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
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
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
In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained.