title: Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.
abstract: The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.
authors.affiliation: 1RIKEN Systems and Structural Biology Center, Yokohama, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Tsuganezawa
authors.forename: Yoshiyasu
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Naoko
authors.initials: N
authors.lastname: Ogawa
authors.forename: Shun
authors.initials: S
authors.lastname: Tsuboi
authors.forename: Norihisa
authors.initials: N
authors.lastname: Okada
authors.forename: Masumi
authors.initials: M
authors.lastname: Mori
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanaka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002232:Carbocyanines; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines; D000071456:Sequestosome-1 Protein; D054852:Small Molecule Libraries / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D048868:Adaptor Proteins, Signal Transducing; C569686:Alexa Fluor 647; D002232:Carbocyanines; D005456:Fluorescent Dyes; C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D054852:Small Molecule Libraries; D005982:Glutathione Transferase
keywords: LC3; autophagy; fluorescence; p62; protein-protein interaction