RESUMEN
Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Polímeros , Unión Proteica , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/metabolismo , Polímeros/química , Humanos , Animales , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Elementos de Respuesta Antioxidante , Neuronas/metabolismo , Neuronas/efectos de los fármacosRESUMEN
Cyclic cell-penetrating peptide 12 (CPP12) is highly efficient for the cytosolic delivery of a variety of cargo molecules into mammalian cells in vitro and in vivo. However, its cytosolic entry efficiency is substantially reduced at lower concentrations or in the presence of serum proteins. In this study, CPP12 analogs were prepared by replacing its hydrophobic residues with amino acids of varying hydrophobicity and evaluated for cellular entry. Substitution of l-3-benzothienylalanine (Bta) for l-2-naphthylalanine (Nal) resulted in CPP12-2, which exhibits up to 3.8-fold higher cytosolic entry efficiency than CPP12, especially at low CPP concentrations; thanks to improved endosomal escape efficiency. CPP12-2 is well suited for the cytosolic delivery of highly potent cargos to achieve biological activity at low concentrations.
Asunto(s)
Péptidos de Penetración Celular , Aminoácidos/metabolismo , Animales , Péptidos de Penetración Celular/química , Citosol/metabolismo , Endosomas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Mamíferos/metabolismoRESUMEN
Intracellular protein-protein interactions (PPIs) are challenging targets for traditional drug modalities. Macrocyclic peptides (MPs) prove highly effective PPI inhibitors in vitro and can be rapidly discovered against PPI targets by rational design or screening combinatorial libraries but are generally impermeable to the cell membrane. Recent advances in MP science and technology are allowing for the development of 'drug-like' MPs that potently and specifically modulate intracellular PPI targets in cell culture and animal models. In this review, we highlight recent progress in generating cell-permeable MPs that enter the mammalian cell by passive diffusion, endocytosis followed by endosomal escape, or as-yet unknown mechanisms.
Asunto(s)
Péptidos Cíclicos , Péptidos , Animales , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Humanos , Mamíferos/metabolismo , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacologíaRESUMEN
The Ras subfamily of small GTPases is mutated in â¼30% of human cancers and represents compelling yet challenging anticancer drug targets owing to their flat protein surface. We previously reported a bicyclic peptidyl inhibitor, cyclorasin B3, which binds selectively to Ras-GTP with modest affinity and blocks its interaction with downstream effector proteins in vitro but lacks cell permeability or biological activity. In this study, optimization of B3 yielded a potent pan-Ras inhibitor, cyclorasin B4-27, which binds selectively to the GTP-bound forms of wild-type and mutant Ras isoforms (KD = 21 nM for KRasG12V-GppNHp) and is highly cell-permeable and metabolically stable (serum t1/2 > 24 h). B4-27 inhibits Ras signaling in vitro and in vivo by blocking Ras from interacting with downstream effector proteins and induces apoptosis of Ras-mutant cancer cells. When administered systemically (i.v.), B4-27 suppressed tumor growth in two different mouse xenograft models at 1-5 mg/kg of daily doses.
Asunto(s)
Antineoplásicos/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Proteínas ras/antagonistas & inhibidores , Animales , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Masculino , Ratones , Ratones Desnudos , Isoformas de Proteínas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Measurements of cellular pH are used to infer information such as stage of cell cycle, presence of cancer and other diseases, as well as delivery or effect of a therapeutic drug. Surface-enhanced Raman spectroscopy (SERS) of nanoparticle-based pH probes have been used to interrogate intracellular pH, with the significant advantage of avoiding photobleaching compared to fluorescent indicators. 4-Mercaptobenzoic acid (MBA) is a commonly used pH-sensitive reporter molecule. Intracellular pH sensing by SERS requires analysis of the observed MBA spectrum and spectral interference can affect the pH determination. Background from common cell containers, imaging too few particles, signal-to-noise ratios, and degradation of reporter molecules are among the factors that may alter appropriate SERS-based pH determination in cells. Here, we have compared common methods of spectral analysis to see how different factors alter the calculated pH in Raman maps of MBA functionalized Au nanostars in SW620 cancer cells. The methods included in our comparison use the relative intensity of the ν(COO-) stretch, chemometric analysis of the ν8a mode, and analyzing the frequency shift of the ν8a mode. These methods show different sensitivity to some of these sources of error in live cell experiments. pH determination based on Raman frequency shift appears to give a more reliable pH determination, though in high signal-to-noise environments, intensity ratios may provide better sensitivity to small changes in pH for cellular imaging.
