Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros

Base de dados
Ano de publicação
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Proteins ; 92(8): 923-932, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38572606

RESUMO

Genetically encoded fluorescent biosensors (GEFBs) proved to be reliable tracers for many metabolites and cellular processes. In the simplest case, a fluorescent protein (FP) is genetically fused to a sensing protein which undergoes a conformational change upon ligand binding. This drives a rearrangement in the chromophore environment and changes the spectral properties of the FP. Structural determinants of successful biosensors are revealed only in hindsight when the crystal structures of both ligand-bound and ligand-free forms are available. This makes the development of new biosensors for desired analytes a long trial-and-error process. In the current study, we conducted µs-long all atom molecular dynamics (MD) simulations of a maltose biosensor in both the apo (dark) and holo (bright) forms. We performed detailed hydrogen bond occupancy analyses to shed light on the mechanism of ligand induced conformational change in the sensor protein and its allosteric effect on the chromophore environment. We find that two strong indicators for distinguishing bright and dark states of biosensors are due to substantial changes in hydrogen bond dynamics in the system and solvent accessibility of the chromophore.


Assuntos
Técnicas Biossensoriais , Ligação de Hidrogênio , Maltose , Simulação de Dinâmica Molecular , Técnicas Biossensoriais/métodos , Maltose/química , Maltose/metabolismo , Regulação Alostérica , Ligantes , Fluorescência , Ligação Proteica , Conformação Proteica
2.
Free Radic Biol Med ; 182: 260-275, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35240292

RESUMO

Camelidae derived single-domain antibodies (sdAbs), commonly known as nanobodies (Nbs), are the smallest antibody fragments with full antigen-binding capacity. Owing to their desirable properties such as small size, high specificity, strong affinity, excellent stability, and modularity, nanobodies are on their way to overtake conventional antibodies in terms of popularity. To date, a broad range of nanobodies have been generated against different molecular targets with applications spanning basic research, diagnostics, and therapeutics. In the field of molecular imaging, nanobody-based probes have emerged as a powerful tool. Radioactive or fluorescently labeled nanobodies are now used to detect and track many targets in different biological systems using imaging techniques. In this review, we provide an overview of the use of nanobodies as molecular probes. Additionally, we discuss current techniques for the generation, conjugation, and intracellular delivery of nanobodies.


Assuntos
Anticorpos de Domínio Único , Anticorpos , Imagem Molecular , Sondas Moleculares , Fagocitose
3.
Pharmaceutics ; 14(8)2022 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-36015197

RESUMO

Avastin® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants' in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA