RESUMO
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.
Assuntos
Receptores de Somatostatina , Somatostatina , Humanos , Receptores de Somatostatina/metabolismo , Glicosilação , Somatostatina/metabolismo , PolissacarídeosRESUMO
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.
Assuntos
Peptídeos/química , Cromatografia Líquida de Alta Pressão , Glicosilação , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Peptídeos/análise , Peptídeos/metabolismo , SolubilidadeRESUMO
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.
Assuntos
Íons/farmacologia , Manganês/farmacologia , Polissacarídeos/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação/efeitos dos fármacos , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Saccharomyces cerevisiae/genética , UDPglucose 4-Epimerase/genética , UDPglucose 4-Epimerase/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Neuropeptide Y (NPY) is a potent orexigenic neuropeptide, and antagonism of NPY Y1 and NPY Y5 receptors (NPYxR) is considered a potentially important anti-obesity drug target. We tested the hypothesis that blockade of the NPY5R will lead to weight loss in humans using MK-0557, a potent, highly selective, orally active NPY5R antagonist. The initial series of experiments reported herein, including a multiple-dose positron-emission tomography study and a 12 week proof-of concept/dose-ranging study, suggested an optimal MK-0557 dose of 1 mg/day. The hypothesis was then tested in a 52 week, multicenter, randomized, double-blind, placebo-controlled trial involving 1661 overweight and obese patients. Although statistically significant at 52 weeks, the magnitude of induced weight loss was not clinically meaningful. These observations provide the first clinical insight into the human NPY-energy homeostatic pathway and suggest that solely targeting the NPY5R in future drug development programs is unlikely to produce therapeutic efficacy.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Cicloexanos/uso terapêutico , Obesidade/tratamento farmacológico , Pirazóis/uso terapêutico , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Compostos de Espiro/uso terapêutico , Administração Oral , Adolescente , Adulto , Idoso , Fármacos Antiobesidade/administração & dosagem , Peso Corporal , Cicloexanos/administração & dosagem , Relação Dose-Resposta a Droga , Método Duplo-Cego , Humanos , Pessoa de Meia-Idade , Estrutura Molecular , Placebos , Tomografia por Emissão de Pósitrons/métodos , Pirazóis/administração & dosagem , Receptores de Neuropeptídeo Y/metabolismo , Sensibilidade e Especificidade , Compostos de Espiro/administração & dosagem , Relação Estrutura-Atividade , Resultado do TratamentoRESUMO
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.
Assuntos
Piridonas/química , Receptores de Somatostatina/antagonistas & inibidores , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Camundongos Obesos , Piridonas/síntese química , Piridonas/farmacocinética , Ratos , Receptores de Somatostatina/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/síntese química , Piridonas/síntese química , Receptores de Somatostatina/antagonistas & inibidores , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Células CHO , Cricetinae , Cricetulus , Descoberta de Drogas , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Piridonas/química , Piridonas/farmacologia , Receptores de Somatostatina/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
We have developed and characterized [(35)S]4a as a potent and selective radioligand for melanin-concentrating hormone 1-receptor (MCH1R). Compound [(35)S]4a showed appreciable specific signals in brain slices prepared from wild-type mice but not from MCH1R deficient mice, confirming the specificity and utility of [(35)S]4a as a selective MCH1R radioligand for ex vivo receptor occupancy assays.
Assuntos
Benzofuranos/química , Receptores de Somatostatina/metabolismo , Compostos de Espiro/química , Sulfonamidas/química , Sequência de Aminoácidos , Animais , Benzofuranos/síntese química , Benzofuranos/farmacologia , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Humanos , Cinética , Camundongos , Camundongos Knockout , Receptores de Somatostatina/antagonistas & inibidores , Receptores de Somatostatina/deficiência , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Sulfonamidas/síntese química , Sulfonamidas/farmacologiaRESUMO
Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC(50) of 54nM. Subsequent optimization led to the identification of several potent derivatives.
Assuntos
Química Farmacêutica/métodos , Imidazóis/química , Receptores de Neuropeptídeo Y/química , Animais , Encéfalo/metabolismo , Ácidos Carboxílicos/química , Líquido Cefalorraquidiano/metabolismo , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Modelos Químicos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Benzimidazóis/química , Receptores de Somatostatina/antagonistas & inibidores , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Benzimidazóis/síntese química , Benzimidazóis/farmacocinética , Ingestão de Alimentos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Somatostatina/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Encéfalo/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos , Receptores de Neuropeptídeo Y/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Líquido Cefalorraquidiano/diagnóstico por imagem , Humanos , Ligantes , Camundongos , Plasma/diagnóstico por imagem , Ligação Proteica , Ratos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Piridinas/química , Receptores de Somatostatina/antagonistas & inibidores , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Células CHO , Cricetinae , Cricetulus , Descoberta de Drogas , Humanos , Piridinas/síntese química , Piridinas/farmacologia , Ratos , Receptores de Somatostatina/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Fármacos Antiobesidade/uso terapêutico , Canais de Potássio Éter-A-Go-Go/metabolismo , Obesidade/tratamento farmacológico , Oximas/química , Oximas/uso terapêutico , Receptores de Somatostatina/antagonistas & inibidores , Animais , Fármacos Antiobesidade/farmacocinética , Fármacos Antiobesidade/farmacologia , Peso Corporal/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oximas/farmacocinética , Oximas/farmacologia , Ligação Proteica , Receptores de Somatostatina/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of spiroindoline-3,4'-piperidine derivatives were synthesized and evaluated for their binding affinities and antagonistic activities at Y5 receptors. Potent Y5 antagonists were tested for their oral bioavailabilities and brain penetration in rats. Some of the antagonists showed good oral bioavailability and/or good brain penetration. In particular, compound 6e was orally bioavailable and brain penetrant, and oral administration of 6e inhibited bPP-induced food intake in rats with a minimum effective dose of 10mg/kg.
