RESUMO
BACKGROUND: Apelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. METHODS: [Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1â¯nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100µM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and ß-arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. RESULTS: In both species, [Pyr1]apelin-13â¯had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting ß-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and ß-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the ß-arrestin assay but was more potent in the cAMP assay. CONCLUSIONS: Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.
Assuntos
Receptores de Apelina/metabolismo , Apelina/metabolismo , Ventrículos do Coração/metabolismo , Peptídeos/metabolismo , beta-Arrestinas/metabolismo , Adulto , Animais , Apelina/química , Apelina/farmacologia , Receptores de Apelina/química , Ligação Competitiva , Células CHO , Colforsina/farmacologia , Misturas Complexas/química , Misturas Complexas/metabolismo , Cricetulus , AMP Cíclico/metabolismo , Feminino , Ventrículos do Coração/química , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Peptídeos/síntese química , Peptídeos/farmacologia , Ligação Proteica , Ratos , Ratos Sprague-DawleyRESUMO
Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.
Assuntos
Hepcidinas/metabolismo , Receptores de Grelina/antagonistas & inibidores , Animais , Cirurgia Bariátrica , Restrição Calórica , Ingestão de Alimentos , Jejum , Feminino , Grelina/antagonistas & inibidores , Grelina/metabolismo , Hormônio do Crescimento/metabolismo , Humanos , Intestino Delgado/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Ligação Proteica , Ratos , Receptores de Grelina/metabolismoRESUMO
Because nonalcoholic steatohepatitis (NASH) is associated with impaired liver regeneration, we investigated the effects of G49, a dual glucagon-like peptide-1/glucagon receptor agonist, on NASH and hepatic regeneration. C57Bl/6 mice fed chow or a methionine and choline-deficient (MCD) diet for 1 week were divided into 4 groups: control (chow diet), MCD diet, chow diet plus G49, and M+G49 (MCD diet plus G49). Mice fed a high-fat diet (HFD) for 10 weeks were divided into groups: HFD and H+G49 (HFD plus G49). Following 2 (MCD groups) or 3 (HFD groups) weeks of treatment with G49, partial hepatectomy (PH) was performed, and all mice were maintained on the same treatment schedule for 2 additional weeks. Analysis of liver function, hepatic regeneration, and comprehensive genomic and metabolic profiling were conducted. NASH was ameliorated in the M+G49 group, manifested by reduced inflammation, steatosis, oxidative stress, and apoptosis and increased mitochondrial biogenesis. G49 treatment was also associated with replenishment of intrahepatic glucose due to enhanced gluconeogenesis and reduced glucose use through the pentose phosphate cycle and oxidative metabolism. Following PH, G49 treatment increased survival, restored the cytokine-mediated priming phase, and enhanced the proliferative capacity and hepatic regeneration ratio in mice on the MCD diet. NASH markers remained decreased in M+G49 mice after PH, and glucose use was shifted to the pentose phosphate cycle and oxidative metabolism. G49 administered immediately after PH was also effective at alleviating the pathological changes induced by the MCD diet. Benefits in terms of liver regeneration were also found in mice fed HFD and treated with G49. CONCLUSION: Dual-acting glucagon-like peptide-1/glucagon receptor agonists such as G49 represent a novel therapeutic approach for patients with NASH and particularly those requiring PH. (Hepatology 2017;65:950-968).
