000K utf8 1100 $c2025 1500 eng 2050 urn:nbn:de:gbv:8:3-2025-00293-4 2051 10.1021/acschembio.4c00812 3000 Miletić, Nebojša 3010 Abdul Azeez, Kamal Rayees 3010 Adhikari, Bikash 3010 Berner, Nicola 3010 Elson, Lewis 3010 Gehrtz, Paul 3010 Hartung, Ingo V 3010 Hoffmann, Lasse 3010 Hoffmann, Marina E 3010 Holzmann, Hanna 3010 Knapp, Stefan 3010 Kuster, Bernhard 3010 Mosler, Thorsten 3010 Müller, Juliane 3010 Müller, Susanne 3010 Němec, Václav 3010 Rathore, Rajeshwari 3010 Schlesiger, Sarah 3010 Schwalm, Martin P 3010 Sivashanmugam, Saran Aswathaman 3010 Weckesser, Janik 3010 Wilhelm, Stephanie 3010 Wolf, Elmar 3010 Đikić, Ivan 4000 Workflow for E3 Ligase Ligand Validation for PROTAC Development$hAmerican Chemical Society (ACS) [Miletić, Nebojša] 4030 $nAmerican Chemical Society (ACS) 4209 Proteolysis targeting chimeras (PROTACs) have gained considerable attention as a new modality in drug discovery. The development of PROTACs has been mainly focused on using CRBN (Cereblon) and VHL (Von Hippel-Lindau ligase) E3 ligase ligands. However, the considerable size of the human E3 ligase family, newly developed E3 ligase ligands, and the favorable druggability of some E3 ligase families hold the promise that novel degraders with unique pharmacological properties will be designed in the future using this large E3 ligase space. Here, we developed a workflow aiming to improve and streamline the evaluation of E3 ligase ligand efficiency for PROTAC development and the assessment of the corresponding "degradable" target space using broad-spectrum kinase inhibitors and the well-established VHL ligand VH032 as a validation system. Our study revealed VH032 linker attachment points that are highly efficient for kinase degradation as well as some of the pitfalls when using protein degradation as a readout. For instance, cytotoxicity was identified as a major mechanism leading to PROTAC- and VHL-independent kinase degradation. The combination of E3 ligase ligand negative controls, competition by kinase parent compounds, and neddylation and proteasome inhibitors was essential to distinguish between VHL-dependent and -independent kinase degradation events. We share here the findings and limitations of our study and hope that this study will provide guidance for future evaluations of new E3 ligase ligand systems for degrader development. 4950 https://doi.org/10.1021/acschembio.4c00812$xR$3Volltext$534 4950 https://nbn-resolving.org/urn:nbn:de:gbv:8:3-2025-00293-4$xR$3Volltext$534 4961 https://macau.uni-kiel.de/receive/macau_mods_00005818 5051 540 5550 Adaptor Proteins, Signal Transducing 5550 Drug Discovery 5550 Humans 5550 Ligands 5550 Protein Kinase Inhibitors 5550 Proteolysis 5550 Ubiquitin-Protein Ligases 5550 Von Hippel-Lindau Tumor Suppressor Protein 5550 Workflow