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High-Throughput Screening for LC3/GABARAP Binders Utilizing the Fluorescence Polarization Assay.

Martin P Schwalm1,2, Johannes Dopfer1,2, Stefan Knapp1,2

  • 1Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|August 8, 2024
PubMed
Summary

Understanding interactions between autophagy modifiers (Atg8-family proteins) and ligands is key for developing autophagy inhibitors. Fluorescence polarization (FP) assays offer a robust method to study these interactions and quantify binding affinities.

Keywords:
Fluorescence polarizationGABARAPHigh-throughput screeningKi determinationLC3LIR motif

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Selective autophagy is a crucial cellular process regulated by Atg8-family proteins.
  • Characterizing interactions between Atg8 proteins and their ligands is vital for understanding autophagy and designing inhibitors.
  • Small molecules and peptides targeting Atg8 proteins hold therapeutic potential.

Purpose of the Study:

  • To introduce the principles and application of fluorescence polarization (FP) assays for studying Atg8-protein interactions.
  • To demonstrate the utility of FP assays in characterizing ligand binding affinity and selectivity.
  • To provide guidance on data analysis and quality control for FP assays.

Main Methods:

  • Fluorescence polarization (FP) assay.
  • Characterization of peptide interactions with human Atg8 proteins (LC3/GABARAPs).
  • Data analysis and quality control protocols for FP assays.

Main Results:

  • FP assays provide rapid, cost-effective, and robust affinity and selectivity data.
  • The study details a case study on peptide interactions with human Atg8 proteins.
  • Proper data analysis ensures accurate calculation of inhibition constants (Ki).

Conclusions:

  • FP assays are a powerful tool for characterizing molecular interactions in selective autophagy.
  • This method facilitates the design and development of targeted autophagy modulators.
  • The described protocols ensure reliable quantification of ligand binding to Atg8 proteins.