%0 Journal Article %A Schwalm, Martin P %A Lenz, Christopher %A Saxena, Krishna %A Klionsky, Daniel J %A Proschak, Ewgenij %A Knapp, Stefan %T Biochemical investigation of LC3/GABARAP-ligand interaction as an important quality measure for LC3/GABARAP-targeting small molecules: addendum to the guidelines (4th edition). %J Autophagy %V 21 %N 9 %@ 1554-8627 %C Abingdon, Oxon %I Taylor & Francis %M DKFZ-2025-00952 %P 2069-2073 %D 2025 %Z ISSN 1554-8635 / 2025 Sep;21(9):2069-2073 %X Targeted protein degradation (TPD) represents a new therapeutic modality that allows the targeting of proteins that are considered undruggable by conventional small molecules. While TPD approaches via the ubiquitin-proteasome system are well established and validated, additional degradation pathways still require rigorous characterization. Here, we focus on macroautophagy/autophagy tethering compounds, a class of small molecules, designed to recruit cargo to LC3/GABARAP proteins for subsequent autophagosome-dependent degradation. We provide guidance for the biophysical and structural characterization of small molecule modulators for studying LC3/GABARAP-ligand interactions. In addition, we discuss potential limitations of autophagy-based TPD systems and emphasize the need for rigorous quality control in the development of LC3/GABARAP-targeting small molecules.Abbreviations: DSF: differential scanning fluorimetry; FP: fluorescence polarization; FRET: Förster/fluorescence resonance energy transfer; HTRF: homogeneous time-resolved fluorescence; ITC: isothermal titration calorimetry; LIR: LC3-interacting region; MGs: molecular glues; NMR: nuclear magnetic resonance; PROTACs: PROteolysis-TArgeting Chimeras; SPR: surface plasmon resonance; TPD: targeted protein degradation; TR-FRET: time-resolved Förster/fluorescence resonance energy transfer; UPS: ubiquitin-proteasome system. %K ATTECs (Other) %K AUTACs (Other) %K Atg8 (Other) %K GABARAP (Other) %K LC3 (Other) %F PUB:(DE-HGF)16 %9 Journal Article %$ pmid:40344429 %R 10.1080/15548627.2025.2498506 %U https://inrepo02.dkfz.de/record/301267