Assuntos
Peptídeo 1 Semelhante ao Glucagon/antagonistas & inibidores , Regeneração Hepática/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores de Glucagon/antagonistas & inibidores , Animais , Biópsia por Agulha , Modelos Animais de Doenças , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Humanos , Imuno-Histoquímica , Peroxidação de Lipídeos , Regeneração Hepática/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Estresse Oxidativo , Distribuição Aleatória , Receptores de Glucagon/administração & dosagem , Resultado do TratamentoRESUMO
Apelin-36 was discovered as the endogenous ligand for the previously orphan receptor APJ. Apelin-36 has been linked to two major types of biological activities: cardiovascular (stimulation of cardiac contractility and suppression of blood pressure) and metabolic (improving glucose homeostasis and lowering body weight). It has been assumed that both of these activities are modulated through APJ. Here, we demonstrate that the metabolic activity of apelin-36 can be separated from canonical APJ activation. We developed a series of apelin-36 variants in which evolutionarily conserved residues were mutated, and evaluated their ability to modulate glucose homeostasis and body weight in chronic mouse models. We found that apelin-36(L28A) retains full metabolic activity, but is 100-fold impaired in its ability to activate APJ. In contrast to its full metabolic activity, apelin-36(L28A) lost the ability to suppress blood pressure in spontaneously hypertensive rats (SHR). We took advantage of these findings to develop a longer-acting variant of apelin-36 that could modulate glucose homeostasis without impacting blood pressure (or activating APJ). Apelin-36-[L28C(30kDa-PEG)] is 10,000-fold less potent than apelin-36 at activating the APJ receptor but retains its ability to significantly lower blood glucose and improve glucose tolerance in diet-induced obese mice. Apelin-36-[L28C(30kDa-PEG)] provides a starting point for the development of diabetes therapeutics that are devoid of the blood pressure effects associated with canonical APJ activation.
Assuntos
Adipocinas/farmacologia , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apelina , Receptores de Apelina , Pressão Sanguínea/efeitos dos fármacos , Camundongos , Ratos , Ratos Endogâmicos SHRRESUMO
Synthetic and natural cyclic peptides provide a testing ground for studying membrane permeability in nontraditional drug scaffolds. Cyclic peptomers, which incorporate peptide and N-alkylglycine (peptoid) residues, combine the stereochemical and geometric complexity of peptides with the functional group diversity accessible to peptoids. We synthesized cyclic peptomer libraries by split-pool techniques, separately permuting side chain and backbone geometry, and analyzed their membrane permeabilities using the parallel artificial membrane permeability assay. Nearly half of the side chain permutations had permeability coefficients (Papp) > 1 × 10-6 cm/s. Some backbone geometries enhanced permeability due to their ability to form more stable intramolecular hydrogen bond networks compared with other scaffolds. These observations suggest that hexameric cyclic peptomers can have good passive permeability even in the context of extensive side chain and backbone variation, and that high permeability can generally be achieved within a relatively wide lipophilicity range.
Assuntos
Permeabilidade da Membrana Celular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacocinética , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologiaRESUMO
It is well established that intramolecular hydrogen bonding and N-methylation play important roles in the passive permeability of cyclic peptides, but other structural features have been explored less intensively. Recent studies on the oral bioavailability of the cyclic heptapeptide sanguinamide A have raised the question of whether steric occlusion of polar groups via ß-branching is an effective, yet untapped, tool in cyclic peptide permeability optimization. We report the structures of 17 sanguinamide A analogues designed to test the relative contributions of ß-branching, N-methylation, and side chain size to passive membrane permeability and aqueous solubility. We demonstrate that ß-branching has little effect on permeability compared to the effects of aliphatic carbon count and N-methylation of exposed NH groups. We highlight a new N-methylated analogue of sanguinamide A with a Leu substitution at position 2 that exhibits solvent-dependent flexibility and improved permeability over that of the natural product.
