(A) Post-translational sites of Lys49 and Lys51on LC3B (PDB ID: 3VTU) are located near LIR peptides. Lys49 is represented in green and Lys51 is represented in magenta. FYCO1 (PDB ID: 5WRD), FUNDC1 (PDB ID: 5GMV), and p62 (PDB ID: 2ZJD) LIR peptides are aligned and are shown in orange. (B) pKa of lysine residues in LC3B predicted by PROPKA. Entry, PDB ID 3VTU; forcefield, CHARMM. (C) Construction of in-house covalent probes library. Covalent warheads that may react with lysine residues were collected from Protein Data Bank (PDB) and similarity search was performed to find probes containing these covalent warheads. (D-E) Chemical structure of the most potent inhibitor (“DC-LC3in”), and determination of the half maximal inhibitory concentration of this compound for the protein-peptide complex by FP assay.