Investigation of the Fuzzy Complex between RSV Nucleoprotein and Phosphoprotein to Optimize an Inhibition Assay by Fluorescence Polarization
| Autor: | Silva Khodjoyan, Deborha Morissette, Fortune Hontonnou, Luis Checa Ruano, Charles-Adrien Richard, Olivier Sperandio, Jean-François Eléouët, Marie Galloux, Philippe Durand, Stéphanie Deville-Foillard, Christina Sizun |
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| Přispěvatelé: | Institut de Chimie des Substances Naturelles (ICSN), Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Collège Doctoral, Sorbonne Université (SU), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), ANR-19-CE18-0012,AntiBronchio,Conception de molécules pour lutter contre le virus de la bronchiolite grâce à une nouvelle cible thérapeutique(2019) |
| Rok vydání: | 2022 |
| Předmět: |
Inorganic Chemistry
Organic Chemistry [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology respiratory syncytial virus phosphoprotein nucleoprotein replication complex fuzzy complex protein–protein interaction PPI inhibition nuclear magnetic resonance fluorescence anisotropy B-cyano BODIPYs General Medicine Physical and Theoretical Chemistry Molecular Biology Spectroscopy Catalysis Computer Science Applications |
| Zdroj: | International Journal of Molecular Sciences; Volume 24; Issue 1; Pages: 569 International Journal of Molecular Sciences International Journal of Molecular Sciences, 2023, 24 (1), pp.569. ⟨10.3390/ijms24010569⟩ |
| ISSN: | 1422-0067 1661-6596 |
| DOI: | 10.3390/ijms24010569 |
| Popis: | International audience; The interaction between Respiratory Syncytial Virus phosphoprotein P and nucleoprotein N is essential for the formation of the holo RSV polymerase that carries out replication. In vitro screening of antivirals targeting the N-P protein interaction requires a molecular interaction model, ideally consisting of a complex between N protein and a short peptide corresponding to the C-terminal tail of the P protein. However, the flexibility of C-terminal P peptides as well as their phosphorylation status play a role in binding and may bias the outcome of an inhibition assay. We therefore investigated binding affinities and dynamics of this interaction by testing two N protein constructs and P peptides of different lengths and composition, using nuclear magnetic resonance and fluorescence polarization (FP). We show that, although the last C-terminal Phe241 residue is the main determinant for anchoring P to N, only longer peptides afford sub-micromolar affinity, despite increasing mobility towards the N-terminus. We investigated competitive binding by peptides and small compounds, including molecules used as fluorescent labels in FP. Based on these results, we draw optimized parameters for a robust RSV N-P inhibition assay and validated this assay with the M76 molecule, which displays antiviral properties, for further screening of chemical libraries. |
| Databáze: | OpenAIRE |
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