RESUMO
We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.
Assuntos
Fármacos Antiobesidade/farmacologia , Imidazóis/farmacologia , Obesidade/tratamento farmacológico , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/antagonistas & inibidores , Administração Oral , Animais , Autorradiografia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dexfenfluramina/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Agonismo Inverso de Drogas , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Imidazóis/administração & dosagem , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/agonistasRESUMO
Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin's effects on food intake are mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp(-/-);Npy(-/-) and Mc3r(-/-);Mc4r(-/-) double knockout mice as well as Npy(-/-) and Agrp(-/-) single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.
Assuntos
Apetite/fisiologia , Neuropeptídeo Y/fisiologia , Hormônios Peptídicos/fisiologia , Proteínas/fisiologia , Proteína Relacionada com Agouti , Animais , Apetite/efeitos dos fármacos , Grelina , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hormônios Peptídicos/farmacologia , Receptor Tipo 3 de Melanocortina/fisiologia , Receptor Tipo 4 de Melanocortina/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Receptores de GrelinaRESUMO
The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.
Assuntos
Lactamas/farmacologia , Hormônios Estimuladores de Melanócitos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 3 de Melanocortina/efeitos dos fármacos , Receptor Tipo 4 de Melanocortina/agonistas , Receptores da Corticotropina/efeitos dos fármacos , alfa-MSH/análogos & derivados , alfa-MSH/farmacologia , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Desenho de Fármacos , Histidina/química , Humanos , Concentração Inibidora 50 , Lactamas/síntese química , Hormônios Estimuladores de Melanócitos/síntese química , Prolina/química , Receptor Tipo 3 de Melanocortina/agonistas , Receptor Tipo 3 de Melanocortina/antagonistas & inibidores , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Receptor Tipo 4 de Melanocortina/efeitos dos fármacos , Receptores da Corticotropina/agonistas , Receptores da Corticotropina/antagonistas & inibidores , Receptores de Melanocortina , Relação Estrutura-Atividade , alfa-MSH/síntese químicaRESUMO
We have designed and synthesized several novel cyclic SHU9119 analogues (Ac-Nle4-[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2) modified in position 6 with nonconventional amino acids. SHU9119 is a high affinity nonselective antagonist at hMC3R and hMC4R with potent agonist activity at hMC1R and hMC5R. We measured the binding affinity and agonist potency of the novel analogues at cloned hMC3R, hMC4R, and hMC5R receptors and identified several selective, high affinity hMC3R and hMC4R antagonists. Compound 4 containing Che substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.48 nM) with 100-fold selectivity over hMC3R antagonist. Analogue 7 with a Cpe substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.51 nM) with a 200-fold selectivity vs the hMC3R. Interestingly, analogue 9 with an Acpc residue in position 6 is a high affinity hMC3R antagonist (IC50 = 2.5 nM) with 100-fold selectivity vs the hMC4R antagonist based on its binding affinities. This compound represents the first cyclic lactam antagonist with high selectivity for the hMC3R vs hMC4R. To understand the possible structural basis responsible for selectivity of these peptides at hMCR3 and hMCR4, we have carried out a molecular modeling study in order to examine the conformational properties of the cyclic peptides modified in position 6 with conformationally restricted amino acids.
Assuntos
Fragmentos de Peptídeos/síntese química , Peptídeos Cíclicos/síntese química , Receptores da Corticotropina/antagonistas & inibidores , alfa-MSH/análogos & derivados , alfa-MSH/síntese química , Animais , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores de Melanocortina , Relação Estrutura-Atividade , alfa-MSH/química , alfa-MSH/farmacologiaRESUMO
We measured plasma concentrations of agouti-related protein (AGRP) in humans and rats and determined whether these were affected by ingestion of a meal after fasting. In 17 healthy human subjects, the mean plasma concentration of AGRP was lower in the fed state than in the fasted state. Two hours after a breakfast meal, AGRP levels dropped by 39%. By contrast, a continued fast for 2 h increased the average AGRP concentration by 73%. In rats with diet-induced obesity, refeeding resulted in a 50% decrease in plasma AGRP concentrations following a fasting-refeeding protocol. Our results support the notion that plasma AGRP may serve as a biomarker for the transition from a fasted to the satiated state.
