RESUMO
Obesity is one of the most prevalent metabolic diseases in the Western world and correlates directly with glucose intolerance and insulin resistance, often culminating in Type 2 Diabetes (T2D). Importantly, our team has recently shown that the TNF superfamily (TNFSF) member protein, TNFSF14, has been reported to protect against high fat diet induced obesity and pre-diabetes. We hypothesized that mimics of TNFSF14 may therefore be valuable as anti-diabetic agents. In this study, we use in silico approaches to identify key regions of TNFSF14 responsible for binding to the Herpes virus entry mediator and Lymphotoxin ß receptor. In vitro evaluation of a selection of optimised peptides identified six potentially therapeutic TNFSF14 peptides. We report that these peptides increased insulin and fatty acid oxidation signalling in skeletal muscle cells. We then selected one of these promising peptides to determine the efficacy to promote metabolic benefits in vivo. Importantly, the TNFSF14 peptide 7 reduced high fat diet-induced glucose intolerance, insulin resistance and hyperinsulinemia in a mouse model of obesity. In addition, we highlight that the TNFSF14 peptide 7 resulted in a marked reduction in liver steatosis and a concomitant increase in phospho-AMPK signalling. We conclude that TNFSF14-derived molecules positively regulate glucose homeostasis and lipid metabolism and may therefore open a completely novel therapeutic pathway for treating obesity and T2D.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/administração & dosagem , Obesidade/complicações , Obesidade/tratamento farmacológico , Peptídeos/administração & dosagem , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/administração & dosagem , Animais , Sítios de Ligação , Glicemia/metabolismo , Simulação por Computador , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/metabolismo , Homeostase/efeitos dos fármacos , Hiperinsulinismo/tratamento farmacológico , Hiperinsulinismo/metabolismo , Hipoglicemiantes/síntese química , Resistência à Insulina , Receptor beta de Linfotoxina/química , Receptor beta de Linfotoxina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , Peptídeos/síntese química , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
Methods to promote myelin regeneration in response to central myelin loss are essential to prevent the progression of clinical disability in demyelinating diseases. The neurotrophin brain-derived neurotrophic factor (BDNF) is known to promote myelination during development via oligodendrocyte expressed TrkB receptors. Here, we use a structural mimetic of BDNF to promote myelin regeneration in a preclinical mouse model of central demyelination. In female mice, we show that selective targeting of TrkB with the BDNF-mimetic enhances remyelination, increasing oligodendrocyte differentiation, the frequency of myelinated axons, and myelin sheath thickness after a demyelinating insult. Treatment with exogenous BDNF exerted an attenuated effect, increasing myelin sheath thickness only. Further, following conditional deletion of TrkB from premyelinating oligodendrocytes, we show the effects of the BDNF-mimetic on oligodendrocyte differentiation and remyelination are lost, indicating these are dependent on oligodendrocyte expression of TrkB. Overall, these studies demonstrate that targeting oligodendrocyte TrkB promotes in vivo remyelination in the brain.SIGNIFICANCE STATEMENT Novel strategies to promote myelin regeneration are required to prevent progressive neurodegeneration and clinical disability in patients with central demyelinating disease. Here, we test whether selectively targeting the TrkB receptor on the myelin-producing oligodendrocytes, can promote remyelination in the brain. Using a structural mimetic of its native ligand, BDNF, we show that stimulation of TrkB enhances remyelination, increasing oligodendrocyte differentiation, the frequency of myelinated axons and thickness of the myelin sheath following a demyelinating insult. Further, we show that these effects are dependent on the phosphorylation of oligodendrocyte expressed TrkB receptors in vivo Overall, we demonstrate that selective targeting of TrkB has therapeutic potential to promote remyelination in the brain.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Encéfalo/efeitos dos fármacos , Doenças Desmielinizantes/tratamento farmacológico , Glicoproteínas de Membrana/agonistas , Terapia de Alvo Molecular , Bainha de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Divisão Celular/efeitos dos fármacos , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Feminino , Bombas de Infusão Implantáveis , Infusões Intraventriculares , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Básica da Mielina/biossíntese , Células-Tronco Neurais/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N-methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N-methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N-methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N-methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N-methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.
