Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.
Proteins
; 89(11): 1508-1521, 2021 11.
Article
em En
| MEDLINE
| ID: mdl-34219271
ABSTRACT
Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Fator de Necrose Tumoral alfa
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Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade
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Fator A de Crescimento do Endotélio Vascular
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Receptores Tipo II do Fator de Necrose Tumoral
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Endotoxinas
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Anti-Inflamatórios
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article