RESUMO
The resurgence of influenza viruses as a significant global threat emphasizes the urgent need for innovative antiviral strategies beyond existing treatments. Here, we present the development and evaluation of a novel super-multivalent sialyllactosylated filamentous phage, termed t-6SLPhage, as a potent entry blocker for influenza A viruses. Structural variations in sialyllactosyl ligands, including linkage type, valency, net charge, and spacer length, were systematically explored to identify optimal binding characteristics against target hemagglutinins and influenza viruses. The selected SLPhage equipped with optimal ligands, exhibited exceptional inhibitory potency in in vitro infection inhibition assays. Furthermore, in vivo studies demonstrated its efficacy as both a preventive and therapeutic intervention, even when administered post-exposure at 2 days post-infection, under 4 lethal dose 50% conditions. Remarkably, co-administration with oseltamivir revealed a synergistic effect, suggesting potential combination therapies to enhance efficacy and mitigate resistance. Our findings highlight the efficacy and safety of sialylated filamentous bacteriophages as promising influenza inhibitors. Moreover, the versatility of M13 phages for surface modifications offers avenues for further engineering to enhance therapeutic and preventive performance.
RESUMO
Nanodiscs containing sialic acid, which binds the hemagglutinin of the influenza virus, rupture the viral envelope and entrap viral ribonucleoproteins in the endolysosome. While nanodiscs are potent antiviral platforms, ganglioside GD1a containing α2,3-sialic acid does not cover all virus strains. When two nanodiscs containing different receptors 6'-sialyllactose and GD1a were mixed, one nanodisc inhibited the function of the other. A nanodisc loaded with two different receptors exhibited a biased activity toward only one receptor precluding the generation of a multifunctional nanodisc. Here, we suggest hetero di-disc, in which two nanodiscs loaded with each receptor were conjugated through protein trans-splicing for a broad-spectrum antiviral. The hetero di-disc showed strong antiviral activity in vitro and in vivo. Our results suggested that hetero di-discs not only expanded the inhibitory spectrum of nanodiscs but also enabled nanodisc-based delivery of multiple ligands without interference.
Assuntos
Influenza Humana , Antivirais/farmacologia , Hemaglutininas , Humanos , Influenza Humana/tratamento farmacológico , Ácido N-Acetilneuramínico/metabolismoRESUMO
Lipid-bilayer nanodiscs (NDs) wrapped in membrane scaffold proteins (MSPs) have primarily been used to study membrane proteins of interest in a physiological environment. Recently, NDs have been employed in broader applications including drug delivery, cancer immunotherapy, bio-imaging, and therapeutic virucides. Here, we developed a method to synthesize a dimeric nanodisc, whose MSPs are circularly end-spliced, with long-term thermal stability and resistance to aggregation. The end-spliced nanodiscs (esNDs) were assembled using MSPs that were self-circularized inside the cytoplasm ofEscherichia colivia highly efficient protein trans-splicing. The esNDs demonstrated a consistent size and 4-5-fold higher stability against heat and aggregation than conventional NDs. Moreover, cysteine residues on trans-spliced circularized MSPs allowed us to modulate the formation of either monomeric nanodiscs (essNDs) or dimeric nanodiscs (esdNDs) by controlling the oxidation/reduction conditions and lipid-to-protein ratios. When the esdNDs were used to prepare an antiviral nanoperforator that induced the disruption of the viral membrane upon contact, antiviral activity was dramatically increased, suggesting that the dimerization of nanodiscs led to cooperativity between linked nanodiscs. We expect that controllable structures, long-term stability, and aggregation resistance of esNDs will aid the development of novel versatile membrane-mimetic nanomaterials with flexible designs and improved therapeutic efficacy.
Assuntos
Antivirais/uso terapêutico , Proteínas de Membrana/uso terapêutico , Nanoestruturas/uso terapêutico , Animais , Antivirais/química , Escherichia coli/genética , Feminino , Bicamadas Lipídicas/química , Bicamadas Lipídicas/uso terapêutico , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nanoestruturas/química , Orthomyxoviridae/efeitos dos fármacos , Trans-Splicing , Envelope Viral/efeitos dos fármacosRESUMO
Influenza, one of the most contagious and infectious diseases, is predominantly transmitted through aerosols, leading to the development of filter-based protective equipment. Though the currently available filters are effective at removing submicron-sized particulates, filter materials with enhanced virus-capture efficiency are still in demand. Coating or chemically modifying filters with molecules capable of binding influenza viruses has received attention as a promising approach for the production of virus-capturing filters. For this purpose, tannic acid (TA), a plant-derived polyphenol, is a promising molecule for filter functionalization because of its antiviral activities and ability to serve as a cost-efficient adhesive for various materials. This study demonstrates the facile preparation of TA-functionalized high-efficiency particulate air (HEPA) filter materials and their efficiency in influenza virus capture. Polypropylene HEPA filter fabrics were coated with TA via a dipping/washing process. The TA-functionalized HEPA filter (TA-HF) exhibits a high in-solution virus capture efficiency of up to 2,723 pfu/mm2 within 10 min, which is almost two orders of magnitude higher than that of non-functionalized filters. This result suggests that the TA-HF is a potent anti-influenza filter that can be used in protective equipment to prevent the spread of pathogenic viruses.
Assuntos
Filtros de Ar/virologia , Filtração/instrumentação , Orthomyxoviridae/química , Taninos/química , Aerossóis/química , Poeira/prevenção & controle , Filtração/métodos , Tamanho da Partícula , Têxteis/virologiaRESUMO
Owing to the emerging resistance to current anti-influenza therapies, strategies for blocking virus-cell interaction with agents that mimic interactions with host cell receptors are garnering interest. In this context, a multivalent presentation of sialyl groups on various types of scaffold materials such as dendrimers, liposomes, nanoparticles, and natural/synthetic polymers has been investigated for the inhibition of influenza A virus infection. However, the development of versatile antiviral agents based on monodisperse scaffolds capable of precise molecular design remains challenging. Whether an anisotropically extended filamentous nanostructure can serve as an effective scaffold for maximum inhibition of viral cell attachment has not been investigated. In this study, the preparation of a series of sialyllactose-conjugated filamentous bacteriophages (SLPhages), with controlled loading levels, ligand valencies, and two types of sialyllactose (α2,3' and α2,6'), is demonstrated. With optimal ligand loading and valency, SLPhages showed inhibitory activity (in vitro) against influenza A viruses at concentrations of tens of picomolar. This remarkable inhibition is due to the strong interaction between the SLPhage and the virus; this interaction is adequately potent to compensate for the cost of the bending and wrapping of the SLPhage around the influenza virus. Our study may open new avenues for the development of filamentous anti-viral agents, in which virus-wrapping or aggregation is the primary feature responsible for the blocking of cell entry.
Assuntos
Vírus da Influenza A , Influenza Humana , Nanopartículas , Antivirais/farmacologia , Humanos , Influenza Humana/tratamento farmacológicoRESUMO
Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and ß-secretase in a concentration-dependent manner. Particularly, ß-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Grafite , Peptídeo Hidrolases , Peptídeos/síntese química , ÓxidosRESUMO
Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity (
Assuntos
Antivirais/farmacologia , Membrana Celular/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas da Matriz Viral/química , Sequência de Aminoácidos , Animais , Antivirais/síntese química , Membrana Celular/química , Membrana Celular/virologia , Cães , Interações Hidrofóbicas e Hidrofílicas , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Concentração Inibidora 50 , Bicamadas Lipídicas/química , Lipossomos/química , Células Madin Darby de Rim Canino , Peptídeos/síntese química , Relação Estrutura-Atividade , Carga Viral/efeitos dos fármacosRESUMO
Membrane-disrupting agents that selectively target virus versus host membranes could potentially inhibit a broad-spectrum of enveloped viruses, but currently such antivirals are lacking. Here, we develop a nanodisc incorporated with a decoy virus receptor that inhibits virus infection. Mechanistically, nanodiscs carrying the viral receptor sialic acid bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscs rupture the viral envelope, trapping viral RNAs inside the endolysosome for enzymatic decomposition. In contrast, liposomes containing a decoy receptor show weak antiviral activity due to the lack of membrane disruption. The nanodiscs inhibit influenza virus infection and reduce morbidity and mortality in a mouse model. Our results suggest a new class of antivirals applicable to other enveloped viruses that cause irreversible physical damage specifically to virus envelope by viruses' own fusion machine. In conclusion, the lipid nanostructure provides another dimension for antiviral activity of decoy molecules.
Assuntos
Antivirais/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , RNA Viral/metabolismo , Células A549 , Animais , Antivirais/química , Antivirais/uso terapêutico , Bioengenharia/métodos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Modelos Animais de Doenças , Cães , Endossomos/metabolismo , Feminino , Humanos , Vírus da Influenza A/fisiologia , Influenza Humana/mortalidade , Influenza Humana/virologia , Bicamadas Lipídicas/química , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Oseltamivir/uso terapêutico , Receptores de Superfície Celular/química , Proteínas Virais/química , Vírion/efeitos dos fármacos , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacosRESUMO
As the M13 bacteriophage, which has integrin binding and calcium binding sites, provides topological cues from the nanofibrous shape and biochemical cues from the Arg-Gly-Asp (RGD) sequence attached to the surface of fibrous phage, it has been recommended as a bioactive component for use in bone tissue engineering. However, although it has good biological activities, its low mechanical properties and low processing ability represent major issues that must be overcome before its use as a tissue engineering substitute. To overcome these issues, we chemically conjugated the M13 bacteriophage and alginate with a cross-linking agent and it was used as a bioactive component on electrospun poly(ε-caprolactone) (PCL) micro/nanofibres. Assessment of the physical properties and in vitro biocompatibility using osteoblast-like cells indicated that the biocomposite supplemented with the conjugated phage/alginate was mechanically enhanced, and the extent of mineralisation of cells on the composite was significantly higher compared to that on the fibrous composites fabricated using physically mixed M13 phage/alginate and RGD-modified alginate. These results indicate that M13 phage-conjugated alginate may have potential to be used as an excellent bioactive component for bone tissue regeneration.
RESUMO
Developing hierarchically structured biomaterials with tunable chemical and physical properties like those found in nature is critically important to regenerative medicine and studies on tissue morphogenesis. Despite advances in materials synthesis and assembly processes, our ability to control hierarchical assembly using fibrillar biomolecules remains limited. Here, we developed a bioinspired approach to create collagen-like materials through directed evolutionary screening and directed self-assembly. We first synthesized peptide amphiphiles by coupling phage display-identified collagen-like peptides to long-chain fatty acids. We then assembled the amphiphiles into diverse, hierarchically organized, nanofibrous structures using directed self-assembly based on liquid crystal flow and its controlled deposition. The resulting structures sustained and directed the growth of bone cells and hydroxyapatite biominerals. We believe these self-assembling collagen-like amphiphiles could prove useful in the structural design of tissue regenerating materials.