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Développement d’une nouvelle approche de criblage et de caractérisation d’interactions ligand-protéine membranaire par chromatographie de faible affinité miniaturisée

Abstract : Fragment-Based Drug Discovery consist of the development of new drugs from fragments (small organic molecules of weak affinity for the target protein) and requires screening methods suitable for the detection of weak affinities (μM-mM). Recently, Weak Affinity Chromatography has been shown to be an interesting alternative approach for fragment screening and consists of immobilizing the target protein on a chromatographic support and then measuring the fragments retention, directly linked to their affinity for the target. To minimize the protein consumption and allow the study of targets difficult to produce, we decided to develop this approach at a miniaturized scale. In practice, monoliths were in-situ synthesized in 75 µm i.d. capillaries and the target model protein was in-situ grafted. Columns were evaluated using frontal analysis with ligands of known affinity for a model protein. Among different combinations, best results were achieved with a streptavidin modified organic monolith which allows the fast immobilization of biotinylated target protein (through the strong streptavidin-biotin interaction). We then focused on a membrane protein, the adenosine A2A receptor (AA2AR), embedded in a biotinylated nanodiscs then grafted on streptavidin columns, consuming less than 1 µg of protein per column. Affinities down to hundreds of µM were quantified, confirming protein activity after immobilization. Ligand identification among fragments illustrates the full potential of ultra-miniaturized weak affinity chromatography for Fragment-Based Drug Discovery towards membrane proteins
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https://tel.archives-ouvertes.fr/tel-02500773
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Submitted on : Friday, March 6, 2020 - 12:38:09 PM
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Lucile Lecas. Développement d’une nouvelle approche de criblage et de caractérisation d’interactions ligand-protéine membranaire par chromatographie de faible affinité miniaturisée. Chimie analytique. Université de Lyon, 2019. Français. ⟨NNT : 2019LYSE1279⟩. ⟨tel-02500773⟩

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