Assuntos
Peptídeos Cíclicos/química , Tiazóis/química , Células CACO-2 , Humanos , Membranas Artificiais , Metilação , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/metabolismo , Permeabilidade , Solubilidade , Relação Estrutura-Atividade , Tiazóis/metabolismo , ÁguaRESUMO
Drug design efforts are turning to a new generation of therapeutic targets, such as protein-protein interactions (PPIs), that had previously been considered "undruggable" by typical small molecules. There is an emerging view that accessing these targets will require molecules that are larger and more complex than typical small molecule drugs. Here, we present a methodology for the discovery of geometrically diverse, membrane permeable cyclic peptide scaffolds based on the synthesis and permeability screening of a combinatorial library, followed by deconvolution of membrane-permeable scaffolds to identify cyclic peptides with good to excellent passive cell permeabilities. We use a combination of experimental and computational approaches to investigate structure-permeability relationships in one of these scaffolds, and uncover structural and conformational factors that govern passive membrane diffusion in a related set of cyclic peptide diastereomers. Further, we investigate the dependency of permeability on side-chain identity of one of these scaffolds through single-point diversifications to show the adaptability of these scaffolds toward development of permeability-biased libraries suitable for bioactivity screens. Overall, our results demonstrate that many novel, cell permeable scaffolds exist beyond those found in extant natural products, and that such scaffolds can be rapidly identified using a combination of synthesis and deconvolution which can, in principle, be applied to any type of macrocyclic template.
Assuntos
Produtos Biológicos , Biomimética , Permeabilidade da Membrana Celular , Técnicas de Química Combinatória , Peptídeos Cíclicos/metabolismo , Células CACO-2 , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
The spider venom peptide Huwentoxin-IV (HwTx-IV) 1 is a potent antagonist of hNav1.7 (IC50 determined herein as 17 ± 2 nM). Nav1.7 is a voltage-gated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. We prepared a number of HwTx-IV analogs as part of a structure-function study into Nav1.7 antagonism. The inhibitory potency of these analogs was determined by automated electrophysiology and is reported herein. In particular, the native residues Glu(1), Glu(4), Phe(6) and Tyr(33) were revealed as important activity modulators and several peptides bearing mutations in these positions showed significantly increased potency on hNav1.7 while maintaining the original selectivity profile of the wild-type peptide 1 on hNav1.5. Peptide 47 (Gly(1), Gly(4), Trp(33)-HwTx) demonstrated the largest potency increase on hNav1.7 (IC50 0.4 ± 0.1 nM).
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Venenos de Aranha/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Venenos de Aranha/síntese química , Venenos de Aranha/química , Aranhas , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/síntese química , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
Melanin-concentrating hormone (MCH) is an important neuropeptide hormone involved in multiple physiological processes. Peptide derivatives of MCH have been developed as tools to aid research including potent radioligands, receptor selective agonists, and potent antagonists. These tools have been used to further understand the role of MCH in physiology, primarily in rodents. However, the tools could also help elucidate the role for MCHR1 and MCHR2 in mediating MCH signaling in higher species.
Assuntos
Hormônios Hipotalâmicos/metabolismo , Melaninas/metabolismo , Peptídeos/metabolismo , Hormônios Hipofisários/metabolismo , Receptores do Hormônio Hipofisário/agonistas , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Humanos , Hormônios Hipotalâmicos/química , Melaninas/química , Dados de Sequência Molecular , Peptídeos/química , Hormônios Hipofisários/química , Salmão/metabolismoRESUMO
Blockade of brain melanin-concentrating hormone 1 receptor (MCH1R) significantly ameliorates fatty liver as well as obesity. However, the mode of action of this effect is unknown. This study examined the effect of a MCH1R antagonist in murine steatohepatitis models with and without obesity and clarified whether these pharmacological effects were attributed to anti-obesity effects. Steatohepatitis with concomitant obese phenotypes was developed after 52-week exposure to a high-fat diet, and steatohepatitis with reduced body weight was developed by exposure to a methionine- and choline-deficient diet for 10 days. Chronic intracerebroventricular infusion of a peptidic MCH1R antagonist reduced hepatic triglyceride contents and ameliorated steatohepatitis on histological observations in both mice models. Improvement of steatohepatitis was concomitant with amelioration of obese phenotypes such as hyperinsulinemia and hyperleptinemia in the case of the obese model, whereas body weight reduction was not associated with amelioration of steatohepatitis by the antagonist in the lean model. Reduction of hepatic gene expressions encoding cytochromes P450 4A was identified by treatment with the antagonist in both the obese and lean models. These results suggest that brain blockade of MCH1R could alleviate steatohepatitis independently from anti-obesity effects. In conclusion, MCH1R antagonist could have a new therapeutic potential for the treatment of human nonalcoholic steatohepatitis.
Assuntos
Gorduras na Dieta/farmacologia , Receptores do Hormônio Hipofisário/antagonistas & inibidores , Animais , Peso Corporal/efeitos dos fármacos , Quimiocina CCL2/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Modelos Animais de Doenças , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/induzido quimicamente , Obesidade/fisiopatologia , Reação em Cadeia da Polimerase , Triglicerídeos/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
Alpha-melanotropin (alphaMSH), Ac-Ser1-Tyr2-Ser3-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2,(1) has been long recognized as an important physiological regulator of skin and hair pigmentation in mammals. Binding of this peptide to the melanocortin receptor 1 (MC1R) leads to activation of tyrosinase, the key enzyme of the melanin biosynthesis pathway. In this study, interactions of the human MC1bR (an isoform of the receptor 1a) with the synthetic cyclic analogs of alphaMSH were studied. These ligands were analogs of MTII, Ac-Nle4-cyclo-(Asp5-His6-D-Phe7-Arg8-Trp9-Lys10)-NH2, a potent pan-agonist at the human melanocortin receptors (hMC1,3-5R). In the structure of MTII, the His6-D-Phe7-Arg8-Trp9 segment has been recognized as "essential" for molecular recognition at the human melanocortin receptors (hMC1,3-5R). Herein, the role of the Trp9 in the ligand interactions with the hMC1b,3-5R has been reevaluated. Analogs with various amino acids in place of Trp9 were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4 and 5 (hMC1b,3-5R). Several of the new peptides were high potency agonists (partial) at hMC1bR (EC50 from 0.5 to 20 nM) and largely inactive at hMC3-5R. The bulky aromatic side chain in position 9, such as that in Trp, was found not to be essential to agonism (partial) of the studied peptides at hMC1bR.
Assuntos
Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Receptor Tipo 1 de Melanocortina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/farmacologia , Ligação Competitiva , AMP Cíclico/análise , AMP Cíclico/biossíntese , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/metabolismo , Receptor Tipo 1 de Melanocortina/química , Receptor Tipo 1 de Melanocortina/classificação , Sensibilidade e Especificidade , Relação Estrutura-Atividade , alfa-MSH/síntese química , alfa-MSH/química , alfa-MSH/isolamento & purificação , alfa-MSH/metabolismoRESUMO
alpha-Melanocyte stimulating hormone (alphaMSH), Ac-Ser(1)-Tyr(2)-Ser(3)-Met(4)-Glu(5)-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), is an endogenous agonist for the melanocortin receptor 1 (MC1R), the receptor found in the skin, several types of immune cells, and other peripheral sites. Three-dimensional models of complexes of this receptor with alphaMSH and its synthetic analog NDP-alphaMSH, Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH(2), have been previously proposed. In those models, the 6-9 segment of the ligand was considered essential for the ligand-receptor interactions. In this study, we probed the role of Trp(9) of NDP-alphaMSH in interactions with hMC1bR. Analogs of NDP-alphaMSH with various amino acids in place of Trp(9) were synthesized and tested in vitro in receptor affinity binding and cAMP functional assays at human melanocortin receptors 1b, 3, 4, and 5 (hMC1b,3-5R). Several new compounds displayed high agonist potency at hMC1bR (EC(50) = 0.5-5 nM) and receptor subtype selectivity greater than 2000-fold versus hMC3-5R. The Trp(9) residue of NDP-alphaMSH was determined to be not essential for molecular recognition at hMC1bR.
Assuntos
Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 1 de Melanocortina/química , Triptofano/fisiologia , alfa-MSH/química , alfa-MSH/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva/efeitos dos fármacos , Humanos , Ligantes , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacologia , Receptor Tipo 1 de Melanocortina/classificação , Relação Estrutura-Atividade , alfa-MSH/análogos & derivadosRESUMO
The melanin-concentrating hormone receptor 1 (MCH-1R) has been recognized as a receptor which mediates effects of the endogenous melanin-concentrating hormone (MCH) on appetite and body weight gain in rodents. In the last several years, a number of hMCH analogs have been designed which were potent and selective ligands for hMCH-1R. These peptidic agonists and antagonists have served as research tools in animal studies that showed a key role of the MCH-1R in the development of obesity and proved that MCH-1R antagonism can produce anti-obesity effects in rodents.
Assuntos
Peptídeos/química , Peptídeos/metabolismo , Receptores do Hormônio Hipofisário/classificação , Receptores do Hormônio Hipofisário/metabolismo , Animais , Humanos , Hormônios Hipotalâmicos/química , Hormônios Hipotalâmicos/metabolismo , Ligantes , Melaninas/química , Melaninas/metabolismo , Peptídeos/farmacologia , Hormônios Hipofisários/química , Hormônios Hipofisários/metabolismo , Receptores do Hormônio Hipofisário/agonistas , Receptores do Hormônio Hipofisário/antagonistas & inibidoresRESUMO
Melanocortin receptors (MC1-5R) and their endogenous ligands (melanocyte-stimulating hormones and adrenocorticotropic hormone) are involved in many physiological processes in humans. Of those receptors, the actions of MC5R are the least understood despite its broad presence in the numerous peripheral tissues and brain. In this study, we describe synthesis and pharmacological properties in vitro (receptor-binding affinity and agonist activity) of several cyclic analogs of alphaMSH which are potent agonists at hMC5R (EC(50) below 1 nM) and of enhanced receptor subtype selectivity (more than 2000-fold versus hMC1b,3R and about 70- to 200-fold versus hMC4R). These compounds are analogs of Ac-Nle(4)-cyclo[Asp(5)-His(6)-D-Nal(2')(7)-Pip(8)-Trp(9)-Lys(10)]-NH(2) (Pip: pipecolic acid) in which His(6) has been replaced with sterically hindered amino acids. They may be useful tools in the elucidation of the MC5R role in skin disorders and in immunomodulatory and in anti-inflammatory actions of alphaMSH.
Assuntos
Receptores da Corticotropina/agonistas , alfa-MSH/agonistas , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Receptores de MelanocortinaRESUMO
The physiological role of melanocortin receptor 5 (MC5R) in humans is not clear despite its broad presence in various peripheral sites and in the brain, cortex, and cerebellum. To differentiate between functions of this receptor and those of the other melanocortin receptors (hMC1,3,4R), peptides with improved receptor subtype selectivity are needed. The endogenous ligands, melanocortins, and their various synthetic analogues are not particularly selective for hMC5R. In this study, cyclic peptides derived from MTII, Ac-Nle-cyclo(Asp-His6-D-Phe7-Arg8-Trp-Lys)-NH2 (a pan-agonist at the melanocortin receptors) were prepared and tested in binding and functional assays on CHO cells expressing hMC1b,3-5R. The analogues included in their structures sterically constrained hydrophobic amino acids in positions 6 (His) and 8 (Arg), and the D-4,4'-biphenyl residue in position 7 (D-Phe). Several of the new compounds were selective potent agonists at hMC5R. They are exemplified by peptide 29, Ac-Nle-cyclo(Asp-Oic6-D-4,4'-Bip7-Pip8-Trp-Lys)-NH2 (Oic=octahydroindole-2-COOH; 4,4'-Bip=4,4'-biphenylalanine; Pip=pipecolic acid) of IC50=0.95 nM and EC50=0.99 nM at hMC5R and selectivity for this receptor with respect to the other melanocortin receptors greater than 5000-fold.
Assuntos
Peptídeos Cíclicos/síntese química , Receptores da Corticotropina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/síntese química , Animais , Ligação Competitiva , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Humanos , Hormônios Estimuladores de Melanócitos/síntese química , Hormônios Estimuladores de Melanócitos/química , Hormônios Estimuladores de Melanócitos/farmacologia , Peptídeos Cíclicos/farmacologia , Ensaio Radioligante , Receptores de Melanocortina , Relação Estrutura-Atividade , alfa-MSH/farmacologiaRESUMO
Alpha-melanotropin, Ac-Ser(1)-Tyr-Ser-Met-Glu-His(6)-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2)(1), is a non-selective endogenous agonist for the melanocortin receptor 5; the receptor present in various peripheral tissues and in the brain, cortex and cerebellum. Most of the synthetic analogs of alphaMSH, including a broadly used and more potent the NDP-alphaMSH peptide, Ac-Ser(1)-Tyr-Ser-Nle(4)-Glu-His(6)-D-Phe(7)-Arg(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2), are also not particularly selective for MC5R. To elucidate physiological functions of the melanocortin receptor 5 in rodents and humans, the receptor subtype selective research tools are needed. We report herein syntheses and pharmacological evaluation in vitro of several analogs of NDP-alphaMSH which are highly potent and specific agonists for the human MC5R. The new linear peptides, of structures and solubility properties similar to those of the endogenous ligand alphaMSH, are exemplified by compound 7, Ac-Ser(1)-Tyr-Ser-Met-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9)-Gly-Lys-Pro-Val(13)-NH(2) (Oic: octahydroindole-2-COOH, 4,4'-Bip: 4,4'-biphenylalanine, Pip: pipecolic acid), shortly NODBP-alphaMSH, which has an IC(50)=0.74 nM (binding assay) and EC(50)=0.41 (cAMP production assay) at hMC5R nM and greater than 3500-fold selectivity with respect to the melanocortin receptors 1b, 3 and 4. A shorter peptide derived from NODBP-alphaMSH: Ac-Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8)-Trp(9) -NH(2) (17) was measured to be an agonist only 10-fold less potent at hMC5R than the full length parent peptide. In the structure of this smaller analog, the Nle-Glu-Oic(6)-D-4,4'-Bip(7)-Pip(8) segment was found to be critical for high agonist potency, while the C-terminal Trp(9) residue was shown to be required for high hMC5R selectivity versus hMC1b,3,4R.
Assuntos
Receptores da Corticotropina/metabolismo , alfa-MSH/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Receptores da Corticotropina/agonistas , Receptores da Corticotropina/genética , Receptores de Melanocortina , Relação Estrutura-Atividade , alfa-MSH/análogos & derivados , alfa-MSH/farmacologiaRESUMO
Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.
Assuntos
Venenos de Abelha/química , Radioisótopos do Iodo/química , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Sequência de Bases , Venenos de Abelha/isolamento & purificação , Venenos de Abelha/metabolismo , Venenos de Abelha/farmacologia , Fenômenos Fisiológicos Celulares , Células Cultivadas , Eletrofisiologia/métodos , Humanos , Rim/citologia , Rim/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Ligação Proteica , Ratos , Fatores de TempoRESUMO
Melanin-concentrating hormone (MCH) is a cyclic orexigenic peptide expressed in the lateral hypothalamus, which plays an important role in regulating energy balance. To elucidate the physiological role of MCH in obesity development, the present study examined the effect of a selective MCH1 receptor (MCH1R) antagonist in the diet-induced obesity mouse model. The MCH1R antagonist has high affinity and selectivity for MCH-1R and potently inhibits intracerebroventricularly injected MCH-induced food intake in Sprague Dawley rats. Chronic intracerebroventricular infusion of the MCH1R antagonist (7.5 microg/d) completely suppressed body weight gain in diet-induced obese mice during the treatment periods and significantly decreased cumulative food intake, by 14%. Carcass analysis showed that the MCH1R antagonist resulted in a selective decrease of body fat in the diet-induced obese mice. In addition, the MCH1R antagonist ameliorated the obesity-related hypercholesterolemia, hyperinsulinemia, hyperglycemia, and hyperleptinemia. These results indicate that MCH has a major role in the development of diet-induced obesity in mice and that a MCH1R antagonist might be a useful candidate as an antiobesity agent.