RESUMO
Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.
Assuntos
Bacteriófagos , Vírus da Influenza A , Internalização do Vírus , Bacteriófagos/metabolismo , Capsídeo/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Ligantes , Ácido N-Acetilneuramínico/farmacologia , Internalização do Vírus/efeitos dos fármacosRESUMO
We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide-modified building block reacts first with an ethynylphosphonite through a Staudinger-phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron-deficient triple bond subsequently undergoes a cysteine-selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine-selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.
Assuntos
Antineoplásicos Imunológicos/química , Cisteína/química , Imunoconjugados/metabolismo , Organofosfonatos/química , Receptor ErbB-2/imunologia , Compostos de Sulfidrila/química , Trastuzumab/química , Antineoplásicos Imunológicos/metabolismo , Cisteína/metabolismo , Humanos , Imunoconjugados/química , Iodoacetamida/química , Iodoacetamida/metabolismo , Maleimidas/química , Maleimidas/metabolismo , Organofosfonatos/metabolismo , Compostos de Sulfidrila/metabolismo , Trastuzumab/metabolismoRESUMO
The title complex, [Cu4I4(C12H27P)4], crystallizes with six mol-ecules in the unit cell and with three independent one-third mol-ecule fragments, completed by application of the relevant symmetry operators, in the asymmetric unit. The tetranuclear copper core shows a tetrahedral geometry (site symmetry 3..). The I atoms also form a tetra-hedron, with Iâ¯I distances of 4.471â (1)â Å. Both tetra-hedra show an orientation similar to that of a pair of self-dual platonic bodies. The edges of the I-tetra-hedral structure are capped to the face centers of the Cu-tetra-hedron and vice versa. The Cufaceâ¯I distances are 2.18â Å (averaged) and the Ifaceâ¯Cu distances are 0.78â Å (averaged). As a geometric consequence of these properties there are eight distorted trigonal-bipyramidal polyhedra evident, wherein each trigonal face builds up the equatorial site and the opposite Cuâ¯I positions form the axial site. As expected, the n-butyl moieties are highly flexible, resulting in large elongations of their anisotropic displacement parameters. Some C atoms of the n-butyl groups were needed to fix alternative discrete disordered positions.
RESUMO
The development of multivalent sialic acid-based inhibitors active against a variety of influenza A virus (IAV) strains has been hampered by high genetic and structural variability of the targeted viral hemagglutinin (HA). Here, we addressed this challenge by employing sialylated polyglycerols (PGs). Efficacy of prototypic PGs was restricted to a narrow spectrum of IAV strains. To understand this restriction, we selected IAV mutants resistant to a prototypic multivalent sialylated PG by serial passaging. Resistance mutations mapped to the receptor binding site of HA, which was accompanied by altered receptor binding profiles of mutant viruses as detected by glycan array analysis. Specifying the inhibitor functionalization to 2,6-α-sialyllactose (SL) and adjusting the linker yielded a rationally designed inhibitor covering an extended spectrum of inhibited IAV strains. These results highlight the importance of integrating virological data with chemical synthesis and structural data for the development of sialylated PGs toward broad anti-influenza compounds.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Glicerol/química , Glicerol/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Polímeros/química , Polímeros/farmacologia , Hemaglutininas/química , Hemaglutininas/metabolismo , Vírus da Influenza A/classificação , Vírus da Influenza A/genética , Estrutura Molecular , Mutação , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.
Assuntos
Allolevivirus/metabolismo , Capsídeo/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/prevenção & controle , Nanopartículas/uso terapêutico , Internalização do Vírus , Células A549 , Animais , Sítios de Ligação , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologia , Ligantes , Células Madin Darby de Rim Canino , Modelos Moleculares , Nanopartículas/metabolismo , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologiaRESUMO
A chemical route to periodic hole arrays in gold films whose holes are loaded with single gold nanoparticles is presented, paving the road to mass production of highly sensitive plasmonic sensors on large areas.