonists.

Continuing medicinal chemistry studies to identify spiropiperidine-derived NPY Y5 receptor antagonists are described. Aryl urea derivatives of a variety of spiropiperidines were tested for their NPY Y5 receptor binding affinities. Of the spiropiperidines so far examined, spiro[3-oxoisobenzofurane-1(3H),4'-piperidine] was a useful scaffold for producing orally active NPY Y5 receptor antagonists. Oral administration of 5c significantly inhibited the Y5 agonist-induced food intake in rats with a minimum effective dose of 3mg/kg. In addition, this compound was efficacious in decreasing body weight in diet-induced obese mice.

Discovery of tetrasubstituted imidazolines as potent and selective neuropeptide Y Y5 receptor antagonists: reduced human ether-a-go-go related gene potassium channel binding affinity and potent antiobesity effect.

A series of novel imidazoline derivatives was synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Optimization of previously reported imidazoline leads, 1a and 1b, was attempted by introduction of substituents at the 5-position on the imidazoline ring and modification of the bis(4-fluorphenyl) moiety. A number of potent derivatives without human ether-a-go-go related gene potassium channel (hERG) activity were identified. Selected compounds, including 2a, were shown to have excellent brain and CSF permeability. Compound 2a displayed a suitable pharmacokinetic profile for chronic in vivo studies and potently inhibited D-Trp(34)NPY-induced acute food intake in rats. Oral administration of 2a resulted in a potent reduction of body weight in a diet-induced obese mouse model.