Assuntos
Amidas/química , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Peptídeos/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Células CACO-2 , Cromatografia Líquida , Humanos , Ligação de Hidrogênio , Espectrometria de Massas , Metilação , Peptídeos/síntese química , Permeabilidade , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Wistar , Solventes/química , Tecnologia Farmacêutica/métodos , TemperaturaRESUMO
Cyclic disulfide-rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X-ray structures of these peptides to assist in drug design applications, but disulfide-rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L- and D-forms of three prototypic cyclic disulfide-rich peptides: SFTI-1 (14-mer with one disulfide bond), cVc1.1 (22-mer with two disulfide bonds), and kB1 (29-mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25â Å to 1.9â Å. Additionally, we obtained the quasi-racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25â Å and 2.3â Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.
Assuntos
Dissulfetos/química , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Desenho de Fármacos , Modelos Moleculares , Dados de Sequência Molecular , EstereoisomerismoRESUMO
The tachykinin neuropeptide substance P (SP) is the canonical agonist peptide for the neurokinin 1 receptor (NK1 R). More recently, it has also been shown to activate the Mas-related G protein-coupled receptor X2 (MRGPRX2) receptor on mast cells (MCs), triggering degranulation and release of inflammatory mediators. SP undergoes rapid C-terminal truncation in vivo by a number of proteases to generate the metabolites SP(1-9)-COOH and in particular SP(1-7)-COOH. While the C terminus of SP is critical for NK1 R activation, studies have shown that the peptide polycationic N terminus is key for MRGPRX2 and mast cell activation. The study thus aimed to determine if the C-terminally truncated metabolites of SP, SP(1-9)-COOH, and SP(1-7)-COOH retained stimulatory activity at MRGPRX2. SP, SP(1-9)-COOH, and SP(1-7)-COOH were synthesized and tested on HEK293 cells expressing NK1 R or MRGPRX2, and LAD2 human mast cells, to determine the activity of SP and its metabolites in Ca2+ mobilization, degranulation, and cytokine assays. As expected from prior studies, both C-terminally truncated SP metabolites had essentially no activity at NK1 R, even at very high concentrations. In contrast, the in vivo metabolite of SP, SP(1-9)-COOH retained ability to activate MRGPRX2 across all parameters tested, albeit with reduced potency compared to intact SP. SP(1-7)-COOH did not produce any significant MRGRPX2 activation. Our results suggest that the SP metabolite, SP(1-9)-COOH, may play a regulatory role through the activation of MRGPRX2. However, given the relatively low potency of both SP and SP(1-9)-COOH at MRGPRX2, additional work is needed to better understand the biological importance of this expanded SP/MRGPRX2 pathway.
Assuntos
Mastócitos , Receptores de Neuropeptídeos , Degranulação Celular , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Substância P/metabolismo , Substância P/farmacologiaRESUMO
A synthetic strategy for conjugating small molecules and peptide-based therapeutics, via a cleavable ester bond, to a lipidated ß3-tripeptide is presented. The drug-loaded ß3-peptide was successfully co-assembled with a functionally inert lipidated ß3-tripeptide to form a hydrogel. Quantitative release of lactose from the hydrogel, by the action of serum esterases, is demonstrated over 28 days. The esterase-mediated sustained release of the bioactive brain-derived neurotrophic factor (BDNF) peptide mimics from the hydrogel resulted in increased neuronal survival and normal neuronal function of peripheral neurons. These studies define a versatile strategy for the facile synthesis and co-assembly of self-assembling ß3-peptide-based hydrogels with the ability to control drug release using endogenous esterases with potential in vivo applications for sustained localized drug delivery.
Assuntos
Esterases/metabolismo , Hidrogéis/farmacologia , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Liberação Controlada de Fármacos , Esterases/sangue , Feminino , Hidrogéis/química , Hidrogéis/metabolismo , Teste de Materiais , Estrutura Molecular , Neurônios/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
The chemical synthesis of cyclic peptides is a well-established area of research. This has been further expanded by development of bio-orthogonal reactions that enable access to peptides of greater structural complexity. One approach utilizes 1,3-dichloroacetone to selectively link free cysteine side-chains with an acetone-like bridge via an SN2 reaction. Here, we have used this reaction to dimerize cyclic peptide monomers to create novel bicyclic dimeric peptides. We investigated a range of reaction parameters to identify the optimal dimerization conditions for our model systems. One of the acetone-linked dimeric peptides was analyzed for proteolytic stability in human serum and was observed to still be fully intact after 48 h. This study provides valuable insights into the application of 1,3-dichloroacetone as a tool in the synthesis of complex, multicyclic peptides.
RESUMO
The p75 splice variant of lens epithelium-derived growth factor (LEDGF) is a 75â kDa protein, which is recruited by the human immunodeficiency virus (HIV) to tether the pre-integration complex to the host chromatin and promote integration of proviral DNA into the host genome. We designed a series of small cyclic peptides that are structural mimics of the LEDGF binding domain, which interact with integrase as potential binding inhibitors. Herein we present the X-ray crystal structures, NMR studies, SPR analysis, and conformational studies of four cyclic peptides bound to the HIV-1 integrase core domain. Although the X-ray studies show that the peptides closely mimic the LEDGF binding loop, the measured affinities of the peptides are in the low millimolar range. Computational analysis using conformational searching and free energy calculations suggest that the low affinity of the peptides is due to mismatch between the low-energy solution and bound conformations.
Assuntos
Integrase de HIV/química , Peptídeos e Proteínas de Sinalização Intercelular/química , Mimetismo Molecular , Peptídeos Cíclicos/química , Cristalografia por Raios X , HIV-1/enzimologia , Conformação Proteica , Análise Espectral/métodosRESUMO
Disulfide-rich macrocyclic peptides are emerging as versatile scaffolds for the development of stable biochemical tools. This potential is due to the combination of their structural stability and range of bioactivities. Here, we explored the activity of these peptides on fibril growth of the hexapeptide Ac-VQIVYK-NH2 (AcPHF6), which is a tau-derived peptide that has been widely used to understand the pathological mechanism of numerous tauopathies, including Alzheimer's disease. Of the cyclic peptides tested, SFTI-1 and kB1 showed an inherent ability to inhibit AcPHF6 fibril formation. Using an end-capping strategy and combining it with a molecular grafting approach, we demonstrated that SFTI-1 could be used as a starting point to design more potent fibril inhibitors. We further identified chemical and structural features of SFTI-1 and its analogues that underpin their inhibitory activity. The ability to inhibit fibril growth using the strategy employed herein supports the 'steric zipper' model of AcPHF6 fibril formation and shows that naturally-occurring cyclic peptides have potential as drug leads or molecular probes for understanding fibril formation.
Assuntos
Oligopeptídeos/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Agregados Proteicos/efeitos dos fármacos , Tauopatias/tratamento farmacológico , Proteínas tau/metabolismo , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/química , Amiloide/metabolismo , Desenho de Fármacos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Alinhamento de Sequência , Tauopatias/metabolismo , Proteínas tau/químicaRESUMO
Despite the widespread use of cyclization as a structure optimization tool in peptide chemistry, little is known about the effect of cyclization on peptide internal dynamics. In this work, we used a combination of multifield NMR relaxation and molecular dynamics techniques to study both monocyclic and polycyclic peptides that have promising biopharmaceutical properties, namely, VH, SFTI-1, and cVc1.1, and their less constrained analogues to study the effects of backbone cyclization (which forms a macrocycle) and disulfide-bond cyclization (which forms internal cycles). We confirmed that backbone cyclization contributes to the rigidity of the monocyclic VH. Interestingly, however, backbone cyclization of the bicyclic SFTI-1 had a limited effect on rigidity, with changes in internal dynamics localized around the ligation site. This suggests that the disulfide bond, which creates an internal cycle, has an insulating effect, protecting the internal cycle from external motional effects. An insulating effect was also observed for the polycyclic cVc1.1: The rigidity of the core was not enhanced by macrocyclization. Additionally, we found that disulfide bonds provide a greater contribution to overall rigidity than macrocyclization. Overall, our results suggest that, although backbone cyclization can improve rigidity, there is a complex interplay between dynamics and cyclization, particularly for polycyclic systems.
