TY  - JOUR
AU  - Miletić, Nebojša
AU  - Weckesser, Janik
AU  - Mosler, Thorsten
AU  - Rathore, Rajeshwari
AU  - Hoffmann, Marina E
AU  - Gehrtz, Paul
AU  - Schlesiger, Sarah
AU  - Hartung, Ingo V
AU  - Berner, Nicola
AU  - Wilhelm, Stephanie
AU  - Müller, Juliane
AU  - Adhikari, Bikash
AU  - Němec, Václav
AU  - Sivashanmugam, Saran Aswathaman
AU  - Elson, Lewis
AU  - Holzmann, Hanna
AU  - Schwalm, Martin P
AU  - Hoffmann, Lasse
AU  - Abdul Azeez, Kamal Rayees
AU  - Müller, Susanne
AU  - Kuster, Bernhard
AU  - Wolf, Elmar
AU  - Đikić, Ivan
AU  - Knapp, Stefan
TI  - Workflow for E3 Ligase Ligand Validation for PROTAC Development.
JO  - ACS chemical biology
VL  - 20
IS  - 2
SN  - 1554-8929
CY  - Washington, DC
PB  - Soc.
M1  - DKFZ-2025-00338
SP  - 507-521
PY  - 2025
N1  - 2025 Feb 21;20(2):507-521
AB  - 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.
LB  - PUB:(DE-HGF)16
C6  - pmid:39932098
DO  - DOI:10.1021/acschembio.4c00812
UR  - https://inrepo02.dkfz.de/record/298895
ER  -