RESUMEN
Orthogonal protection of amino acid side chains in solid phase peptide synthesis allows for selective deprotection of side chains and the formation of cyclic peptides on resin. Cyclizations are useful as they may improve the activity of the peptide or improve the metabolic stability of peptides in vivo. One cyclization method often used is the formation of a lactam bridge between an amine and a carboxylic acid. It is desirable to perform the cyclization on resin as opposed to in solution to avoid unwanted side reactions; therefore, a common strategy is to use -Alloc and -OAllyl protecting groups as they are compatible with Fmoc solid phase peptide synthesis conditions. Alloc and -OAllyl may be removed using Pd(PPh3 )4 and phenylsilane in DMF. This method can be problematic as the reaction is most often performed at room temperature under argon gas. It is not usually done at higher temperatures because of the fear of poisoning the palladium catalyst. As a result, the reaction is long and reagent-intensive. Herein, we report the development of a method in which the -Alloc/-OAllyl groups are removed using a microwave synthesizer under atmospheric conditions. The reaction is much faster, allowing for the removal of the protecting groups before the catalyst is oxidized, as well as being less reagent-intensive. This method of deprotection was tested using a variety of amino acid sequences and side chain protecting groups, and it was found that after two 5-min deprotections at 38°C, all -Alloc and -OAllyl groups were removed with >98% purity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Fluorenos/química , Péptidos Cíclicos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Secuencia de Aminoácidos , Ácidos Carboxílicos/química , Catálisis , Ciclización , Ésteres , Lactamas/química , Microondas , Paladio/química , Péptidos Cíclicos/aislamiento & purificación , Silanos/químicaRESUMEN
Deltorphins are naturally occurring peptides produced by the skin of the giant monkey frog (Phyllomedusa bicolor). They are δ-opioid receptor-selective agonists. Herein, we report the design and synthesis of a peptide, Tyr-d-Ala-(pI)Phe-Glu-Ile-Ile-Gly-NH2 3 (GATE3-8), based on the [d-Ala(2)]deltorphin II template, which is δ-selective in in vitro radioligand binding assays over the µ- and κ-opioid receptors. It is a full agonist in [(35)S]GTPγS functional assays and analgesic when administered supraspinally to mice. Analgesia of 3 (GATE3-8) is blocked by the selective δ receptor antagonist naltrindole, indicating that the analgesic action of 3 is mediated by the δ-opioid receptor. We have established a radioligand in which (125)I is incorporated into 3 (GATE3-8). The radioligand has a KD of 0.1 nM in Chinese hamster ovary (CHO) cells expressing the δ receptor. Additionally, a series of peptides based on 3 (GATE3-8) was synthesized by incorporating various halogens in the para position on the aromatic ring of Phe(3). The peptides were characterized for binding affinity at the µ-, δ-, and κ-opioid receptors, which showed a linear correlation between binding affinity and the size of the halogen substituent. These peptides may be interesting tools for probing δ-opioid receptor pharmacology.
Asunto(s)
Analgésicos Opioides/síntesis química , Analgésicos Opioides/farmacología , Péptidos/síntesis química , Péptidos/farmacología , Receptores Opioides delta/metabolismo , Analgésicos Opioides/química , Animales , Células CHO , Cromatografía Líquida de Alta Presión , Cricetulus , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Halogenación , Masculino , Ratones , Oligopéptidos/química , Dolor/tratamiento farmacológico , Dolor/metabolismo , Péptidos/química , Ensayo de Unión Radioligante , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismoRESUMEN
The melanocortin 2 (MC(2)) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC(2) receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC(2) receptor responsible for these characteristics, we created chimeras between MC(2) and MC(4) receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC(2) but not MC(4) receptors. Replacing the first transmembrane segment of the MC(2) receptor with the corresponding region from the MC(4) receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC(4) receptor with the corresponding regions from the MC(2) receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC(2) receptors with MC(4) sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC(2) receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC(2) receptor as important for MRAP dependent-signal transduction and ligand specificity.
Asunto(s)
Proteínas de la Membrana/metabolismo , Receptor de Melanocortina Tipo 2/química , Receptor de Melanocortina Tipo 2/metabolismo , Receptor de Melanocortina Tipo 4/química , Receptor de Melanocortina Tipo 4/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Ligandos , Ratones , Transporte de Proteínas , Receptor de Melanocortina Tipo 2/genética , Receptor de Melanocortina Tipo 4/genética , Proteínas Recombinantes de Fusión/genética , Transducción de Señal , Especificidad por SustratoRESUMEN
The melanocortin system consists of five seven-transmembrane spanning G-protein coupled receptors (MC1-5) that are stimulated by endogenous agonists and antagonized by the only two known endogenous antagonists of GPCRs, agouti and agouti-related protein (AGRP). These receptors have been associated with many physiological functions, including the involvement of the MC4R in feeding behavior and energy homeostasis, making this system an attractive target for the treatment of obesity. Small-molecule mimetics of endogenous ligands may result in the development of compounds with properties more suitable for use as therapeutic agents. The research presented herein involves the synthesis and analysis of 12 melanocortin receptor agonists using the 1,4-benzodiazepine-2,5-dione template and is the first report of these derivatives as melanocortin receptor agonists. Structure-activity relationship studies using this privileged structure template has resulted in molecules with molecular weights around 400 that possess nanomolar agonist potency at the melanocortin receptors examined in this study.
Asunto(s)
Benzodiazepinas/síntesis química , Receptores de Melanocortina/agonistas , Benzodiazepinas/química , Benzodiazepinas/farmacología , Línea Celular , Genes Reporteros , Humanos , Ligandos , Relación Estructura-Actividad , beta-Galactosidasa/genéticaRESUMEN
A novel microwave-assisted solid-phase peptide synthesis utilizing N-Fmoc-protected(alpha-aminoacyl)benzotriazoles was applied in the preparation of tri-, tetra-, penta-, hexa-, and heptapeptides in 71% average crude yield.
Asunto(s)
Microondas , Péptidos/química , Péptidos/síntesis química , Triazoles/química , Triazoles/síntesis química , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Modelos Moleculares , Conformación MolecularRESUMEN
The melanocortin receptor system consists of endogenous agonists, antagonists, G-protein coupled receptors, and auxiliary proteins that are involved in the regulation of complex physiological functions such as energy and weight homeostasis, feeding behavior, inflammation, sexual function, pigmentation, and exocrine gland function. Herein, we report the structure-activity relationship (SAR) of a new chimeric hAGRP-melanocortin agonist peptide template Tyr-c[beta-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) that was characterized using amino acids previously reported in other melanocortin agonist templates. Twenty peptides were examined in this study, and six peptides were selected for (1)H NMR and computer-assisted molecular modeling structural analysis. The most notable results include the identification that modification of the chimeric template at the His position with Pro and Phe resulted in ligands that were nM mouse melanocortin-3 receptor (mMC3R) antagonists and nM mouse melanocortin-4 receptor (mMC4R) agonists. The peptides Tyr-c[beta-Asp-His-DPhe-Ala-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) and Tyr-c[beta-Asp-His-DNal(1')-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) resulted in 730- and 560-fold, respectively, mMC4R versus mMC3R selective agonists that also possessed nM agonist potency at the mMC1R and mMC5R. Structural studies identified a reverse turn occurring in the His-DPhe-Arg-Trp domain, with subtle differences observed that may account for the differences in melanocortin receptor pharmacology. Specifically, a gamma-turn secondary structure involving the DPhe(4) in the central position of the Tyr-c[beta-Asp-Phe-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH(2) peptide may differentiate the mixed mMC3R antagonist and mMC4R agonist pharmacology.
Asunto(s)
Oligopéptidos/síntesis química , Péptidos Cíclicos/síntesis química , Proteínas/química , Receptores de Melanocortina/agonistas , Receptores de Melanocortina/antagonistas & inhibidores , alfa-MSH/química , Proteína Relacionada con Agouti , Animales , Unión Competitiva , Línea Celular , Diseño de Fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular , Espectroscopía de Resonancia Magnética , Ratones , Modelos Moleculares , Oligopéptidos/química , Oligopéptidos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Estructura Secundaria de Proteína , Receptor de Melanocortina Tipo 3/antagonistas & inhibidores , Receptor de Melanocortina Tipo 4/agonistas , Relación Estructura-ActividadRESUMEN
The melanocortin pathway consists of endogenous agonists, antagonists, G-protein coupled receptors (GPCRs), and auxiliary proteins. This pathway has been identified to participate physiologically in numerous biological pathways including energy homeostasis, pigmentation, sexual function, inflammation, cardiovascular function, adrenal function, sebaceous gland lipid production, just to list a few. During this past decade, a clear link between the melanocortin-4 receptor (MC4R) and obesity, in both mice and humans via the regulation of food intake and energy homeostasis, has made this pathway the target of many academic and industrial research endeavors in attempts to develop potent and selective MC4R small molecules as anti-obesity therapeutic agents. Herein, we attempt to summarize the known proteins that constitute the melanocortin system and discuss advances in peptide and non-peptide drug discovery.