Asunto(s)
Células/química , Concentración de Iones de Hidrógeno , Espectrometría Raman/métodos , Benzoatos/química , Línea Celular Tumoral , Humanos , Nanopartículas del Metal/química , Plata/química , Compuestos de Sulfhidrilo/químicaRESUMEN
Intracellular delivery of biological agents such as peptides, proteins, and nucleic acids generally rely on the endocytic pathway as the major uptake mechanism, resulting in their entrapment inside the endosome and lysosome. The recent discovery of cell-penetrating molecules of exceptionally high endosomal escape and cytosolic delivery efficiencies and elucidation of their mechanism of action represent major breakthroughs in this field. In this Topical Review, we provide an overview of the recent progress in understanding and enhancing the endosomal escape process and the new opportunities opened up by these recent findings.
Asunto(s)
Portadores de Fármacos/metabolismo , Endosomas/metabolismo , Animales , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Humanos , Fusión de Membrana , Ósmosis , Péptidos/química , Péptidos/metabolismo , Preparaciones Farmacéuticas/administración & dosificaciónRESUMEN
A novel family of amphiphilic ionic carbosilane dendrons containing fatty acids at the focal point were synthesized and characterized. They spontaneously self-assembled in aqueous solution into micelles both in the absence and presence of salt, as confirmed by surface tension, conductivity, and DLS measurements. Dendron based micelles have spherical shapes and increase in size on decreasing dendron generation. These dendritic micelles have been demonstrated to be able to form complexes with therapeutic macromolecules such as siRNA and show a high loading capacity for drugs such as procaine, suggesting their potential use as nanocarriers for therapeutics.
RESUMEN
This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.
Asunto(s)
Dendrímeros/química , Neoplasias/terapia , Fósforo/química , ARN Interferente Pequeño/genética , Tratamiento con ARN de Interferencia/métodos , Silanos/química , Transfección/métodos , Apoptosis , Supervivencia Celular , Dicroismo Circular , Dendrímeros/toxicidad , Electroforesis en Gel de Agar , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Células HeLa , Heparina/química , Humanos , Luz , Microscopía Electrónica de Transmisión , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Conformación de Ácido Nucleico , Tamaño de la Partícula , Fósforo/toxicidad , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Dispersión de Radiación , Silanos/toxicidad , Espectrometría de Fluorescencia , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
Novel hybrids of fluorescein-labeled poly(ethylene glycol)-modified single-walled carbon nanotubes (SWCNTs) with nucleic acids were prepared. 5'-Pyrene conjugates of oligodeoxyribonucleotides were used to construct the noncovalent hybrids, with the pyrene residues acting as anchor groups, immobilizing an oligonucleotide on the SWCNT surface. The hybrid formation characteristics were studied using ζ-potential measurements and adsorption isotherm plots. Transmission electron microscopy (TEM) of the samples stained with contrast agents proved that the pyrene conjugates of oligonucleotides were adsorbed onto the surfaces of the functionalized SWCNTs. On the basis of the MTT assay, the functionalized SWCNTs and their hybrids with oligonucleotides exhibited low toxicity toward HeLa, KB-3-1, and KB-8-5 cells. A TEM study of ultrathin sections of cells treated with SWCNTs revealed that the nanotubes directly interacted with the cellular surface.