RESUMEN
Dengue is an important arboviral infectious disease for which there is currently no specific cure. We report gemini-like (geminoid) alkylated amphiphilic peptides containing lysines in combination with glycines or alanines (C15H31C(O)-Lys-(Gly or Ala)nLys-NHC16H33, shorthand notation C16-KXnK-C16 with X = A or G, and n = 0-2). The representatives with 1 or 2 Ala inhibit dengue protease and human furin, two serine proteases involved in dengue virus infection that have peptides with cationic amino acids as their preferred substrates, with IC50 values in the lower µM range. The geminoid C16-KAK-C16 combined inhibition of DENV2 protease (IC50 2.3 µM) with efficacy against replication of wildtype DENV2 in LLC-MK2 cells (EC50 4.1 µM) and an absence of toxicity. We conclude that the lysine-based geminoids have activity against dengue virus infection, which is based on their inhibition of the proteases involved in viral replication and are therefore promising leads to further developing antiviral therapeutics, not limited to dengue.
Asunto(s)
Antivirales , Virus del Dengue , Furina , Inhibidores de Proteasas , Replicación Viral , Antivirales/farmacología , Dengue/tratamiento farmacológico , Virus del Dengue/efectos de los fármacos , Virus del Dengue/fisiología , Furina/antagonistas & inhibidores , Humanos , Péptido Hidrolasas , Péptidos/farmacología , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Nature's enzymes are an ongoing source of inspiration for scientists. The complex processes behind their selectivity and efficiency is slowly being unraveled, and these findings have spawned many biomimetic catalysts. However, nearly all focus on the conversion of small molecular substrates. Nature itself is replete with inventive catalytic systems which modify, replicate, or decompose entire polymers, often in a processive fashion. Such processivity can, for example, enhance the rate of catalysis by clamping to the polymer substrate, which imparts a large effective molarity. Reviewed herein are the various strategies for processivity in nature's arsenal and their properties. An overview of what has been achieved by chemists aiming to mimic one of nature's greatest tricks is also included.
Asunto(s)
Enzimas/química , Catálisis , Polímeros/química , Especificidad por SustratoRESUMEN
The synthesis and properties of gemini surfactants of the type (R(1)(CO)-Lys(H)-NH)2(CH2)n are reported. For a spacer length of n = 6, the hydrophobic acyl tail was varied in length (R(1) = C8, C10, C12, C14, C16, and C18) and, for R(1) = C18, the degree of unsaturation. For R(1)(CO) = oleoyl (C18:1 Z) the spacer length (n = 2-8) and the stereochemistry of the lysine building block were varied; a 'half-gemini' derivative with a single oleoyl tail and head group was also prepared. The potential of the gemini surfactants to transfer polynucleotides across a cell membrane was investigated by transfection of HeLa cells with beta-galactosidase, both in the presence and absence of the helper lipid DOPE. Oleoyl was found to be by far the best hydrophobic tail for this biological activity, whereas the effect of the lysine stereochemistry was less pronounced. The effect of an optimum spacer length (n = 6) was observed only in the absence of helper lipid. The most active surfactant, i.e. the one with oleoyl chains and n = 6, formed liposomes with sizes in the range of 60-350 nm, and its lipoplex underwent a transition from a lamellar to a hexagonal morphology upon lowering the pH from 7 to 3.
Asunto(s)
Técnicas de Transferencia de Gen , Liposomas/química , Lisina/química , Tensoactivos/química , Cationes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Lípidos/química , Liposomas/síntesis química , Liposomas/ultraestructura , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Modelos Químicos , Estructura Molecular , Fosfatidiletanolaminas/química , Plásmidos/genética , Dispersión del Ángulo Pequeño , Espectrometría de Masa por Ionización de Electrospray , Tensoactivos/síntesis química , Transfección/métodos , Difracción de Rayos X , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The described protocol is a simple and easily implemented method for making dynamic micropatterns for cell culture. It is based on the use of a surface coating material (azido-PLL-g-PEG (APP)) that initially repels cells, but which can be made strongly adherent by addition of a small functional peptide (BCN-RGD) to the cell culture medium. The method can be applied to trigger the adhesion, migration, or shape change of single cells or of populations of cells, and it can be used to create patterned cocultures. The entire process can be subdivided into three main parts. The first part describes the creation of patterned APP substrates. The second part describes cell seeding and "click" triggering of cell adhesion; the final part describes variations that allow the overlay of multiple patterns or the creation of patterned cocultures. The APP coating of substrates and the triggering of adhesion only involves treating the surface with aqueous stock solutions, allowing any biology lab to adopt this technique.
Asunto(s)
Forma de la Célula , Técnicas de Cocultivo/métodos , Animales , Adhesión Celular , Química Clic , Indicadores y Reactivos , Microtecnología , Péptidos/química , Polímeros/química , Propiedades de SuperficieRESUMEN
With the advent of supramolecular chemistry and later nanotechnology a great deal of research has been focused on new types of molecular structures, which are not held together by covalent bonds but by non-covalent mechanical interactions. Examples include the catenane, rotaxane, and knot interlocked structures. The design and synthesis of these architectures is an art by itself and as such is worth being reviewed. In this tutorial review we will focus, however, on the functional aspects of interlocked molecules and discuss how these can find applications, e.g. as artificial muscles, as molecular valves, as components of electronic devices, and as catalysts.
Asunto(s)
Sustancias Macromoleculares/química , Estructura Molecular , Nanotecnología/métodos , Órganos Artificiales , Catálisis , Electrónica , Microscopía de Fuerza Atómica , Músculos , Rotaxanos/química , Relación Estructura-ActividadRESUMEN
In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.
Asunto(s)
Complejos de Coordinación/química , ADN/química , Oligopéptidos/química , Secuencia de Bases , Catálisis , Daño del ADN , Microscopía de Fuerza Atómica , Modelos Moleculares , Oxidación-ReducciónRESUMEN
There's an APP for that: cell-repellent APP (azido-[polylysine-g-PEG]) is used to create substrates for spatially controlled dynamic cell adhesion. The simple addition of a functional peptide to the culture medium rapidly triggers cell adhesion. This highly accessible yet powerful technique allows diverse applications, demonstrated through tissue motility assays, patterned coculturing and triggered cell shape change.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacología , Azidas/química , Compuestos Bicíclicos con Puentes/química , Adhesión Celular/efectos de los fármacos , Células HeLa , Humanos , Oligopéptidos/química , Polietilenglicoles/química , Polilisina/química , Propiedades de Superficie , Ingeniería de TejidosRESUMEN
We report the design, synthesis, and characterization of a novel type of cationic lipopeptide, gemini-like amphiphilic peptides or 'geminoids'. As an example, the SPKR peptide, inspired by biological nucleic acid binding motifs, was appended with unsaturated (oleoyl/oleyl) alkyl tails. The compound shows remarkable DNA and siRNA delivery, without lysogenic helper lipid, in a variety of cells, with a moderate cytotoxic effect. It aggregates to nanoparticles that combine with DNA to lipoplexes, which undergo a change from lamellar to the more lysogenic hexagonal packing upon lowering the pH. The versatility of the chemical approach allowed us to study peptides related to SPKR, and to establish that the Pro and at least one of the cationic (Lys, Arg) residues are essential for the biological activity.
Asunto(s)
ADN/administración & dosificación , Portadores de Fármacos/química , Técnicas de Transferencia de Gen , Oligopéptidos/química , ARN Interferente Pequeño/administración & dosificación , Tensoactivos/química , Aminoácidos/química , Animales , Cationes , ADN/genética , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Lípidos/química , Liposomas , Modelos Moleculares , Nanopartículas/química , Ácidos Oléicos/química , Oligopéptidos/genética , Tamaño de la Partícula , Plásmidos , ARN Interferente Pequeño/genética , Salmón , Dispersión del Ángulo Pequeño , Transfección , Difracción de Rayos XRESUMEN
Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS(.+). Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Nanoestructuras/química , Polímeros/síntesis química , Catálisis , Proteínas Fúngicas , Gluconatos/metabolismo , Glucosa/metabolismo , Glucosa Oxidasa/síntesis química , Glucosa Oxidasa/metabolismo , Peroxidasa de Rábano Silvestre/síntesis química , Peroxidasa de Rábano Silvestre/metabolismo , Cinética , Lactonas , Lipasa/síntesis química , Lipasa/metabolismo , Nanoestructuras/ultraestructura , Oxidación-Reducción , Polímeros/químicaRESUMEN
The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at the N-terminus of enzymes, e.g. horseradish peroxidase (HRP) and the red fluorescent protein DsRed. The effective introduction of azides was verified by mass spectrometry, after which the azido-proteins were used in Cu(I)-catalyzed [3 + 2] cycloaddition reactions. Azido-HRP retained its catalytic activity after conjugation of a small molecule. This modified protein could also be successfully immobilized on the surface of an acetylene-covered polymersome. Azido-DsRed was coupled to an acetylene-bearing protein allowing it to act as a fluorescent label, demonstrating the wide applicability of the diazo transfer procedure.