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1.
Peptides ; 131: 170347, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32569606

RESUMO

The peptide hormone adrenomedullin (ADM) consists of 52 amino acids and plays a pivotal role in the regulation of many physiological processes, particularly those of the cardiovascular and lymphatic system. Like calcitonin (CT), calcitonin gene-related peptide (CGRP), intermedin (IMD) and amylin (AMY), it belongs to the CT/CGRP family of peptide hormones, which despite their low little sequence identity share certain characteristic structural features as well as a complex multicomponent receptor system. ADM, IMD and CGRP exert their biological effects by activation of the calcitonin receptor-like receptor (CLR) as a complex with one of three receptor activity-modifying proteins (RAMP), which alter the ligand affinity. Selectivity within the receptor system is largely mediated by the amidated C-terminus of the peptide hormones, which bind to the extracellular domains of the receptors. This enables their N-terminus consisting of a disulfide-bonded ring structure and a helical segment to bind within the transmembrane region and to induce an active receptor confirmation. ADM is expressed in a variety of tissues in the human body and is fundamentally involved in multitude biological processes. Thus, it is of interest as a diagnostic marker and a promising candidate for therapeutic interventions. In order to fully exploit the potential of ADM, it is necessary to improve its pharmacological profile by increasing the metabolic stability and, ideally, creating receptor subtype-selective analogs. While several successful attempts to prolong the half-life of ADM were recently reported, improving or even retaining receptor selectivity remains challenging.

2.
J Med Chem ; 63(5): 2358-2371, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-31589041

RESUMO

G-protein-coupled receptors like the human Y1 receptor (hY1R) are promising targets in cancer therapy due to their high overexpression on cancer cells and their ability to internalize together with the bound ligand. This mechanism was exploited to shuttle boron atoms into cancer cells for the application of boron neutron capture therapy (BNCT), a noninvasive approach to eliminate cancer cells. A maximized number of carboranes was introduced to the hY1R-preferring ligand [F7,P34]-NPY by solid phase peptide synthesis. Branched conjugates loaded with up to 80 boron atoms per peptide molecule exhibited a maintained receptor activation profile, and the selective uptake into hY1R-expressing cells was demonstrated by internalization studies. In order to ensure appropriate solubility in aqueous solution, we proved the need for eight hydroxyl groups per carborane. Thus, we suggest the utilization of bis-deoxygalactosyl-carborane building blocks in solid phase peptide synthesis to produce selective boron delivery agents for BNCT.

3.
J Org Chem ; 85(3): 1446-1457, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31813224

RESUMO

Boron neutron capture therapy (BNCT) allows the selective elimination of malignant tumor cells without affecting healthy tissue. Although this binary radiotherapy approach has been known for decades, BNCT failed to reach the daily clinics to date. One of the reasons is the lack of selective boron delivery agents. Using boron loaded peptide conjugates, which address G protein-coupled receptors overexpressed on tumor cells allow the intracellular accumulation of boron. The gastrin-releasing peptide receptor (GRPR) is a well-known target in cancer diagnosis and can potentially be used for BNCT. Here, we present the successful introduction of multiple bis-deoxygalactosyl-carborane building blocks to the GRPR-selective ligand [d-Phe6, ß-Ala11, Ala13, Nle14]Bn(6-14) (sBB2L) generating peptide conjugates with up to 80 boron atoms per molecule. Receptor activation was retained, metabolic stability was increased, and uptake into PC3 cells was proven without showing any intrinsic cytotoxicity. Furthermore, undesired uptake into liver cells was suppressed by using l-deoxygalactosyl modified carborane building blocks. Due to its high boron loading and excellent GRPR selectivity, this conjugate can be considered as a promising boron delivery agent for BNCT.

4.
J Pept Sci ; 25(12): e3224, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31743956

RESUMO

The gastrin-releasing peptide receptor (GRPR) is part of the bombesin receptor family and a well-known target in cancer diagnosis and therapy. In the last decade, promising results have been achieved by using peptide-drug conjugates, which allow selective targeting of GRPR expressing tumor cells. Most ligands, however, have been antagonists even though agonists can lead to higher tumor uptake owing to their internalization. So far, only a few studies focused on the identification of small GRPR-selective agonists that are metabolically stable. Here, we developed novel bombesin analogs with high selectivity for the GRPR and improved blood plasma stability. The most promising analog [d-Phe6 , ß-Ala11 , NMe-Ala13 , Nle14 ]Bn(6-14) displays an activity of 0.3nM at the GRPR, a more than 4000-fold selectivity over the other two bombesin receptors and more than 75% stability in human blood plasma after 24 hours. This analog is proposed as a promising drug shuttle for the intracellular delivery of different payloads in targeted tumor therapy approaches.


Assuntos
Bombesina/farmacologia , Neurotransmissores/farmacologia , Receptores da Bombesina/agonistas , Bombesina/análogos & derivados , Bombesina/sangue , Células Cultivadas , Estabilidade de Medicamentos , Humanos , Neurotransmissores/sangue , Neurotransmissores/química
5.
ChemMedChem ; 14(21): 1849-1855, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31442005

RESUMO

We describe two synthetic amino acids with inverted side chain stereochemistry, which induce opposite biological activity. Phe4 is an important part of the activation motif of ghrelin, and in short peptide inverse agonists such as KwFwLL-NH2 , the aromatic core is necessary for inactivation of the receptor. To restrict indole/phenyl mobility and simultaneously strengthen the interaction between peptide and receptor, we exchanged the natural monoaryl amino acids for diaryl amino acids derived from tryptophan. By standard solid-phase peptide synthesis, each of them was inserted into ghrelin or in the aromatic core of the inverse agonist. Both ghrelin analogues showed nanomolar activity, indicating sufficient space to accommodate the additional side chain. In contrast, diaryl amino acids in the inverse agonist had considerable influence on receptor signaling. Whereas the introduction of Wsf maintains inverse agonism of the peptide, Wrf shifts the receptor more to active states and can induce agonism depending on its introduction site.

6.
Structure ; 27(3): 537-544.e4, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30686667

RESUMO

The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.


Assuntos
Grelina/química , Grelina/metabolismo , Receptores de Grelina/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica
7.
J Pept Sci ; 25(3): e3147, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30680847

RESUMO

Adrenomedullin (ADM) is a vasoactive peptide hormone of 52 amino acids and belongs to the calcitonin peptide superfamily. Its vasodilative effects are mediated by the interaction with the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor (GPCR), associated with the receptor activity modifying protein 2 (RAMP2) and functionally described as AM-1 receptor (AM1 R). A disulfide-bonded ring structure consisting of six amino acids between Cys16 and Cys21 has been shown to be a key motif for receptor activation. However, the specific structural requirements remain to be elucidated. To investigate the influence of ring size and position of additional functional groups that replace the native disulfide bond, we generated ADM analogs containing thioether, thioacetal, alkane, and lactam bonds between amino acids 16 and 21 by Fmoc/t-Bu solid phase peptide synthesis. Activity studies of the ADM disulfide bond mimetics (DSBM) revealed a strong impact of structural parameters. Interestingly, an increased ring size was tolerated but the activity of lactam-based mimetics depended on its position within the bridging structure. Furthermore, we found the thioacetal as well as the thioether-based mimetics to be well accepted with full AM1 R activity. While a reduced selectivity over the calcitonin gene-related peptide receptor (CGRPR) was observed for the thioethers, the thioacetal was able to retain a wild-type-like selectivity profile. The carbon analog in contrast displayed weak antagonistic properties. These results provide insight into the structural requirements for AM1 R activation as well as new possibilities for the development of metabolically stabilized analogs for therapeutic applications of ADM.


Assuntos
Adrenomedulina/química , Adrenomedulina/farmacologia , Dissulfetos/química , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/metabolismo , Adrenomedulina/síntese química , Dissulfetos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
8.
J Pept Sci ; 24(10): e3119, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30168238

RESUMO

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron-containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein-coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor-directed boron uptake. In this study, we present the generation of short, boron-rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super-agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta-carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron-loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.


Assuntos
Boro/farmacologia , Peptídeos/síntese química , Receptores de Grelina/agonistas , Boro/química , Terapia por Captura de Nêutron de Boro , Portadores de Fármacos , Células HEK293 , Humanos , Oligopeptídeos/química , Peptídeos/química
9.
ChemMedChem ; 13(17): 1797-1805, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29979487

RESUMO

Adrenomedullin (ADM) is a peptide hormone of the calcitonin gene-related peptide (CGRP) family. It is involved in the regulation of cardiovascular processes such as angiogenesis, vasodilation, and the reduction of oxidative stress. ADM mediates its effects by activation of the ADM-1 and -2 receptors (AM1 R/AM2 R), but also activates the CGRP receptor (CGRPR) with reduced potency. It binds to the extracellular domains of the receptors with its C-terminal binding motif (residues 41-52). The activation motif, consisting of a disulfide-bonded ring structure (residues 16-21) and an adjacent helix (residues 22-30), binds to the transmembrane region and stabilizes the receptor conformation in the active state. While it was shown that the binding motif of ADM guides AM1 R selectivity, there is little information on the activation motif itself. Here, we demonstrate that Thr22 of ADM contributes to the selectivity. By using solid-phase peptide synthesis and cAMP-based signal transduction, we studied the effects of analogues in the activation motif of ADM on AM1 R and CGRPR activity. Our results indicate that Thr22 terminates the α-helix and orients the ring segment by hydrogen bonding. Using olefin stapling, we showed that the α-helical arrangement of the ring segment leads to decreased AM1 R activity, but does not affect CGRPR activation. These results demonstrate that the conformation of the ring segment of ADM has a strong impact on the selectivity within the receptor system.


Assuntos
Adrenomedulina/farmacologia , Proteína Semelhante a Receptor de Calcitonina/antagonistas & inibidores , Cardiotônicos/farmacologia , Treonina/química , Adrenomedulina/química , Proteína Semelhante a Receptor de Calcitonina/metabolismo , Cardiotônicos/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-Atividade
10.
Bioorg Med Chem ; 26(10): 2759-2765, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395804

RESUMO

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market.


Assuntos
Técnicas de Química Sintética/métodos , Descoberta de Drogas/métodos , Lipídeos/química , Peptídeos/química , Polímeros/química , Animais , Humanos , Lipídeos/síntese química , Lipídeos/farmacocinética , Lipídeos/farmacologia , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/farmacocinética , Peptídeos/farmacologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Polietilenoglicóis/farmacologia , Polímeros/síntese química , Polímeros/farmacocinética , Polímeros/farmacologia , Técnicas de Síntese em Fase Sólida/métodos
11.
Int J Mol Sci ; 18(4)2017 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-28379199

RESUMO

The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.


Assuntos
Proteínas de Transporte/metabolismo , Imagem Molecular/métodos , Pirimidinas/síntese química , Pirimidinas/metabolismo , Animais , Células CHO , Cricetulus , Halogenação , Humanos , Ligantes , Estrutura Molecular , Tomografia por Emissão de Pósitrons/métodos , Ligação Proteica , Pirimidinas/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo
12.
Sci Rep ; 7: 46128, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28387359

RESUMO

The expression, functional reconstitution and first NMR characterization of the human growth hormone secretagogue (GHS) receptor reconstituted into either DMPC or POPC membranes is described. The receptor was expressed in E. coli. refolded, and reconstituted into bilayer membranes. The molecule was characterized by 15N and 13C solid-state NMR spectroscopy in the absence and in the presence of its natural agonist ghrelin or an inverse agonist. Static 15N NMR spectra of the uniformly labeled receptor are indicative of axially symmetric rotational diffusion of the G protein-coupled receptor in the membrane. In addition, about 25% of the 15N sites undergo large amplitude motions giving rise to very narrow spectral components. For an initial quantitative assessment of the receptor mobility, 1H-13C dipolar coupling values, which are scaled by molecular motions, were determined quantitatively. From these values, average order parameters, reporting the motional amplitudes of the individual receptor segments can be derived. Average backbone order parameters were determined with values between 0.56 and 0.69, corresponding to average motional amplitudes of 40-50° of these segments. Differences between the receptor dynamics in DMPC or POPC membranes were within experimental error. Furthermore, agonist or inverse agonist binding only insignificantly influenced the average molecular dynamics of the receptor.


Assuntos
Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Receptores de Grelina/metabolismo , Dimiristoilfosfatidilcolina/química , Grelina/metabolismo , Humanos , Fosfatidilcolinas/química , Receptores de Grelina/agonistas , Receptores de Grelina/química , Proteínas Recombinantes/metabolismo
13.
J Pept Sci ; 23(7-8): 472-485, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28150464

RESUMO

Adrenomedullin (ADM) is a 52-amino acid multifunctional peptide, which belongs to the calcitonin gene-related peptide (CGRP) superfamily of vasoactive peptide hormones. ADM exhibits a significant vasodilatory potential and plays a key role in various regulatory mechanisms, predominantly in the cardiovascular and lymphatic system. It exerts its effects by activation of the calcitonin receptor-like receptor associated with one of the receptor activity-modifying proteins 2 or 3. ADM was first isolated from human phaeochromocytoma in 1993. Numerous studies revealed a widespread distribution in various tissues and organs, which is reflected by its multiple physiological roles in health and disease. Because of its anti-inflammatory, anti-apoptotic and proliferative properties, ADM exhibits potent protective functions under diverse pathological conditions, but it is also critically involved in tumor progression. ADM has therefore raised great interest in therapeutic applications and several clinical trials already revealed promising results. However, because the receptor activation mode has not yet been fully elucidated, a rational design of potent and selective ligands is still challenging. Detailed information on the binding mode of ADM from a recently reported crystal structure as well as efforts to improve its plasma stability and bioavailability may help to overcome these limitations in the future. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.


Assuntos
Adrenomedulina/metabolismo , Adrenomedulina/genética , Animais , Calcitonina/genética , Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/genética , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo
14.
ChemMedChem ; 11(21): 2378-2384, 2016 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-27558296

RESUMO

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h-1 kg-1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential.

15.
J Med Chem ; 59(12): 5695-705, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27166982

RESUMO

The 52 amino acid peptide hormone adrenomedullin (ADM) plays a major role in the development and regulation of the cardiovascular and lymphatic system and has therefore gained significant interest for clinical applications. Because adrenomedullin exhibits low metabolic stability, enhancement of the plasma half-life is essential for peptide-based drug design. Fluorescently labeled ADM analogues synthesized by Fmoc/t-Bu solid phase peptide synthesis were used to analyze their enzymatic degradation and specific fragmentation pattern in human blood plasma. The determination of important cleavage sites allowed the development of selectively modified peptides in a rational approach. By combination of palmitoylation, lactam-bridging, and Nα-methylation, ADM analogues protected from enzymatic cleavage in human blood were developed and revealed an explicitly elongated half-life of 5 days in comparison to the wild-type in vitro. This triple-modification did not alter the selectivity of the analogues at the AM1 receptor, highlighting their potential for therapeutic applications.


Assuntos
Adrenomedulina/metabolismo , Adrenomedulina/sangue , Adrenomedulina/química , Adrenomedulina/farmacologia , Células Cultivadas , Estabilidade de Medicamentos , Células HEK293 , Meia-Vida , Humanos , Estrutura Molecular , Receptores de Adrenomedulina/agonistas , Receptores de Adrenomedulina/metabolismo , Relação Estrutura-Atividade
16.
Sci Rep ; 6: 21025, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26868142

RESUMO

Three-kinase mitogen-activated protein kinase (MAPK) signaling cascades are present in virtually all eukaryotic cells. MAPK cascades are organized by scaffold proteins, which assemble cognate kinases into productive signaling complexes. Arrestin-3 facilitates JNK activation in cells, and a short 25-residue arrestin-3 peptide was identified as the critical JNK3-binding element. Here we demonstrate that this peptide also binds MKK4, MKK7, and ASK1, which are upstream JNK3-activating kinases. This peptide is sufficient to enhance JNK3 activity in cells. A homologous arrestin-2 peptide, which differs only in four positions, binds MKK4, but not MKK7 or JNK3, and is ineffective in cells at enhancing activation of JNK3. The arrestin-3 peptide is the smallest MAPK scaffold known. This peptide or its mimics can regulate MAPKs, affecting cellular decisions to live or die.


Assuntos
Ativadores de Enzimas , Proteína Quinase 10 Ativada por Mitógeno/metabolismo , Peptídeos , beta-Arrestina 1/química , beta-Arrestina 2/química , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/síntese química , Ativadores de Enzimas/química , Ativadores de Enzimas/farmacologia , Humanos , Proteína Quinase 10 Ativada por Mitógeno/genética , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia
17.
Biopolymers ; 106(1): 101-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26566778

