Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

Autor: Claire Raingeval, Claire Demesmay, Renaud Wagner, Lucie Hartmann, Lydia N. Caro, Vincent Dugas, Sarah Mohamed-Bouteben, Lucile Lecas, Isabelle Krimm
Přispěvatelé: Separative Methods - Techniques séparatives, Institut des Sciences Analytiques (ISA), Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Biotechnologie et signalisation cellulaire (BSC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Centre de RMN à très hauts champs de Lyon (CRMN), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Strasbourg (UNISTRA)-Institut de recherche de l'Ecole de biotechnologie de Strasbourg (IREBS)-Centre National de la Recherche Scientifique (CNRS), Separative Methods - Techniques séparatives (TechSep), Université de Lyon-Université de Lyon-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Low protein
Receptor
Adenosine A2A
02 engineering and technology
Protein-ligand interaction
[CHIM.THER]Chemical Sciences/Medicinal Chemistry
Ligands
01 natural sciences
Biochemistry
Proof of Concept Study
Chromatography
Affinity
Analytical Chemistry
Affinity chromatography
[CHIM.ANAL]Chemical Sciences/Analytical chemistry
Drug Discovery
Weak affinity chromatography
Environmental Chemistry
Humans
[CHIM]Chemical Sciences
Binding site
Organic Chemicals
Integral membrane protein
Spectroscopy
ComputingMilieux_MISCELLANEOUS
G protein-coupled receptor
Miniaturization
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry
Molecular Biology/Structural Biology [q-bio.BM]
Chemistry
[CHIM.ORGA]Chemical Sciences/Organic chemistry
010401 analytical chemistry
Membranes
Artificial
021001 nanoscience & nanotechnology
Small molecule
0104 chemical sciences
Immobilized Proteins
Membrane protein
Biophysics
Nanoparticles
Target protein
Nanodisc
0210 nano-technology
Protein Binding
Zdroj: Analytica Chimica Acta
Analytica Chimica Acta, Elsevier Masson, 2020, 1113, pp.26-35. ⟨10.1016/j.aca.2020.03.062⟩
Analytica Chimica Acta, 2020, 1113, pp.26-35. ⟨10.1016/j.aca.2020.03.062⟩
ISSN: 0003-2670
DOI: 10.1016/j.aca.2020.03.062⟩
Popis: Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 μg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.
Databáze: OpenAIRE
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