New insights on the interaction mechanism of rhTNFα with its antagonists Adalimumab and Etanercept
| Autor: | Raquel Montesino, Pedro José Neira, Jorge R. Toledo, Frank Camacho, Oliberto Sánchez, María Angélica Contreras, Alaín González, Jannel Acosta, Luis Macaya |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
medicine.medical_treatment
Protomer Pharmacology Biochemistry Epitope Etanercept Protein–protein interaction 03 medical and health sciences 0302 clinical medicine Western blot Protein purification Adalimumab medicine Humans Molecular Biology 030304 developmental biology 0303 health sciences medicine.diagnostic_test Tumor Necrosis Factor-alpha Microscale thermophoresis Chemistry Cell Biology Cytokine 030220 oncology & carcinogenesis Tumor necrosis factor alpha medicine.drug |
| Zdroj: | Biochemical Journal. 477:3299-3311 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bcj20200568 |
| Popis: | TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS–PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (10−8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|