Assuntos
Química Farmacêutica/métodos , Indóis/administração & dosagem , Indóis/farmacologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/química , Administração Oral , Aminas/química , Animais , Disponibilidade Biológica , Encéfalo/efeitos dos fármacos , Desenho de Fármacos , Concentração Inibidora 50 , Isocianatos/química , Modelos Químicos , Biblioteca de Peptídeos , Ratos , Ureia/químicaRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Piperidinas/química , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Compostos de Espiro/química , Ureia/análogos & derivados , Administração Oral , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacologia , Ingestão de Alimentos , Humanos , Camundongos , Piperidinas/síntese química , Piperidinas/farmacologia , Ratos , Receptores de Neuropeptídeo Y/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Ureia/síntese química , Ureia/farmacologia , Redução de PesoRESUMO
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.
Assuntos
Piperidinas/química , Receptores de Somatostatina/antagonistas & inibidores , Compostos de Espiro/química , Linhagem Celular , Descoberta de Drogas , Humanos , Piperidinas/síntese química , Piperidinas/farmacologia , Receptores de Somatostatina/metabolismo , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Imidazolinas/química , Piridonas/química , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Cães , Descoberta de Drogas , Humanos , Imidazolinas/síntese química , Imidazolinas/farmacocinética , Camundongos , Piridonas/síntese química , Piridonas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Fármacos Antiobesidade/química , Fármacos Antiobesidade/farmacologia , Indóis/química , Indóis/farmacologia , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismo , Animais , Fármacos Antiobesidade/síntese química , Fármacos Antiobesidade/farmacocinética , Disponibilidade Biológica , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Indóis/síntese química , Indóis/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/agonistas , Compostos de Espiro/síntese química , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Amidas/síntese química , Benzofuranos/síntese química , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Compostos de Espiro/síntese química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Administração Oral , Amidas/farmacologia , Animais , Benzofuranos/farmacologia , Encéfalo/metabolismo , Líquido Cefalorraquidiano/metabolismo , Estabilidade de Medicamentos , Ingestão de Alimentos/efeitos dos fármacos , Ratos , Compostos de Espiro/farmacologiaRESUMO
Despite the well-established role of histamine as an anorexigenic neurotransmitter, the role of histamine H(3) receptors in feeding behavior is controversial. Herein we investigated the role of histamine H(3) receptor on several orexigenic agents in mice. Thioperamide (histamine H(3) receptor inverse agonist) inhibited neuropeptide Y- and nociceptin-induced hyperphagia but had no effect on U-50488 (opioid kappa-receptor agonist)-induced hyperphagia. In contrast, imetit (histamine H(3) receptor agonist) inhibited U-50488-induced hyperphagia but augmented neuropeptide Y-induced hyperphagia while it did not alter nociceptin-induced hyperphagia. These results indicate distinctive roles of histamine H(3) receptors in various orexigenic pathways.
Assuntos
Apetite/fisiologia , Hiperfagia/fisiopatologia , Receptores Histamínicos H3/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida , Animais , Apetite/efeitos dos fármacos , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H3/farmacologia , Hiperfagia/induzido quimicamente , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeo Y , Peptídeos Opioides , Piperidinas/farmacologia , Receptores Histamínicos H3/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , NociceptinaRESUMO
Syntheses and structure-activity relationships of a novel class of 2-[3-oxospiro[isobenzofuran-1(3H),1'-cyclohexan]-4'-yl]benzimidazole NPY Y5 receptor antagonists are described. Optimization of the lead compound 2a by incorporating substituents into the 5-position or into both the 5- and 6-positions of the benzimidazole core part led to the identification of 5-(5-methyl-1,2,4-oxadiazol-2-yl)benzimidazole (2r: IC(50)=3.3 nM) and 5-(2-methyltetrazol-5-yl)benzimidazole (2u: IC(50)=5.9 nM), both of which are potent, selective, and orally bioavailable Y5 receptor antagonists.