RESUMO

Subtle changes in the sequence at the N-terminus and in the aromatic core of hexapeptidic ghrelin receptor inverse agonists can switch behavior from inverse agonism to agonism, but the C-terminal role of the sequence is unclear. Thus, analogs of the ghrelin receptor inverse agonist KbFwLL-NH2 (b = ß-(3-benzothienyl)-d-alanine) were synthesized by solid phase peptide synthesis in order to identify the influence of aromaticity, charge, and hydrophobicity. Potency and efficacy of the hexapeptides were evaluated in inositol triphosphate turnover assays. Notably, modifications directly at the C-terminal Leu(6) could influence peptide efficacy leading to decreased constitutive activity. High hydrophobicity at the C-terminal position was of importance for elevated inverse agonist activity, the introduction of charged amino acids led to decreased potency. In contrast, structure-activity relationship studies of Leu(5) located closer to the aromatic core revealed an agonism-inducing position. These findings imply that amino acids with possible cation-π or π-π interactions and a suitable orientation at the C-terminus of the aromatic core induce agonism. Receptor binding studies showed that most peptides bind to the receptor at a concentration of 1 µM and modification directly at the C-terminus is generally more accepted than Leu(5) substitution. Interestingly, this observation is not dependent on the type of modification. These studies reveal another switch region of the short ghrelin receptor ligand pointing out the sensitivity of the ghrelin receptor binding pocket.


Assuntos
Oligopeptídeos/química , Receptores de Grelina/antagonistas & inibidores , Animais , Células COS , Chlorocebus aethiops , Oligopeptídeos/síntese química , Receptores de Grelina/agonistas , Receptores de Grelina/química
18.
Chem Biol ; 22(11): 1431-1436, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26548612

RESUMO

Ghrelin receptor (GhrR) is a promising drug target because of its central role in energy homeostasis. GhrR, known for high constitutive activity, is thought to display multi-state conformations during activation and signaling. We used genetically encoded unnatural amino acids and bioorthogonal labeling reactions to engineer multiple fluorescent donor-acceptor pairs to probe ligand-directed structural changes in GhrR. We demonstrate how conformational dynamics of a G-protein-coupled receptor can be measured in reconstituted systems.


Assuntos
Ligantes , Receptores de Grelina/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Azidas/química , Reação de Cicloadição , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Compostos Organometálicos/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Conformação Proteica , Receptores de Grelina/genética , Receptores de Grelina/metabolismo
19.
J Med Chem ; 58(10): 4180-93, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25905598

RESUMO

Ghrelin and Y2 receptors play a central role in appetite regulation inducing opposite effects. The Y2 receptor induces satiety, while the ghrelin receptor promotes hunger and weight gain. However, the food regulating system is tightly controlled by interconnected pathways where redundancies can lead to poor efficacy and drug tolerance when addressing a single molecule. We developed a multitarget strategy to synthesize dual peptides simultaneously inhibiting the ghrelin receptor and stimulating the Y2 receptor. Dual peptides showed dual activity in vitro, and one compound induced a slight diminution of food intake in a rodent model of obesity. In addition, stability studies in rats revealed different behaviors between the dual peptide and its corresponding monomers. The Y2 receptor agonist was unstable in blood, while the dual peptide showed an intermediate stability compared to that of the highly stable ghrelin receptor inverse agonist.


Assuntos
Fármacos Antiobesidade/farmacologia , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Grelina , Peptídeos/química , Peptídeos/farmacologia , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores de Grelina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Fármacos Antiobesidade/química , Ligação Competitiva , Células COS , Chlorocebus aethiops , Desenho de Fármacos , Feminino , Humanos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Terapia de Alvo Molecular , Peptídeos/síntese química , Receptores de Grelina/agonistas
20.
PLoS One ; 10(3): e0122444, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25803439

RESUMO

The peptide hormone ghrelin activates the growth hormone secretagogue receptor 1a, also known as the ghrelin receptor. This 28-residue peptide is acylated at Ser3 and is the only peptide hormone in the human body that is lipid-modified by an octanoyl group. Little is known about the structure and dynamics of membrane-associated ghrelin. We carried out solid-state NMR studies of ghrelin in lipid vesicles, followed by computational modeling of the peptide using Rosetta. Isotropic chemical shift data of isotopically labeled ghrelin provide information about the peptide's secondary structure. Spin diffusion experiments indicate that ghrelin binds to membranes via its lipidated Ser3. Further, Phe4, as well as electrostatics involving the peptide's positively charged residues and lipid polar headgroups, contribute to the binding energy. Other than the lipid anchor, ghrelin is highly flexible and mobile at the membrane surface. This observation is supported by our predicted model ensemble, which is in good agreement with experimentally determined chemical shifts. In the final ensemble of models, residues 8-17 form an α-helix, while residues 21-23 and 26-27 often adopt a polyproline II helical conformation. These helices appear to assist the peptide in forming an amphipathic conformation so that it can bind to the membrane.


Assuntos
Membrana Celular/metabolismo , Biologia Computacional/métodos , Grelina/química , Grelina/metabolismo , Modelos Moleculares , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
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