Assuntos
Proteínas/análise , Adulto , Proteína Relacionada com Agouti , Análise de Variância , Animais , Ingestão de Alimentos/fisiologia , Jejum/sangue , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Leptina/sangue , Masculino , Obesidade/sangue , Obesidade/fisiopatologia , Ratos , Fatores de TempoRESUMO
The melanocortin receptors are involved in several important physiological functions. The potent and enzymatically stable analogues MT-II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2)) and SHU9119 (Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2)) are important ligands of these receptors but are relatively nonselective. To differentiate between the physiological functions of these receptors, agonists, and antagonists with improved receptor selectivities are needed. We report here analogues of the well-characterized antagonist SHU9119 in which we replaced His(6) with conformationally constrained amino acids. By this structure-activity study we discovered two important compounds, PG-901 (Ac-Nle(4)-c[Asp(5)-Pro(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and PG-911 (Ac-Nle(4)-c[Asp(5)-Hyp(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), characterized to be full agonists at the hMC5R (EC(50) = 0.072 nM and 0.031 nM, respectively), but full antagonists at the hMC3R and the hMC4R. We also demonstrated that the relative stereochemistry of the amino acid at the 6-position is critical for activity, and could play an important role in potency as well as in selectivity for the melanocortin receptors.
Assuntos
Desenho de Fármacos , Hormônios Estimuladores de Melanócitos/química , Hormônios Estimuladores de Melanócitos/farmacologia , Peptídeos Cíclicos/farmacologia , Substituição de Aminoácidos , Humanos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Receptor Tipo 3 de Melanocortina , Receptores da Corticotropina/agonistas , Receptores da Corticotropina/antagonistas & inibidores , Receptores da Corticotropina/metabolismo , Receptores de Melanocortina , Relação Estrutura-AtividadeRESUMO
To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.
Assuntos
Alanina/química , Receptores da Corticotropina/metabolismo , gama-MSH/síntese química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Dados de Sequência Molecular , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores de Melanocortina , Relação Estrutura-Atividade , gama-MSH/química , gama-MSH/metabolismoRESUMO
Peptide Ac-Nle(4)-cyclo(5beta-->10epsilon)(Asp(5)-His(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), compound 1, a cyclic derivative of alpha-melanotropin, is a nonselective high affinity antagonist at human melanocortin receptors 3 and 4, and an agonist at melanocortin receptors 1 and 5. To differentiate between the physiological functions of these receptors, antagonists with improved receptor selectivity are needed. In this study, analogues of compound 1 without Ac-Nle(4) or His(6) and/or the amino group of Asp(5) were prepared and tested in binding assays and in functional assays on CHO cells expressing hMC3-5R. Several of these peptides were to be selective, high affinity hMC-4R antagonists. The most interesting was compound 10, named MBP10, cyclo(6beta-->10epsilon)(succinyl(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), an antagonist (IC(50) = 0.5 nM) with 125-fold selectivity over hMC-3R (and of >300-fold selectivity over MC-1RB). This compound had no agonist activity at hMC-3R or hMC-4R and only weak agonist activity at hMC-5R. Examination of the sequences of these new peptides revealed that the D-(2')Nal(7)-Arg(8)-Trp(9) segment of peptide 1 forms the "essential core" required for high affinity and high selectivity of analogues of peptide 1 at hMC-4R, but the "extended core", His(6)-D-(2')Nal(7)-Arg(8)-Trp(9), is necessary for the maximum affinity for hMC-3R and hMC-5R.
Assuntos
Peptídeos Cíclicos/síntese química , Receptores da Corticotropina/antagonistas & inibidores , alfa-MSH/metabolismo , Animais , Ligação Competitiva , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , AMP Cíclico/metabolismo , Humanos , Conformação Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/metabolismo , Receptores de Melanocortina , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Human melanin-concentrating hormone (hMCH) is a potent but nonselective agonist at human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R, respectively). To determine the structural features of this neuropeptide which are necessary for efficient binding to and activation of the receptors, Ala-substituted, open-chain, and truncated analogues were synthesized and tested in the binding assays in CHO cells expressing hMCH-1R and hMCH-2R, and in functional assays measuring the level of intracellular calcium mobilization in human HEK-293 cells expressing these receptors. A compound consisting merely of the cyclic core of hMCH with the Arg attached to the N-terminus of the disulfide ring was found to activate both hMCH-1R and hMCH-2R about as effectively as full-length hMCH. Thus, the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) appears to constitute the "active core" that is necessary for agonist potency at hMCH-1R and hMCH-2R. A potent and approximately 4-fold more selective agonist at hMCH-1R than at hMCH-2R is also reported.
Assuntos
Hormônios Hipotalâmicos/química , Hormônios Hipotalâmicos/fisiologia , Melaninas/química , Melaninas/fisiologia , Fragmentos de Peptídeos/fisiologia , Hormônios Hipofisários/química , Hormônios Hipofisários/fisiologia , Receptores do Hormônio Hipofisário/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Hormônios Hipotalâmicos/metabolismo , Isomerismo , Melaninas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/fisiologia , Hormônios Hipofisários/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/agonistasRESUMO
alpha-Melanotropin (alphaMSH) and several of its derivatives are potent but not selective agonists at melanocortin receptors 3, 4, and 5 present in the brain (MC3-5R). To differentiate between the physiological role of hMC-4R (believed to be involved in regulation of energy balance) from those of melanocortin receptors 3 and 5, potent and receptor-specific agonists are needed. Therefore, the cyclic derivatives of alphaMSH of a general structure, cyclo(X-His-d-Phe-Arg-Trp-Y)-NH(2), where X is succinic acid or an omega-amino-carboxylic acid, and Y is an alpha,omega-di-amino-carboxylic acid or an omega-carboxy-alpha-amino acid, were prepared and tested in binding assays and in cAMP assays on CHO cells expressing hMC3-5R. Several of the 21-membered or larger lactams turned out to be potent and hMC-4R-selective agonists. For instance, cyclo(CO-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Dab)-NH(2) (Dab: 2,4-di-amino-butyric acid) was a potent agonist at hMC-4R (EC(50) = 4 nM) with 55-fold selectivity over hMC-3R and greater than 1000-fold selectivity over hMC-5R. Another potent and selective compound was cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2): EC(50) about 1 nM at hMC-4R, with 90-fold selectivity over hMC-3R and greater than 2000-fold selectivity over hMC-5R.
Assuntos
Receptores de Peptídeos/metabolismo , alfa-MSH/agonistas , Animais , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Peptídeos/síntese química , Peptídeos/farmacologia , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/metabolismo , Receptores de MelanocortinaRESUMO
A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4'-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.
Assuntos
Cálcio/metabolismo , Indóis/metabolismo , Hormônios Peptídicos , Peptídeos/metabolismo , Piperidinas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Compostos de Espiro/metabolismo , Sítios de Ligação/fisiologia , Células Cultivadas , Grelina , Humanos , Indóis/química , Concentração Inibidora 50 , Luminescência , Mimetismo Molecular , Peptídeos/química , Piperidinas/síntese química , Ligação Proteica/fisiologia , Receptores de Grelina , Compostos de Espiro/químicaRESUMO
Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.
Assuntos
Encéfalo/metabolismo , Cromossomos Humanos Par 22 , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Receptores do Hormônio Hipofisário/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Células COS , Membrana Celular/fisiologia , Chlorocebus aethiops , Mapeamento Cromossômico , Cricetinae , Feminino , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Oncorhynchus keta , Especificidade de Órgãos , Hipófise/química , Hipófise/fisiologia , Ensaio Radioligante , Receptores Acoplados a Proteínas G , Receptores do Hormônio Hipofisário/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
To study the effect of NPY deletion on the regulation of its receptors in the NPY knockout (NPY KO) mice, the expression and binding of NPY receptors were investigated by in situ hybridization and receptor autoradiography using (125)I-[Leu(31),Pro(34)]PYY and (125)I-PYY(3-36) as radioligands. A 6-fold increase in Y2 receptor mRNA was observed in the CA1 region of the hippocampus in NPY KO mice, but a significant change could not be detected for Y1, Y4, Y5 and y6 receptors. Receptor binding reveals a 60-400% increase of Y2 receptor binding in multiple brain areas. A similar increase in Y1 receptor binding was seen only in the hypothalamus. These results demonstrate the NPY receptor expression is altered in mice deficient for its natural ligand.
Assuntos
Regulação da Expressão Gênica , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Regulação para Cima , Animais , Ligação Competitiva , Encéfalo/metabolismo , Membrana Celular/metabolismo , Hipocampo/metabolismo , Hipotálamo , Hibridização In Situ , Ligantes , Masculino , Camundongos , Camundongos Knockout , Ligação Proteica , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.
Assuntos
Receptores Odorantes/farmacologia , Receptores Odorantes/fisiologia , Animais , Humanos , Ligantes , Receptores Odorantes/isolamento & purificação , Transdução de SinaisRESUMO
Neuromedin U (NMU) is a family of peptides found in the gut and the central nervous system [Neuroscience 25 (1988) 797; Biochem. Biophys. Res. Commun. 130 (1985) 1078]. While several peripheral activities such as uterus stimulating and hypertensive effects have been described for NMU [Biochem. Biophys. Res. Commun. 130 (1985) 1078], its role in the CNS remains poorly understood. Recently, we reported the identification of two receptors for NMU (NMU1R and NMU2R), and demonstrated that NMU may play a role in regulating feeding behavior. The central effect of NMU is likely mediated primarily via NMU2R, since NMU1R is detectable only in the periphery, but not in the brain [Nature 406 (2000) 70]. In this report, we describe detailed mapping of NMU2R mRNA expression in the rat brain by in situ hybridization. The most intense signals were observed in the ependymal cell layer along the wall of the third ventricle in the hypothalamus, CA1 region of the hippocampus, indusium griseum and septohippocampal nucleus. Moderate expression was detected in the hypothalamic paraventricular nucleus, dorsal raphe nucleus as well as a number of other brain structures. The presence of NMU2R in the hypothalamus is consistent with its role in energy balance. Significant levels of expression of NMU2R elsewhere in the brain may suggest additional physiological functions for this neuropeptide.
