RESUMO
The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET-M33DIM) and evaluated as a candidate drug for infections due to multidrug-resistant (MDR) Gram-negative pathogens. SET-M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration [MICs], 1.5-11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11-22 µM). It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC50 was as much as 22 times better than that of SET-M33, a peptide with the same amino-acid sequence, but synthesized in tetra-branched form (638 vs 28 µM). In in vivo and ex vivo experiments, SET-M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge. It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/farmacologia , Pneumonia Bacteriana/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Animais , Antibacterianos/síntese química , Farmacorresistência Bacteriana Múltipla , Feminino , Hospedeiro Imunocomprometido , Lipopolissacarídeos , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa , Células RAW 264.7 , Testes de ToxicidadeRESUMO
The tumor-specific tetrabranched peptide NT4 binds membrane sulfate glycosaminoglycans and receptors belonging to the low density lipoprotein receptor-related protein (LRP) family, like LRP6, which are overexpressed in cancer. The binding occurs through a multimeric positively-charged motif of NT4 that interacts with negatively charged motives in both glycosaminoglycans and LRP receptors. LRP6 has an essential function in canonical Wnt signaling, acting together with receptors of the Frizzled family as coreceptor for Wnt ligands. The extracellular domain of LRP6 contains four YWTD ß-propellers, which are fundamental for interactions with ligands, such as Wnt and Wnt inhibitors. To investigate the molecular interactions between the NT4 peptide and LRP6 receptor, we synthesized a library of epitope mapping peptides reproducing the YWTD ß-propeller 3 and 4 of LRP6. The peptides that showed to bind NT4 represented the portion of LRP6 located on the top face of ß-propeller 3 and contained negatively charged residues, including glutamic acid-708 which is known to be involved in Wnt3a interaction. The results pave the way for a possible development of peptide inhibitors of Wnt3a pathway to be used as drugs in oncology.
Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neurotensina/metabolismo , Humanos , Ligantes , Neurotensina/análogos & derivados , Neurotensina/síntese química , Ressonância de Plasmônio de Superfície/métodos , Via de Sinalização WntRESUMO
The development of selective tumor targeting agents to deliver multiple units of chemotherapy drugs to cancer tissue would improve treatment efficacy and greatly advance progress in cancer therapy. Here we report a new drug delivery system based on a tetrabranched peptide known as NT4, which is a promising cancer theranostic by virtue of its high cancer selectivity. We developed NT4 directly conjugated with one, two, or three units of paclitaxel and an NT4-based nanosystem, using NIR-emitting quantum dots, loaded with the NT4 tumor-targeting agent and conjugated with paclitaxel, to obtain a NT4-QD-PTX nanodevice designed to simultaneously detect and kill tumor cells. The selective binding and in vitro cytotoxicity of NT4-QD-PTX were higher than for unlabeled QD-PTX when tested on the human colon adenocarcinoma cell line HT-29. NT4-QD-PTX tumor-targeted nanoparticles can be considered promising for early tumor detection and for the development of effective treatments combining simultaneous therapy and diagnosis.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Paclitaxel , Peptídeos , Pontos Quânticos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HT29 , Humanos , Paclitaxel/química , Paclitaxel/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Pontos Quânticos/química , Pontos Quânticos/uso terapêuticoRESUMO
BACKGROUND: Near-infrared quantum dots (NIR QDs) are a new class of fluorescent labels with excellent bioimaging features, such as high fluorescence intensity, good fluorescence stability, sufficient electron density, and strong tissue-penetrating ability. For all such features, NIR QDs have great potential for early cancer diagnosis, in vivo tumor imaging and high resolution electron microscopy studies on cancer cells. RESULTS: In the present study we constructed NIR QDs functionalized with the NT4 cancer-selective tetrabranched peptides (NT4-QDs). We observed specific uptake of NT4-QDs in human cancer cells in in vitro experiments and a much higher selective accumulation and retention of targeted QDs at the tumor site, compared to not targeted QDs, in a colon cancer mouse model. CONCLUSIONS: NIR QDs labelled with the tetrabranched NT4 peptide have very promising performance for selective addressing of tumor cells in vitro and in vivo, proving rising features of NT4-QDs as theranostics.
Assuntos
Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Imagem Óptica/métodos , Peptídeos/química , Pontos Quânticos/química , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Raios Infravermelhos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Peptídeos/metabolismo , Pontos Quânticos/metabolismo , Pontos Quânticos/ultraestruturaRESUMO
Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 µM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all-l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.
Assuntos
Proteínas de Anfíbios/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Mucosa Respiratória/microbiologia , Proteínas de Anfíbios/química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/química , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Humanos , Lipopolissacarídeos , Microscopia Confocal , Infecções por Pseudomonas/microbiologiaRESUMO
M33 is a branched peptide currently under preclinical characterization for the development of a new antibacterial drug against gram-negative bacteria. Here, we report its pegylation at the C-terminus of the three-lysine-branching core and the resulting increase in stability to Pseudomonas aeruginosa elastase. This protease is a virulence factor that acts by destroying peptides of the native immune system. Peptide resistance to this protease is an important feature for M33-Peg activity against Pseudomonas.
Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/metabolismo , Elastase Pancreática/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Antibacterianos/síntese química , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/genética , Polietilenoglicóis/químicaRESUMO
We report the identification of a new human tumor necrosis factor-alpha (TNF-α) specific peptide selected by competitive panning of a phage library. Competitive elution of phages was obtained using the monoclonal antibody adalimumab, which neutralizes pro-inflammatory processes caused by over-production of TNF-α in vivo, and is used to treat severe symptoms of rheumatoid arthritis. The selected peptide was synthesized in monomeric and branched form and analyzed for binding to TNF-α and competition with adalimumab and TNF-α receptors. Results of competition with TNF-α receptors in surface plasmon resonance and melanoma cells expressing both TNF receptors make the peptide a candidate compound for the development of a novel anti-TNF-α drug.
Assuntos
Biblioteca de Peptídeos , Peptídeos/farmacologia , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Humanos , Ligação Proteica/efeitos dos fármacos , Técnicas de Síntese em Fase Sólida , Ressonância de Plasmônio de SuperfícieRESUMO
The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Células Epiteliais/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Acinetobacter baumannii/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Brônquios/citologia , Linhagem Celular , Citrobacter freundii/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Fragmentos de Peptídeos/farmacologia , Pseudomonas/efeitos dos fármacos , Mucosa Respiratória/citologiaRESUMO
Receptors for endogenous regulatory peptides, like the neuropeptide neurotensin, are overexpressed in several human cancers and can be targets for peptide-mediated tumor-selective therapy. Peptides, however, have the main drawback of an extremely short half-life in vivo. We showed that neurotensin and other endogenous peptides, when synthesized as dendrimers, retain biological activity and become resistant to proteolysis. Here, we synthesized the neurotensin functional fragment NT(8-13) in a tetrabranched form linked to different units for tumor therapy or diagnosis. Fluorescent molecules were used to monitor receptor binding and internalization in HT29 human adenocarcinoma cells and receptor binding in HT29 tumor xenografts in nude mice. Linking of chemotherapic molecules like chlorin e6 and methotrexate to dendrimers resulted in a dramatic increase in drug selectivity, uptake of which by target cells became dependent on peptide receptor binding. When nude mice carrying human tumor xenografts were treated with branched NT(8-13)-methotrexate, a 60% reduction in tumor growth was observed with respect to mice treated with the free drug.
Assuntos
Neoplasias Experimentais/tratamento farmacológico , Neurotensina/síntese química , Neurotensina/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada , Células HT29 , Humanos , Metotrexato/química , Metotrexato/uso terapêutico , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Neurotensina/química , Receptores de Neurotensina/metabolismoRESUMO
Heparan sulfate proteoglycans, HSPGs, modulate major transformations of cancer cells, leading to tumor growth, invasion and metastasis. HSPGs also regulate neo-angiogenesis which prompts cancer progression and metastatic spread. A different aspect of heparin and analogs is their prominent role in the coagulation of blood. The interplay between coagulation and metastasis is being actively studied: anticoagulants such as heparin-derivatives have anticancer activity and procoagulants, such as thrombin, positively modulate proliferation, migration and invasion. The branched peptide NT4 binds to HSPGs and targets selectively cancer cells and tissues. For this, it had been extensively investigated in the last years and it proved to be efficient as chemotherapeutic and tumor tracer in in vivo models of cancer. We investigated the effects of the branched peptide in terms of modulation of angiogenesis and invasiveness of cancer cells. NT4 proved to have a major impact on endothelial cell proliferation, migration and tube formation, particularly when induced by FGF2 and thrombin. In addition, NT4 had important effects on aggressive tumor cells migration and invasion and it also had an anticoagulant profile.The peptide showed very interesting evidence of interference with tumor invasion pathways, offering a cue for its development as a tumor-targeting drug, and also for its use in the study of links between coagulation and tumor progression involving HSPGs.
Assuntos
Anticoagulantes/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteoglicanas de Heparan Sulfato/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/prevenção & controle , Peptídeos/farmacologia , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Células MCF-7 , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Peptídeos/química , Peptídeos/uso terapêuticoRESUMO
Cancer-selective tetra-branched peptides, named NT4, can be coupled to different functional units for cancer cell imaging or therapy. NT4 peptides specifically bind to lipoprotein receptor-related proteins (LRP) receptors and to heparan sulfate chains on membrane proteoglycans and can be efficiently internalized by cancer cells expressing these membrane targets. Since binding and internalization of NT4 peptides is mediated by specific NT4 receptors on cancer cell membranes and this may allow drug resistance produced by drug membrane transporters to be by-passed, we tested the ability of drug-armed NT4 to by-pass drug resistance in cancer cell lines. We found that MTX-conjugated NT4 allows drug resistance to be by-passed in MTX-resistant human breast cancer cells lacking expression of folate reduced carrier. NT4 peptides appear to be extremely promising cancer-selective targeting agents that can be exploited as theranostics in personalized oncological applications.
RESUMO
A synthetic antimicrobial peptide was identified as a possible candidate for the development of a new antibacterial drug. The peptide, SET-M33L, showed a MIC90 below 1.5 µM and 3 µM for Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively. In in vivo models of P. aeruginosa infections, the peptide and its pegylated form (SET-M33L-PEG) enabled a survival percentage of 60-80% in sepsis and lung infections when injected twice i.v. at 5 mg/Kg, and completely healed skin infections when administered topically. Plasma clearance showed different kinetics for SET-M33L and SET-M33L-PEG, the latter having greater persistence two hours after injection. Bio-distribution in organs did not show significant differences in uptake of the two peptides. Unlike colistin, SET-M33L did not select resistant mutants in bacterial cultures and also proved non genotoxic and to have much lower in vivo toxicity than antimicrobial peptides already used in clinical practice. The characterizations reported here are part of a preclinical development plan that should bring the molecule to clinical trial in the next few years.
Assuntos
Antibacterianos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Klebsiella pneumoniae/fisiologia , Pneumonia/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/fisiologia , Sepse/tratamento farmacológico , Pele/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Colistina/uso terapêutico , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Pele/microbiologia , Pele/patologiaRESUMO
The structure of peptide p6.7, a mimotope of the nicotinic receptor ligand site that binds alpha-bungarotoxin and neutralizes its toxicity, was compared to that of the acetylcholine binding protein. The central loop of p6.7, when complexed with alpha-bungarotoxin, fits the structure of the acetylcholine binding protein (AChBP) ligand site, whereas peptide terminal residues seem to be less involved in toxin binding. The minimal binding sequence of p6.7 was confirmed experimentally by synthesis of progressively deleted peptides. Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new alpha-bungarotoxin binding peptides on the basis of their dissociation kinetic rate. The tetra-branched forms of the resulting high-affinity peptides were effective as antidotes in vivo at a significantly lower dose than the tetra-branched lead peptide.
Assuntos
Antídotos/síntese química , Bungarotoxinas/metabolismo , Peptídeos/síntese química , Receptores Nicotínicos/metabolismo , Sequência de Aminoácidos , Animais , Antídotos/metabolismo , Sítios de Ligação , Bungarotoxinas/química , Bungarotoxinas/toxicidade , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Desenho de Fármacos , Cinética , Camundongos , Modelos Moleculares , Mimetismo Molecular , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores Nicotínicos/química , Alinhamento de SequênciaRESUMO
Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy, and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively, and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo. NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Portadores de Fármacos/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Peptídeos/farmacologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Portadores de Fármacos/química , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Paclitaxel/química , Peptídeos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In previous papers we demonstrated that tetrabranched peptides containing the sequence of human neurotensin, NT4, are much more selective than native monomeric analogues for binding to different human cancer cells and tissues. We show here that the much higher binding of NT4 peptides, with respect to native neurotensin, to either cancer cell lines or human cancer surgical samples is generated by a switch in selectivity toward additional membrane receptors, which are specifically expressed by different human cancers. We demonstrate that the branched structure provides NT4 with ability to bind heparin and receptors belonging to the low density lipoprotein receptor (LDLR) family, known to be involved in cancer biology. Systematic modification of neurotensin sequence in NT4 peptides led to identification of a multimeric positively charged motif, which mediates interaction with both heparin and endocytic receptors. Our findings provide the molecular basis for construction of cancer theranostics with high cancer selectivity.
Assuntos
Glicosaminoglicanos/metabolismo , Neurotensina/metabolismo , Receptores de Superfície Celular/metabolismo , Sulfatos/metabolismo , Linhagem Celular Tumoral , Citocinas/química , Citocinas/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Heparitina Sulfato/metabolismo , Humanos , Midkina , Modelos Moleculares , Neurotensina/química , Ligação Proteica/efeitos dos fármacos , Conformação ProteicaRESUMO
The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.