RESUMEN
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.
Asunto(s)
Fármacos Antiobesidad/farmacología , Imidazoles/farmacología , Obesidad/tratamiento farmacológico , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Administración Oral , Animales , Autorradiografía , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dexfenfluramina/farmacología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Agonismo Inverso de Drogas , Ingestión de Alimentos/efectos de los fármacos , Humanos , Imidazoles/administración & dosificación , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Unión Proteica , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB2/agonistasRESUMEN
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.
Asunto(s)
Apetito/fisiología , Neuropéptido Y/fisiología , Hormonas Peptídicas/fisiología , Proteínas/fisiología , Proteína Relacionada con Agouti , Animales , Apetito/efectos de los fármacos , Ghrelina , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Hormonas Peptídicas/farmacología , Receptor de Melanocortina Tipo 3/fisiología , Receptor de Melanocortina Tipo 4/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores de GhrelinaRESUMEN
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.
Asunto(s)
Lactamas/farmacología , Hormonas Estimuladoras de los Melanocitos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 3/efectos de los fármacos , Receptor de Melanocortina Tipo 4/agonistas , Receptores de Corticotropina/efectos de los fármacos , alfa-MSH/análogos & derivados , alfa-MSH/farmacología , Animales , Células CHO , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Diseño de Fármacos , Histidina/química , Humanos , Concentración 50 Inhibidora , Lactamas/síntesis química , Hormonas Estimuladoras de los Melanocitos/síntesis química , Prolina/química , Receptor de Melanocortina Tipo 3/agonistas , Receptor de Melanocortina Tipo 3/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/efectos de los fármacos , Receptores de Corticotropina/agonistas , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Melanocortina , Relación Estructura-Actividad , alfa-MSH/síntesis químicaRESUMEN
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.
Asunto(s)
Fragmentos de Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Receptores de Corticotropina/antagonistas & inhibidores , alfa-MSH/análogos & derivados , alfa-MSH/síntesis química , Animales , Células CHO , Cricetinae , AMP Cíclico/biosíntesis , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Melanocortina , Relación Estructura-Actividad , alfa-MSH/química , alfa-MSH/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas/análisis , Adulto , Proteína Relacionada con Agouti , Análisis de Varianza , Animales , Ingestión de Alimentos/fisiología , Ayuno/sangre , Humanos , Péptidos y Proteínas de Señalización Intercelular , Leptina/sangre , Masculino , Obesidad/sangre , Obesidad/fisiopatología , Ratas , Factores de TiempoRESUMEN
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.
Asunto(s)
Alanina/química , Receptores de Corticotropina/metabolismo , gamma-MSH/síntesis química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Datos de Secuencia Molecular , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Melanocortina , Relación Estructura-Actividad , gamma-MSH/química , gamma-MSH/metabolismoRESUMEN
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.