RESUMO
To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.
Assuntos
Receptores de Peptídeos/metabolismo , alfa-MSH/análogos & derivados , Proteína Relacionada com Agouti , Sequência de Aminoácidos , Aminoácidos Cíclicos/genética , Sítios de Ligação , Cisteína/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo , Receptor Tipo 4 de Melanocortina , Receptores de Peptídeos/genética , Proteínas Recombinantes/metabolismo , Treonina/genética , alfa-MSH/química , alfa-MSH/metabolismoRESUMO
The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.
Assuntos
Hormônios Peptídicos , Peptídeos/química , Receptores de Superfície Celular/agonistas , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Grelina , Humanos , Medições Luminescentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Relação Estrutura-AtividadeRESUMO
The growth hormone secretagogue receptor (GHS-R) is involved in the regulation of pulsatile GH release. However, until recently, natural endogenous ligands for the receptor were unknown. We fractionated porcine hypothalamic extracts and assayed fractions for activity on HEK293 cells expressing GHS-R and aequorin. A partial agonist was isolated and identified using microspray tandem mass spectrometry as adenosine. GHS-R activation by adenosine and synthetic adenosine agonists is inhibited by the GHS-R selective antagonists L-765,867, D-Lys(3)-GHRP-6, and by theophylline and XAC. Cross desensitization of the GHS-R occurs with both MK-0677 and adenosine. Ligand binding and site directed mutagenesis studies show that adenosine binds to a binding site that is distinct from the previously characterized MK-0677 and GHRP-6 binding pocket. We propose, that adenosine is a physiologically important endogenous GHS-R ligand and speculate that GHS-R ligands modulate dopamine release from hypothalamic neurons.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Receptores de Superfície Celular/agonistas , Receptores Acoplados a Proteínas G , Adenosina/antagonistas & inibidores , Adenosina/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Equorina/metabolismo , Animais , Sítios de Ligação , Extratos Celulares , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Dopamina/metabolismo , Humanos , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Indóis/farmacologia , Ligantes , Medições Luminescentes , Espectrometria de Massas , Modelos Biológicos , Mutagênese Sítio-Dirigida , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Grelina , Compostos de Espiro/farmacologia , Suínos , Teofilina/farmacologiaRESUMO
Genetic and pharmacological studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiological function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r(-/-)) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-month Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. (Feed efficiency is the ratio of weight gain to food intake.) Consistent with increased fat mass, Mc3r(-/-) mice are hyperleptinaemic and male Mc3r(-/-) mice develop mild hyperinsulinaemia. Mc3r(-/-) mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r(-/-) mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.
Assuntos
Tecido Adiposo/metabolismo , Peso Corporal , Receptores da Corticotropina/genética , Receptores da Corticotropina/fisiologia , Fatores Etários , Animais , Southern Blotting , Temperatura Corporal , Calorimetria , Corticosterona/biossíntese , Comportamento Alimentar , Feminino , Genótipo , Glucose/biossíntese , Humanos , Hiperinsulinismo/genética , Hibridização In Situ , Insulina/biossíntese , Leptina/biossíntese , Masculino , Camundongos , Camundongos Knockout , Modelos Genéticos , Atividade Motora , Obesidade/genética , Oligopeptídeos/farmacologia , Fenótipo , Isoformas de Proteínas , Receptor Tipo 3 de Melanocortina , Receptor Tipo 4 de Melanocortina , Receptores da Corticotropina/química , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Recombinação Genética , Tiroxina/biossíntese , Fatores de Tempo , Distribuição Tecidual , alfa-MSH/análogos & derivadosRESUMO
Recent developments in the quest for control of human obesity include the discovery of hormones, neuropeptides, receptors and transcription factors involved in feeding behavior, metabolic rate and adipocyte development. As a result, obesity research is quickly developing a level of sophistication that is expected to yield new treatment approaches. Even though newly approved clinical interventions are being tested in the market place, the obesity epidemic continues to face numerous unmet clinical needs and awaits the development and implementation of safe and highly effective pharmacotherapy.