RESUMO
Chemical modifications to proteins have wide applications. They may be used in, for example, the production of biopharmaceuticals and fluorescent probes. Despite their importance, highly regioselective chemical protein modifications are often challenging to achieve. We have developed two highly selective methods for protein acylation using poly-His tags inserted either at the N-terminus or in combination with a specific Lys residue. For this, we used an N-terminal Gly-His6 (Gly-His tag) or the sequence Hism-Lys-Hisn (Lys-His tag), respectively. The Gly-His tag directed the acylation to the N-terminal Nα-amine when reacted with 4-methoxyphenyl esters to yield stable conjugates. Next, the Lys-His tag was developed to allow modifications at the C-terminus or in loop regions of proteins. This gave a high selectivity of acylation of the designated Lys Nε-amine in the tag over native Lys residues in the protein under mild conditions. Here, we describe the synthesis of aromatic esters carrying different functionalities and reactivity tuning substituents on the phenol. The expression of poly-His tagged proteins, and the procedure for the highly selective peptide and protein acylations are detailed in this contribution. The versatility of these methods has been demonstrated by the attachment of different functionalities such as fluorophores, biotin, and azides to different proteins and an antibody.
Assuntos
Histidina , Peptídeos , Proteínas , Acilação , Peptídeos/química , Histidina/química , Proteínas/química , Ésteres/químicaRESUMO
DNA-stabilized silver nanoclusters (DNA-AgNCs) are biocompatible emitters with intriguing properties. However, they have not been extensively used for bioimaging applications due to the lack of structural information and hence predictable conjugation strategies. Here, a copper-free click chemistry method for linking a well-characterized DNA-AgNC to molecules of interest is presented. Three different peptides and a small protein, human insulin, were tested as labeling targets. The conjugation to the target compounds was verified by MS, HPLC, and time-resolved anisotropy measurements. Moreover, the spectroscopic properties of DNA-AgNCs were found to be unaffected by the linking reactions. For DNA-AgNC-conjugated human insulin, fluorescence imaging studies were performed on Chinese hamster ovary (CHO) cells overexpressing human insulin receptor B (hIR-B). The specific staining of the CHO cell membranes demonstrates that DNA-AgNCs are great candidates for bioimaging applications, and the proposed linking strategy is easy to implement when the DNA-AgNC structure is known.
Assuntos
Nanopartículas Metálicas , Prata , Humanos , Cricetinae , Animais , Prata/química , Células CHO , Química Click , Nanopartículas Metálicas/química , Cricetulus , DNA/química , Insulina , Peptídeos , Espectrometria de FluorescênciaRESUMO
Chemical modification of peptides and proteins, such as PEGylation and lipidation, creates conjugates with new properties. However, they are typically not dynamic or stimuli-responsive. Self-assembly controlled by a stimulus will allow adjusting properties directly. Here, we report that conjugates of oligogalacturonic acids (OGAs), isolated from plant-derived pectin, are Ca2+-responsive. We report the conjugation of OGA to human insulin (HI) to create new glyco-insulins. In addition, we coupled OGA to model peptides. We studied their self-assembly by dynamic light scattering, small-angle X-ray scattering, and circular dichroism, which showed that the self-assembly to form nanostructures depended on the length of the OGA sequence and Zn2+ and Ca2+ concentrations. Subcutaneous administration of OGA12-HI with Zn2+ showed a stable decrease in blood glucose over a longer period of time compared to HI, despite the lower receptor binding affinity.
Assuntos
Insulina , Peptídeos , Humanos , Glicemia , Dicroísmo Circular , Insulina/química , Peptídeos/química , Cálcio/metabolismoRESUMO
Chiral communications exist in secondary structures of foldamers and copolymers via a network of noncovalent interactions within effective intermolecular force (IMF) range. It is not known whether long-range chiral communication exists between macromolecular tertiary structures such as peptide coiled-coils beyond the IMF distance. Harnessing the high sensitivity of single-molecule force spectroscopy, we investigate the chiral interaction between covalently linked DNA duplexes and peptide coiled-coils by evaluating the binding of a diastereomeric pair of three DNA-peptide conjugates. We find that right-handed DNA triple helices well accommodate peptide triple coiled-coils of the same handedness, but not with the left-handed coiled-coil stereoisomers. This chiral communication is effective in a range (<4.5 nm) far beyond canonical IMF distance. Small-angle X-ray scattering and molecular dynamics simulation indicate that the interdomain linkers are tightly packed via hydrophobic interactions, which likely sustains the chirality transmission between DNA and peptide domains. Our findings establish that long-range chiral transmission occurs in tertiary macromolecular domains, explaining the presence of homochiral pairing of superhelices in proteins.
Assuntos
DNA/química , Substâncias Macromoleculares/química , Simulação de Acoplamento Molecular , Domínios Proteicos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Proteínas/química , EstereoisomerismoRESUMO
Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His6 segment. This tag promoted acylation of the N-terminal Nα -amine resulting in stable conjugates. Herein, we report the peptide sequences Hisn -Lys-Hism , which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys Nϵ -amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.
Assuntos
Lisina , Proteínas , Acilação , Sequência de Aminoácidos , Peptídeos/químicaRESUMO
The use of C-terminal peptide thioesters and hydrazides in synthetic protein chemistry has inspired the search for optimal solid-phase peptide synthesis (SPPS) strategies for their assembly. However, peptide thioesters are not directly accessible by conventional Fmoc-SPPS owing to the nucleophilicity of the secondary amine required for Fmoc removal. Here, we report the mild and effective activation of the pGlu linker and a new safety-catch linker that was used for the convenient synthesis of peptide thioesters and hydrazides via efficient amide-to-imide activation followed by nucleophilic displacement.
Assuntos
Amidas , Técnicas de Síntese em Fase Sólida , Ésteres , Imidas , PeptídeosRESUMO
Glioblastoma (GBM) is a devastating cancer with basically no curative treatment. Even with aggressive treatment, the median survival is disappointing 14 months. Surgery remains the key treatment and the postoperative survival is determined by the extent of resection. Unfortunately, the invasive growth with irregular infiltrating margins complicates an optimal surgical resection. Precise intraoperative tumor visualization is therefore highly needed and molecular targeted near-infrared (NIR) fluorescence imaging potentially constitutes such a tool. The urokinase-type Plasminogen Activator Receptor (uPAR) is expressed in most solid cancers primarily at the invading front and the adjacent activated peritumoral stroma making it an attractive target for targeted fluorescence imaging. The purpose of this study was to develop and evaluate a new uPAR-targeted optical probe, IRDye800CW-AE344, for fluorescence guided surgery (FGS). Methods: In the present study we characterized the fluorescent probe with regard to binding affinity, optical properties, and plasma stability. Further, in vivo imaging characterization was performed in nude mice with orthotopic human patient derived glioblastoma xenografts, and we performed head-to-head comparison within FGS between our probe and the traditional procedure using 5-ALA. Finally, the blood-brain barrier (BBB) penetration was characterized in a 3D BBB spheroid model. Results: The probe effectively visualized GBM in vivo with a tumor-to-background ratio (TBR) above 4.5 between 1 to 12 h post injection and could be used for FGS of orthotopic human glioblastoma xenografts in mice where it was superior to 5-ALA. The probe showed a favorable safety profile with no evidence of any acute toxicity. Finally, the 3D BBB model showed uptake of the probe into the spheroids indicating that the probe crosses the BBB. Conclusion: IRDye800CW-AE344 is a promising uPAR-targeted optical probe for FGS and a candidate for translation into human use.
Assuntos
Glioblastoma , Indóis , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais , Imagem Óptica , Peptídeos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular Tumoral , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Glioblastoma/cirurgia , Xenoenxertos , Humanos , Indóis/química , Indóis/farmacologia , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/cirurgia , Peptídeos/química , Peptídeos/farmacologiaRESUMO
Preparative reversed-phase HPLC is the established method for the purification of peptides, but has significant limitations. We systematically investigated the use of high-performance reversed-phase flash chromatography (HPFC) to rapidly purify laboratory-scale quantities of crude, synthetic peptides and chemically modified insulins. We demonstrated these methods for a diverse set of peptides, including short, medium, and long peptides. Depending on the purity profile of the peptide, HPFC can be used either as the sole purification method, or as a pre-purification method prior to final HPLC purification. Furthermore, HPFC is suitable for the purification of peptides that are not fully in solution. We provide guidelines for the HPFC of synthetic peptides and small proteins, including the choice of columns, eluents, and gradients. We believe that HPFC is a valuable alternative to HPLC purification of peptides and small proteins.