Assuntos
Simulação de Dinâmica Molecular , Peptídeos/química , Ciclização , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Peptídeos/isolamento & purificaçãoRESUMO
An increasing number of macrocyclic peptides that cross biological membranes are being reported, suggesting that it might be possible to develop peptides into orally bioavailable therapeutics; however, current understanding of what makes macrocyclic peptides cell permeable is still limited. Here, we synthesized 62 cyclic hexapeptides and characterized their permeability using in vitro assays commonly used to predict in vivo absorption rates, i.e. the Caco-2 and PAMPA assays. We correlated permeability with experimentally measured parameters of peptide conformation obtained using rapid methods based on chromatography and nuclear magnetic resonance spectroscopy. Based on these correlations, we propose a model describing the interplay between peptide permeability, lipophilicity and hydrogen bonding potential. Specifically, peptides with very high permeability have high lipophilicity and few solvent hydrogen bond interactions, whereas peptides with very low permeability have low lipophilicity or many solvent interactions. Our model is supported by molecular dynamics simulations of the cyclic peptides calculated in explicit solvent, providing a structural basis for the observed correlations. This prospective exploration into biomarkers of peptide permeability has the potential to unlock wider opportunities for development of peptides into drugs.
Assuntos
Simulação de Dinâmica Molecular , Peptídeos Cíclicos/farmacocinética , Células CACO-2 , Humanos , Modelos Biológicos , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Relação Quantitativa Estrutura-AtividadeRESUMO
Cyclic peptides are increasingly being recognized as valuable templates for drug discovery or design. To facilitate efforts in the structural characterization of cyclic peptides, we explore the use of pulse-field gradient experiments as a convenient and noninvasive approach for characterizing their diffusion properties in solution. We present diffusion coefficient measurements of five cyclic peptides, including dichC, SFTI-1, cVc1.1, kB1, and kB2. These peptides range in size from six to 29 amino acids and have various therapeutically interesting activities. We explore the use of internal standards, such as dioxane and acetonitrile, to evaluate the hydrodynamic radius from the diffusion coefficient, and show that 2,2-dimethyl-2-silapentane-5-sulfonic acid, a commonly used chemical shift reference, can be used as an internal standard to avoid spectral overlap issues and simplify data analysis. The experimentally measured hydrodynamic radii correlate with increasing molecular weight and in silico predictions. We further applied diffusion measurements to characterize the self-association of kB2 and showed that it forms oligomers in a concentration-dependent manner, which may be relevant to its mechanism of action. Diffusion coefficient measurements appear to have broad utility in cyclic peptide structural biology, allowing for the rapid characterization of their molecular shape in solution.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Difusão , Dados de Sequência MolecularRESUMO
Recently disulfide-rich head-to-tail cyclic peptides have attracted the interest of medicinal chemists owing to their exceptional thermal, chemical and enzymatic stability brought about by their constrained structures. Here we review current trends in the field of peptide-based pharmaceuticals and describe naturally occurring cyclic disulfide-rich peptide scaffolds, discussing their pharmaceutically attractive properties and benefits. We describe how we can utilise these stable frameworks to graft and/or engineer pharmaceutically interesting epitopes to increase their selectivity and bioactivity, opening up new possibilities for addressing 'difficult' pharmaceutical targets.