Workflow for E3 Ligase Ligand Validation for PROTAC Development
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.
Authors
- Ingo V. Hartung (ORCID: https://orcid.org/0000-0001-8750-679X)
- H. Holzmann
- Bikash Adhikari (ORCID: https://orcid.org/0000-0002-9064-0356)
- Paul Gehrtz
- Nicola Berner
- Stefan Knapp (ORCID: https://orcid.org/0000-0001-5995-6494)
- Marina E. Hoffmann (ORCID: https://orcid.org/0000-0002-5381-3043)
- Rajeshwari Rathore
- Stephanie Wilhelm (ORCID: https://orcid.org/0000-0002-1066-6565)
- Václav Němec (ORCID: https://orcid.org/0000-0001-6900-1368)
- Thorsten Mosler (ORCID: https://orcid.org/0000-0003-1800-2680)
- Kamal R. Abdul Azeez (ORCID: https://orcid.org/0000-0001-9985-0336)
- Martin P. Schwalm (ORCID: https://orcid.org/0000-0002-1252-1829)
- Janik Weckesser (ORCID: https://orcid.org/0000-0001-5100-2776)
- Bernhard Küster (ORCID: https://orcid.org/0000-0002-9094-1677)
- Susanne Müller (ORCID: https://orcid.org/0000-0003-2402-4157)
- Sarah Schlesiger (ORCID: https://orcid.org/0000-0001-7880-3936)
- Lewis Elson (ORCID: https://orcid.org/0009-0004-6142-3653)
- Elmar Wolf (ORCID: https://orcid.org/0000-0002-5299-6335)
- Ivan Đikić (ORCID: https://orcid.org/0000-0001-8156-9511)
- Lasse Hoffmann
- Juliane Müller (ORCID: https://orcid.org/0000-0003-2126-6521)
- Saran Aswathaman Sivashanmugam
- Nebojša Miletić
Institutions
- Goethe University Frankfurt (DE)
- Center for Global Development (US)
- Merck KGaA, Darmstadt (Germany) (DE)
- German Cancer Research Center (DE)
- Christian-Albrechts-Universität zu Kiel (DE)
- Medicina (PL)
- Institute of Biochemistry (RO)
- Frankfurt Cancer Institute (DE)
- Deutsches Konsortium für Translationale Krebsforschung (DE)
- Technical University of Munich (DE)
Publication Details
- Journal
- ACS Chemical Biology
- Published
- 2025-02-11
- DOI
- https://doi.org/10.1021/acschembio.4c00812
- Citations
- 13
- Primary Topic
- Protein Degradation and Inhibitors
- Type
- article
- Field-Weighted Citation Impact
- 10.48
Funders
- Deutsches Krebsforschungszentrum
- Deutsche Forschungsgemeinschaft
- Bundesministerium für Bildung und Forschung
- Fraunhofer-Gesellschaft
- Hessisches Ministerium für Wissenschaft und Kunst
- Technische Universität München
- Deutsche Krebshilfe
- Innovative Medicines Initiative
- Deutschen Konsortium für Translationale Krebsforschung
- LOEWE-Zentrum für Translationale Medizin und Pharmakologie