Assuntos
Insulinas/isolamento & purificação , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Humanos , Insulinas/química , Peptídeos/análise , Ácidos Esteáricos/químicaRESUMO
DNA nanostructures have been designed and used in many different applications. However, the use of nucleic acid scaffolds to promote the self-assembly of artificial protein mimics is only starting to emerge. Herein five coiled-coil peptide structures were templated by the hybridization of a d-DNA triplex or its mirror-image counterpart, an l-DNA triplex. The self-assembly of the desired trimeric structures in solution was confirmed by gel electrophoresis and small-angle X-ray scattering, and the stabilizing synergy between the two domains was found to be chirality-independent but orientation-dependent. This is the first example of using a nucleic acid scaffold of l-DNA to template the formation of artificial protein mimics. The results may advance the emerging POC-based nanotechnology field by adding two extra dimensions, that is, chirality and polarity, to provide innovative molecular tools for rational design and bottom-up construction of artificial protein mimics, programmable materials and responsive nanodevices.
Assuntos
DNA/química , Nanotecnologia/métodos , Modelos Moleculares , Nanoestruturas/química , Nanotecnologia/instrumentação , Hibridização de Ácido Nucleico , Peptídeos/química , Domínios ProteicosRESUMO
Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 µmol/kg) and L1P3V8 (6 µmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model.
Assuntos
Proteínas de Transporte/administração & dosagem , Transtornos Heredodegenerativos do Sistema Nervoso/tratamento farmacológico , Oligopeptídeos/administração & dosagem , Peptídeos/administração & dosagem , Peptídeos/metabolismo , RNA Mensageiro/antagonistas & inibidores , Administração Intranasal , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Transporte/farmacocinética , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada/métodos , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/farmacocinética , Peptídeos/farmacocinética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHHn- for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-Hisn tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.
Assuntos
Dipeptídeos/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Acilação , Sequência de Aminoácidos , Azidas/química , Biotina/metabolismo , Ésteres/metabolismo , Corantes Fluorescentes/química , Gluconatos/metabolismo , Lactonas/metabolismo , Peptídeos/química , Polietilenoglicóis/química , Processamento de Proteína Pós-TraducionalRESUMO
Two highly specific biomolecular recognition events, nucleic acid duplex hybridization and DNA-peptide recognition in the minor groove, were coalesced in a miniature ensemble for the first time by covalently attaching a natural AT-hook peptide motif to nucleic acid duplexes via a 2'-amino-LNA scaffold. A combination of molecular dynamics simulations and ultraviolet thermal denaturation studies revealed high sequence-specific affinity of the peptide-oligonucleotide conjugates (POCs) when binding to complementary DNA strands, leveraging the bioinformation encrypted in the minor groove of DNA duplexes. The significant cooperative DNA duplex stabilization may pave the way toward further development of POCs with enhanced affinity and selectivity toward target sequences carrying peptide-binding genetic islands.
Assuntos
DNA/química , Oligonucleotídeos/química , Peptídeos/química , Sítios de Ligação , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Estudo de Prova de Conceito , Conformação ProteicaRESUMO
Bariatric surgery is currently the most effective treatment of obesity, which has spurred an interest in developing pharmaceutical mimetics. It is thought that the marked body weight-lowering effects of bariatric surgery involve stimulated secretion of appetite-regulating gut hormones, including glucagon-like peptide 1. We here report that intestinal expression of secretin is markedly upregulated in a rat model of Roux-en-Y gastric bypass, suggesting an additional role of secretin in the beneficial metabolic effects of Roux-en-Y gastric bypass. We therefore developed novel secretin-based peptide co-agonists and identified a lead compound, GUB06-046, that exhibited potent agonism of both the secretin receptor and glucagon-like peptide 1 receptor. Semi-acute administration of GUB06-046 to lean mice significantly decreased cumulative food intake and improved glucose tolerance. Chronic administration of GUB06-046 to diabetic db/db mice for 8 weeks improved glycemic control, as indicated by a 39% decrease in fasting blood glucose and 1.6% reduction of plasma HbA1c levels. Stereological analysis of db/db mice pancreata revealed a 78% increase in beta-cell mass after GUB06-046 treatment, with no impact on exocrine pancreas mass or pancreatic duct epithelial mass. The data demonstrate beneficial effects of GUB06-046 on appetite regulation, glucose homeostasis, and beta-cell mass in db/db mice, without proliferative effects on the exocrine pancreas and the pancreatic duct epithelium. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Índice Glicêmico/efeitos dos fármacos , Obesidade/tratamento farmacológico , Peptídeos/administração & dosagem , Secretina/química , Animais , Cirurgia Bariátrica , Proliferação de Células , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Obesidade/metabolismo , Obesidade/cirurgia , Peptídeos/farmacologia , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores dos Hormônios Gastrointestinais/agonistas , Secretina/metabolismoRESUMO
Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases.
Assuntos
Encéfalo/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Lipopeptídeos/farmacocinética , RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Drosophila melanogaster , Células HEK293 , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Masculino , Peptídeos/genética , Peptídeos/metabolismo , RNA/metabolismo , Ratos Sprague-DawleyRESUMO
Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We recently showed that membrane shape/curvature can in itself mediate the recruitment of lipidated proteins. However, exactly how membrane curvature and composition synergize remains largely unexplored. Here we investigated how three critical structural parameters of lipids, namely acyl chain saturation, headgroup size, and acyl chain length, modulate the capacity of membrane curvature to recruit lipidated proteins. As a model system we used the lipidated minimal membrane anchor of the GTPase, N-Ras (tN-Ras). Our data revealed complex synergistic effects, whereby tN-Ras binding was higher on planar DOPC than POPC membranes, but inversely higher on curved POPC than DOPC membranes. This variation in the binding to both planar and curved membranes leads to a net increase in the recruitment by membrane curvature of tN-Ras when reducing the acyl chain saturation state. Additionally, we found increased recruitment by membrane curvature of tN-Ras when substituting PC for PE, and when decreasing acyl chain length from 14 to 12 carbons (DMPC versus DLPC). However, these variations in recruitment ability had different origins, with the headgroup size primarily influencing tN-Ras binding to planar membranes whereas the change in acyl chain length primarily affected binding to curved membranes. Molecular field theory calculations recapitulated these findings and revealed lateral pressure as an underlying biophysical mechanism dictating how curvature and composition synergize to modulate recruitment of lipidated proteins. Our findings suggest that the different compositions of cellular compartments could modulate the potency of membrane curvature to recruit lipidated proteins and thereby synergistically regulate the trafficking and sorting of lipidated proteins.
Assuntos
Genes ras , Lipossomos/química , Modelos Moleculares , Fosfatidilcolinas/química , Pressão , Ligação Proteica , Propriedades de SuperfícieRESUMO
The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the ß1 adrenergic receptor and the ß2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.
Assuntos
Membrana Celular/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Animais , Membrana Celular/química , Imagem Óptica , Células PC12 , Fragmentos de Peptídeos/farmacologia , Peptídeo YY/farmacologia , Ratos , Receptores Acoplados a Proteínas G/agonistas , TermodinâmicaRESUMO
The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12â nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.
Assuntos
Oligonucleotídeos/química , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Catálise , Dicroísmo Circular , Cobre/química , Reação de Cicloadição , Eletroforese em Gel de Poliacrilamida , Hibridização de Ácido Nucleico , Desnaturação Proteica , Estrutura Secundária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.
Assuntos
Oligonucleotídeos Antissenso/química , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Química Click , DNA/genética , DNA/metabolismo , Humanos , Oligonucleotídeos/sangue , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/sangue , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Biblioteca de Peptídeos , Peptídeos/sangue , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , RNA/genética , RNA/metabolismoRESUMO
Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide-conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide-conjugated liposomes induced only cytotoxicity at the highest concentration in non-activated or activated endothelial cells, as well as in co-culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide-conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5ß1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co-culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide-conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.
Assuntos
Sistemas de Liberação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Integrinas/metabolismo , Monócitos/efeitos dos fármacos , Oligopeptídeos/química , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/imunologia , Relação Dose-Resposta a Droga , Endocitose , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Lipopolissacarídeos/farmacologia , Lipossomos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Microscopia de Fluorescência , Terapia de Alvo Molecular , Monócitos/imunologia , Monócitos/metabolismo , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides.